Molecular Biology Notes PDF

Molecular Diology exam prep Cell composition and structure We have 5 areas of study within cell study: Cytology, Cytochemistry, Cytophysiology, Cytogenetics and Cytopathology Cytology De nition: The study of cells, their structure, and their function. Key Focus: Examines cell morphology (...

Molecular Diology exam prep Cell composition and structure We have 5 areas of study within cell study: Cytology, Cytochemistry, Cytophysiology, Cytogenetics and Cytopathology Cytology De nition: The study of cells, their structure, and their function. Key Focus: Examines cell morphology (shape, size, and arrangement) and how cells are organized within tissues. Example: Studying blood cells under a microscope to identify normal versus abnormal cells. Cytochemistry De nition: The branch of cytology that focuses on the chemical composition of cells and their biochemical processes. Key Focus: Uses staining techniques to detect speci c molecules (like DNA, proteins, or enzymes) within cells. Example: Staining a cell nucleus to observe DNA or enzymes to study metabolic activity. Cytophysiology De nition: The study of cell function, including how cells carry out their physiological processes. Key Focus: Explores how cells grow, divide, communicate, and maintain homeostasis. Example: Investigating how insulin triggers glucose uptake in cells. Cytogenetics De nition: The study of the genetic material within cells, particularly chromosomes, and how they in uence inheritance and cellular functions. Key Focus: Analyzing chromosomal structure and abnormalities to understand genetic disorders. Example: Examining chromosomes to diagnose Down syndrome or detecting translocations in cancer. fi fi fi fi fl fi Cytopathology De nition: The study of disease-related changes in cells, particularly their structure and function. Key Focus: Identifying cellular abnormalities to diagnose diseases like cancer or infections. Example: Performing a Pap smear to detect precancerous changes in cervical cells. The cell Cells are the smallest living structural and functional unit that compromise all organisms. They are formed by the division of other cells. A large part of the cells contain genetic information which is passed on to daughter cells during cell division. All cells are made up of the same chemical compounds. All metabolic processes necessary for life occur in cells. We have to types of cells prokaryotic and eukaryotic: fi Eukaryotic organisms: We have two types of eukaryotic organisms: Single cell which are Protozoa, some algae and fungi Multi cell which are plants, animals and fungi Components of eukaryotic cells: A eukaryotic cell needs to consists of the following components which all have speci c function: Cytoplasm CytoskeletonY Nucleus v Endoplasmic reticulum - Mitochondria - Golgi apparatus ~ Lysosomes - Peroxisomes ~ Prokaryotic cell components: Prokaryotic cells contain: Cell membrane - Cell wall v Capsule also called mucus v Flagella also called cilia - Cytosine v Nucleotid which as the same function of cell nucleus ~ Ribosomes - Plasmids - Cell components All organisms are composed of two types of chemicals: Inorganic and organic Organic Molecules De nition: Organic molecules contain carbon atoms bonded to hydrogen (C-H bonds) and often include other elements like oxygen, nitrogen, phosphorus, and sulfur. Key Characteristics: ○ They are typically larger and more complex than inorganic molecules. ○ Found in living organisms and are essential for life processes. ○ They form the basis of biological macromolecules like carbohydrates, proteins, lipids, and nucleic acids. Examples: ○ Carbohydrates (e.g., glucose, C₆H₁₂O₆) – Provide energy. ○ Proteins (e.g., enzymes) – Perform various functions in the body. ○ DNA/RNA – Carry genetic information. ○ Lipids (e.g., fats, oils) – Store energy and make up cell membranes. 2. Inorganic Molecules De nition: Inorganic molecules typically do not contain carbon-hydrogen (C-H) bonds (though some exceptions exist, like carbon dioxide, CO₂). Key Characteristics: ○ They are often smaller and simpler. ○ Found in both living and non-living things. ○ Play vital roles in physiological processes but do not form the structure of living organisms. Examples: ○ Water (H₂O) – Essential for all life; makes up 70-80% of the body. ○ Salts (e.g., NaCl) – Help in nerve conduction and maintaining osmotic balance. ○ Minerals (e.g., calcium, Ca²⁺; magnesium, Mg²⁺) – Strengthen bones and support enzyme function. ○ Gases (e.g., oxygen, O₂; carbon dioxide, CO₂) – Crucial for respiration and cellular metabolism. fi fi Cells: The inorganic components that build cells include chemical elements such as: Macroelements Micro elements Trace elements Ultra race elements Water They also contain chemical elements such as: Water is the mail component of every organism. On average 70-80% of the content of a living cell. It’s essential for the proper functioning of the body. It’s a great solvent for many chemical compounds and reactions. It can also be a substrate and product of many chemical reactions. The reason behind why water is so great in molecular biology is because of its chemical structure and properties. Water: It’s has one oxygen atom and two hydrogen atoms A specialized dipole dipole force known as a hydrogen bond exists between the oxygen and hydrogen. This bond is inherently polarized due to the difference in electronegativity between two atoms. When we have an uneven distribution of charges the water molecule becomes depolarized The attraction of hydrogen atoms by oxygen atoms causes water molecules to combine into larger groups Carbon atom: The nucleus contains 6 protons and 6 neutrons and two shells one with 2 electrons and the second one with 4 electrons. A carbon atom has 4 valence electrons and 4 vacancies for electrons It has a unique role because of its ability to form a strong covalent bond with other carbon atoms Carbon can join to form: chains, branched structures and rings Organic compounds consist of carbon atoms bonded to at least one other element. Such as hydrogen and oxygen and nitrogen, sulfur and phosphorus Organic components: We have four major families of small organic which are containing carbon and hydrogen molecules: saccharides Fatty acids Amino acids Nucleotides They all are carbon compounds that contain up to 30 carbon atoms Are found free in solution in the cytoskeleton Carbohydrates: Saccharides which contain carbon, hydrogen and oxygen Energy: Glycogen in animals Starch in plants Structure: Cellulose in cell walls of plants Chitin in the cell walls in fungi Ribose and deoxyribose sugars of DNA and RNA Modi ers of protein Transport: Glucose in animals and humans Sucrose in plants fi Fatty acids Contains an even number of carbon atoms 14 to 24 They have a carboxyl group connected to a hydrocarbon chain which is fat The shorter the chain, the more uid the fatty acids is called saturated fatty acids and have only single bonds Unsaturated fatty acids have one or more double bonds Lipids Esters of fatty acids bonded to alcohols Examples: Glycerol Spingosine Higher monohydric alcohol They are insoluble in water due to low ability to polarize under the in uence of water They are composed of 4 rings which are called sterane. The most simple lipids are esters formed from alcohols and fatty acids: Fats and oils triglycerides Waxes which are esters with non glycerol alcohols The more complex lipids are made of an alcohol, fatty acids and molecules such as phosphoric acid (phospholipid) and carbohydrate (glycolipid) Lipids are the building blocs of biological membranes They also have a form of energetical storage such as subcutaneous tissue in hibernators and seeds in plants fl Lipid function: Is serves as a form of protection in form of fat. We can nd it anywhere in the body. In plants and leaves it protects from exessive water loss in the form of wax coverings In seals from low temperatures Cell composition and structure: The internal cell environment is separated f from the external environment by a cell membrane or by an additional cell wall which we nd in bacteria and plant cells The internal envirorment of the cell is known as the cytoplasm containing cytoskeleton and organelles Organelles are either membrane bound or non membrane bound All the organelles are surrounded by a cell/plasma membrane. All cell membranes, both extracellular and intracellular consist of lipids, proteins and sugars. Functions of the cell membrane: ❑ protect from physical, chemical and biological factors ❑ react to chemical, thermal and mechanical stimuli ❑ enzymatic- catalysis of various metabolic reactions ❑ regulate transport of substances into and out of the cell ❑ maintain the balance of osmotic pressure between the inside and the outside of the cell fi Cytoplasm: The cytoplasm is like a gel-like solution inside the cell. It has particles that: Are too small to settle down (won't sink). Are too big to completely dissolve in water. The cytoplasm has two parts: 1. Dispersive phase: Mostly water (90% of the cytoplasm). 2. Dispersed phase: Particles oating in the water, made up of: 1. 9% organic compounds (e.g., proteins, sugars). 2. 1% minerals (e.g., salts). Functions: ○ Fill the cell and give shape ○ Envirorment for suspending cell organelles ○ Site of metabolic reaction ○ Move organelles and transport substances in the cell due to movement of the cytoplasm The cytoplasm is exible and thick because it has a lot of proteins. It can exist in two forms: 1. Semi-liquid (sol) – more uid. 2. Semi-solid (gel) – thicker and rmer. The cytoplasm can move in different ways: Rotationally: Moves around a central vacuole. Circulating: Moves between organelles. Pulsating: Moves back and forth in different directions. Fountaining: Flows around two vacuoles in opposite directions. fl fl fl fi  Cytoskeleton: It’s the skeleton inside the cell. It’s a system of laments with three families of protein Intermediate laments: Diameter form 8 to 10 nm Tissue speci c proteins such as keratin They give cells resistance to mechanical damage, stretching and crushing Help maintain a speci c cell shape Build the cell nuclear laminate Microtubules: Diamenter of about 25nm Composed of tubulin They have several function such as: Building centrioles and mitosis spindle, Transport within the cell due, Formation of cilia and agella In cells that do not divide such as neurons, microtubules group together in a region called the centrosome, Actin laments: Diamenter of about 7 nm Made out of actin Several functions such as: Provide mechanical support for the cell and various cell organelles. Involved in the movement of cytoplasms and organelles Movement and cell chape change Contraction of muscle cells fl fi fi fi fi fi  Cell structure: Organelles Membrane bound organelles are divided into: - Double membrane bound Single membrane bound Non membrane bound Organelles surrounded by a double membrane are: Nucleus, genetic information Mitochondria, the site of cellular respiration Chloroplasts, a group of organelles in plants Organelles surrounded by a single cell membrane: Golgi apparatus, modi es proteins Lysosomes, digestive enzymes Peroxisomes, compounds to breakdown peroxides ER, Network of channels and attened cisternae site of protein production Vacuoles, animal cell: waste product:::: Plant cell: garbage bins of the cell and water balance Non-membrane bound organelles: ❑ cell wall - outer covering of some non-animal cells ❑ cytoskeleton - provides cell structure ❑ ribosomes - location of protein synthesis ❑ centrosome / microtubule organizing center – contain centrioles and microtubules important in cell division ❑ centriole – cylindrical organelle involved in spindle bers creation in cell division fi fi fl Nucleus: One nucleus (monokaryocytes): Most cells. Two nuclei (bikaryocytes): Some specialized cells. Multiple nuclei (polykaryocytes): Certain cells, like muscle cells. No nucleus: Red blood cells (erythrocytes) and outer skin cells (stratum corneum). Size and shape: Depends on the cell type, age, and activity. Can be round, oval, or fragmented. Takes up about 10% of the cell's volume. Position: Usually in the center of the cell. Sometimes near the cell membrane, depending on the cell type. The cell nucleus can be in three different states: ❑ interphase– between/preparation of cell division ❑ mitotic - during cell division ❑ metabolic – present in cells in the resting or G0 phase; directs metabolic processes, maintenance functions Interphase: Nuclear Envelope: A membrane that surrounds and protects the nucleus. Nuclear Matrix (Nucleoplasm): The uid inside the nucleus. Nucleolus: The part of the nucleus that makes ribosomes. Chromatin: The DNA and proteins inside the nucleus, which can be: Condensed (Heterochromatin): Tightly packed, less active. Dispersed (Euchromatin): Loosely packed, more active in making RNA. Nucleolus The nucleus usually contains one nucleolus, unseparated from the nucleoplasm (no membrane). It consists of fragments of ve chromosomes, containing DNA responsible for the synthesis of ribosomal RNA (rRNA) and ribosomal subunits. These regions are called nucleolar organizers (NOR) In humans, there are 10 NORs, which are located on the short fl fi Functions of the nucleus: Functions of the nucleus: ❑ site of DNA synthesis - replication of genetic information before nuclear division ❑ site of synthesis of RNA from DNA (transcription) ❑ site of formation of ribosomes– the structures responsible for protein synthesis (translation The endoplasmic reticulum: The **endoplasmic reticulum (ER)** is a network of single-layer membranes that form interconnected cisternae, channels, and vesicles within the cell. It plays a critical role in increasing the cell’s internal surface area, dividing the cytoplasm into compartments, and directing the transport of organelles, substrates, and products. The **smooth ER** lacks ribosomes and is responsible for the synthesis of lipids and steroids, detoxi cation of harmful substances, and internal transport within the cell. In contrast, the **rough ER** has ribosomes on its surface, which are involved in protein synthesis, modi cation, and quality control. The rough ER also connects the outer nuclear membrane to the cell membrane and other organelles, ensuring ef cient communication and transport within the cell. Ribosomes: Ribosomes are cellular structures composed of ribosomal RNA (rRNA) and proteins. In eukaryotic cells, there are two main types of ribosomes: **free ribosomes**, which oat in the cytoplasm and produce proteins that function within the cytosol, and **bound ribosomes**, which are attached to the endoplasmic reticulum and produce proteins that undergo post- translational modi cations and are exported from the cell. Additionally, ribosomes in mitochondria and chloroplasts are smaller and resemble bacterial ribosomes. Each ribosome consists of two subunits: one small and one large. Ribosomes are classi ed based on size, with prokaryotic ribosomes being smaller (70S) and eukaryotic ribosomes being larger (80S). These structural differences re ect their specialized roles in protein synthesis. fi fi fl Mitochondria: The number of **mitochondria** in a cell varies depending on the organism, cell type, and its energy needs. Mitochondria range in size from 2 to 8 μm and can quickly change shape and size, appearing lamentous, granular, or branched. They replicate by dividing from existing mitochondria. The number of mitochondria differs by cell type: epidermal cells have 2-6, sperm cells 20-50, liver cells 1,000-2,500, skeletal muscle bers up to 1,600, skin cells ~2,000, nerve cells ~10,000, and ova more than 100,000. Structurally, mitochondria have a **two-layer membrane**. The smooth outer membrane allows passive transport of many substances, while the inner membrane, with folds called cristae, selectively allows compounds to pass via facilitated diffusion. Between these membranes is the intermembrane space, and inside is the mitochondrial matrix, which contains double-stranded circular mitochondrial DNA (mtDNA), 70S ribosomes, and enzymes for ATP production. Each mitochondrion has 4-10 mtDNA molecules organized into protein complexes called nucleoids. Functionally, mitochondria are critical for **aerobic respiration**, including the Krebs cycle and electron transport chain, to produce ATP, the energy currency of the cell. They deliver ATP throughout the cytoplasm and can move within the cell. Interestingly, mitochondria divide in a manner similar to bacteria, re ecting their evolutionary origin. fi fl Golgi apparatus: The **Golgi apparatus** is a cell organelle made up of 3-20 highly attened, arched structures called **cisternae**, along with separating vesicles. It has three functional regions: the **cis cisternae** (facing the nucleus and endoplasmic reticulum, where vesicles with materials for processing enter), the **medial cisternae** (middle layer), and the **trans cisternae** (facing the cell membrane, where processed products are released). The Golgi apparatus receives materials from the perinuclear endoplasmic reticulum, the cell membrane, and endosomes. Its key functions include: - **Post-translational modi cation** of proteins and lipids for export. - Linking carbohydrates to proteins, fats, and nucleosides. - Sulfation of proteins and proteoglycans. - Recycling cell membrane components after endocytosis. Additionally, secretory proteins, hydrolases, and some integral membrane proteins are synthesized in precursor forms. The Golgi apparatus processes and matures these precursors through controlled proteolysis, as seen in the well-known mechanisms of **proalbumin** and **preproinsulin** maturation. fi Lysosomes: **Lysosomes** are membrane-bound vesicles involved in intracellular digestion, varying in shape and size depending on the cell type and function. For example, lysosomes in macrophages are several microns in size, while those in hepatocytes and neurons are 0.5 to 1 μm. In hepatocytes and broblasts, lysosomes occupy ~0.5% of the cytoplasm, increasing to ~2.5% in macrophages. Each lysosome contains about 40 **hydrolytic enzymes** (acid hydrolases) that function in acidic conditions (pH 5), maintained by a proton pump (H⁺-ATPase). The lysosomal membrane is resistant to these enzymes due to specialized proteins. Lysosomes are classi ed as: 1. **Primary lysosomes**: Formed in the rough ER and Golgi apparatus, they contain inactive enzymes. 2. **Secondary lysosomes**: Formed by the fusion of primary lysosomes with other vesicles, such as: - **Autophagosomes**: Contain the cell’s own components (e.g., damaged organelles). - **Endosomes**: Contain material taken up by endocytosis, which can be subdivided into **phagosomes** (solid particles) and **pinosomes** ( uids). Secondary lysosomes are further divided into: - **Autolysosomes**: Involved in autophagy (removal of damaged organelles) and autolysis (digestion of dying or dead cells). - **Heterolysosomes**: Formed by fusion with endocytosed material. Byproducts of lysosomal digestion, such as simple sugars, amino acids, and nucleotides, are released into the cytoplasm for reuse in cellular processes. fi fi fl Perixosomes: **Peroxisomes** are small, oval or spherical organelles enclosed by a single membrane, with diameters ranging from 0.2 to 1.8 μm. Their number, shape, and function vary depending on the cell type, tissue, developmental stage, and cellular stress. They are most abundant in liver, kidney, and nervous tissue. The granular matrix inside peroxisomes may contain a crystalline core called the **nucleoid**, which varies by species and tissue type. Peroxisomes are involved in over 60 **catabolic and anabolic processes**, including: - **Detoxi cation**: Breaking down toxic compounds, like converting ethanol into acetaldehyde. - **Fatty acid oxidation**: Breaking long-chain fatty acids (C22) into shorter chains (C8) via β- oxidation and modifying branched fatty acids through α-oxidation. - **Synthesis**: Producing cholesterol, bile acids, and plasmalogens (essential for the myelin sheath in neurons). Hydrogen peroxide (H₂O₂), a byproduct of fatty acid and alcohol oxidation, is broken down within peroxisomes by **catalase** or **peroxidases** to prevent toxicity. Peroxisomes form either **de novo** (from vesicles budding off the ER and mitochondria) or by division of pre-existing peroxisomes. During **de novo formation**, vesicles mature into peroxisomes by recruiting enzymes, peroxins, and membrane proteins. Division of existing peroxisomes involves the formation of a tubular structure, a constricting ring, and the production of two daughter peroxisomes. fi Centrosome: Structure found near the cell nucleus and the Golgi The centrosome is made up of two cylindrical structures called centrioles which are composed of microtubules. Before a cell divides, the centrosome duplicates itself, forming two centrosomes (each with two centrioles). These centrosomes then move to opposite ends of the cell to help organize and guide cell division. Plant cells: Plant cell contains additional components: Living plastic components such as plastids Dead no plastic components Cell wall and vacuole Plastids: **Plastids** are oval-shaped organelles surrounded by a double membrane. They contain their own DNA and ribosomes, allowing them to function independently in some ways. All plastids develop from precursor structures called **proplastids**. There are three main types of plastids: 1. **Chloroplasts**: Contain the green pigment **chlorophyll**, enabling **photosynthesis** to occur. 2. **Chromoplasts**: Contain pigments like **xanthophyll** and **carotenoids**, which give color to fruits, owers, and leaves. 3. **Leucoplasts**: Colorless plastids with storage functions, divided into: - **Proteinoplasts**: Store proteins in the form of aleurone grains. - **Amyloplasts**: Store carbohydrates as starch grains. - **Lipidoplasts**: Store fats. Plastids play diverse roles in energy production, pigmentation, and storage, depending on their type. fl Cell wall Plant cells have a multi-layered cell wall composed mainly of cellulose or chitin. Cellulose is a polymer of glucose, made of carbon, hydrogen, and oxygen, while chitin is a polymer of N- acetylglucosamine, which contains nitrogen along with carbon, hydrogen, and oxygen. There are two types of cell walls: the primary cell wall, made of cellulose and pectin, which forms during cell growth, and the secondary cell wall, made of cellulose and lignin, which forms after growth is completed. The cell wall serves several important functions: it provides shape and rigidity to the cell, limits further growth, and protects the cell from mechanical damage, infections caused by bacteria, fungi, and viruses, as well as excessive water loss. Plant vacuole The vacuole is a membrane-bound structure in plant cells, surrounded by a single membrane called the tonoplast. Inside the vacuole is a uid called cell sap, which contains several components: about 90% water, various ions (such as potassium, sodium, calcium, magnesium, zinc, sulfate, and chloride), proteins (including aleurone grains and amino acids), sugars (such as glucose, fructose in fruit, and sucrose in sugar beets), and organic acids. The vacuole serves several important functions, including maintaining cell rmness through turgor pressure, storing reserve materials, and collecting unnecessary metabolic products. fl Endosymbiotic theory Prokaryotic cells, which originated about 3.5 billion years ago, predate eukaryotic cells, which appeared around 1.7 billion years ago. The endosymbiotic theory explains the origin of mitochondria and chloroplasts in eukaryotic cells, proposing that these organelles evolved from prokaryotic cells. Several pieces of evidence support this theory: mitochondria and chloroplasts contain circular DNA similar in structure and size to bacterial DNA, they replicate by division rather than being formed anew by the cell, and their ribosomes resemble bacterial ribosomes. Additionally, mitochondria and chloroplasts use N-formylmethionine as the rst amino acid in protein synthesis, just like bacteria. Cell metabolism Metabolism encompasses all biochemical reactions occurring within the cells of living organisms. It involves the circulation of matter, energy, and information that enables vital processes such as growth, reproduction, movement, and response to stimuli. Metabolic processes occur in two directions: anabolism and catabolism. Anabolism refers to the synthesis of complex organic compounds from simpler ones, requiring an input of energy. The energy is stored in the chemical bonds of the products, which contain more energy than the substrates. Catabolism, on the other hand, involves the breakdown of complex compounds into simpler products, releasing energy that is stored in energy carriers like ATP. Cellular respiration is an example of catabolic activity where organic compounds are broken down into inorganic molecules, such as CO₂ and H₂O, to release energy. Glycolysis, the rst step of aerobic respiration, occurs in the cytoplasm, breaking glucose into pyruvic acid in a 1:2 ratio without producing ATP. In the mitochondria, pyruvic acid undergoes further oxidation into end products, such as water and carbon dioxide, releasing energy. I ntracellular respiration: Lecture two: The cell membrane Membrane structure: Everything is build according to the same scheme. They always contain: lipids, proteins, sugars which are bound to lipids or proteins. Membrane lipids are divided into three groups based on their chemical structure: Phospholipids Sphingolipids Sterols Phospholipids Phospholipids are a type of lipid made up of four main components: two fatty acids, glycerol (a type of alcohol), phosphoric acid, and a functional group attached to the phosphate. The functional group determines the speci c properties of the phospholipid. Common functional groups that can be linked to the phosphate include ethanolamine, choline, inositol, and serine. fi Sphingolipids Sphingolipids are a type of lipid composed of sphingosine (a long-chain amino alcohol), a fatty acid, and, in some cases, phosphoric acid. They also include a functional group, such as ethanolamine, choline, or serine. Sphingolipids are divided into two subgroups: sphingomyelins and glycolipids. Sphingomyelins consist of sphingosine, a fatty acid, phosphoric acid, and a functional group like serine, ethanolamine, or choline. These lipids are essential components of brain matter, neural tissue, and the myelin sheath that surrounds nerve endings. Glycolipids Glycolipids are lipids composed of three main components: sphingosine, a fatty acid, and one or more sugar molecules. The simplest glycolipids, called cerebrosides, contain a single sugar molecule, such as glucose or galactose. More complex glycolipids, known as gangliosides, can contain up to seven sugar residues. Sterols Sterols are alcohols that belong to the steroid family. The most signi cant sterol in animals is cholesterol. Cholesterol is a cyclic compound that features a branched side chain. Structure of membrane lipids Membrane lipids typically contain one or two fatty acid residues, which usually have an even number of carbon atoms (commonly 16 to 18). At least one bond in these fatty acid residues may be unsaturated, causing the fatty acid to take up more space due to its shape. Membrane lipid molecules are amphiphilic, meaning they have two distinct regions: a hydrophilic ("water-loving") polar head and a hydrophobic ("water-fearing") nonpolar tail. The hydrophilic head, depending on its structure, can either carry an electric charge or act as an electric dipole. Despite structural variations, all membrane lipids share the common feature of having a hydrophilic head and one or two hydrophobic tails. Lipid bilayer The cell membrane consists of a phospholipid bilayer, made up of two layers of phospholipids. In this structure, the hydrophilic (polar) heads are positioned on the surface of the bilayer, facing the aqueous environments inside and outside the cell, while the hydrophobic (nonpolar) hydrocarbon chains are oriented towards the interior of the bilayer. The surrounding water prevents the membrane lipids from escaping the bilayer, maintaining the membrane's integrity. Membrane proteins Membrane proteins are classi ed based on how they interact with the lipid bilayer: integral, peripheral, and surface proteins. **Integral membrane proteins** are embedded within the plasma membrane and include: monotopic proteins (attached to one side of the membrane), transmembrane proteins (spanning the entire bilayer), and polytopic proteins (crossing the membrane multiple times). Their structure is stabilized by interactions between the hydrophobic amino acid side chains and the hydrophobic hydrocarbon tails of the lipids, while the hydrophilic regions face inward, allowing polar molecules and water to pass. Integral proteins that do not fully penetrate the membrane are classi ed as outer monolayer, inner monolayer, or internal membrane proteins located within the hydrophobic region. **Peripheral membrane proteins** are located on the inner or outer surfaces of the membrane and are attached by electrostatic, hydrogen, or van der Waals forces. **Surface proteins** are found only on the membrane's outer surface and are linked via anchor motifs like protein loops or lipids. Membrane proteins serve various roles: **transport proteins** facilitate the movement of substances across the membrane, **structural proteins** connect cells or attach them to the extracellular matrix, **receptor proteins** participate in signaling pathways, and **enzymatic proteins** catalyze reactions on or within the cell. fi  Glycolipids and glycoproteins Some proteins and lipids in the outer layer of the cell membrane are covalently attached to sugars. Most membrane proteins bind to short sugar chains, called oligosaccharides, to form glycoproteins, while others attach to long polysaccharide chains to form proteoglycans. A single protein can attach to multiple sugar chains, but a single lipid attaches to only one sugar chain to form a glycolipid. These sugar chains, found only on the outer surface of the membrane, form a protective layer called the carbohydrate layer or glycocalyx. The glycocalyx plays key roles in protecting the cell surface, recognizing other cells, forming cell- to-cell contacts, and organizing cells into larger groups. Cell membrane properties The cell membrane exhibits several key features: selective permeability, uidity, asymmetry, and heterogeneity. Its primary function is to act as a barrier that regulates the passage of molecules. Small nonpolar molecules like oxygen and carbon dioxide, as well as small uncharged polar molecules like water and ethanol, can passively diffuse through the lipid bilayer. However, larger uncharged molecules like glucose and amino acids, and ions or electrically charged molecules, cannot diffuse through it. The membrane is an elastic, two-dimensional uid, and its uidity is in uenced by its composition, including cholesterol content (reduces uidity) and unsaturated fatty acids (increases uidity). The lipid bilayer's elasticity allows it to bend and return to its original shape. Lipid molecules in the membrane can perform various movements, including segmental ( exion), rotational, and lateral (within the same layer). Transverse movement, or " ip- op," where lipids move between layers, is rare and can be either uncatalyzed or enzyme-catalyzed by ippases. Factors affecting membrane uidity include the length of hydrocarbon chains, the number of unsaturated bonds, cholesterol levels, and temperature. Membrane integral proteins can undergo rotational and lateral movements, but these are slower due to their larger size, and they do not perform transverse " ip- op" movements. fl fl fl fl fl fl fl fl fl fl Restriction of lateral movement of proteins. The lateral movement of membrane proteins can be restricted by their attachment to various structures. These include the cell cortex, which is located inside the cell; extracellular matrix molecules, which are found outside the cell; and proteins present on the surface of neighboring cells. Membrane asymmetry Membrane asymmetry refers to the difference in lipid and protein composition between the two layers, or lea ets, of the cell membrane. The outer layer is primarily composed of phosphatidylcholines and sphingomyelin, along with surface proteins, glycolipids, and glycoproteins. In contrast, the inner layer mainly contains lipids with electrically charged polar heads, such as phosphatidylserine, and lipids that can form hydrogen bonds, like phosphatidylethanolamine. Membrane heterogeneity The cell membrane is heterogeneous, meaning it is not uniform in structure. Most of the membrane is composed of a lipid bilayer, which includes phospholipids, cholesterol, glycolipids, and proteins. In addition to this bilayer, there are specialized structures called lipid rafts and caveolae. Lipid rafts are dynamic, at areas of the membrane that are rich in cholesterol and sphingolipids, and play roles in signaling and transport. Caveolae, on the other hand, are bottle-shaped invaginations of the membrane, also rich in cholesterol, sphingolipids, and caveolin, and are involved in signaling, endocytosis, and transcytosis. However, lipid rafts and caveolae are not found in the membranes of lymphocytes, erythrocytes, or nerve cells. fl fl  Membrane transport: Passive transport: Osmosis is the movement of water molecules across a cell membrane from an area of high water concentration to an area of low water concentration. Water molecules can diffuse directly through the lipid bilayer, but this process is slow. To speed up water transport, some cells have specialized channels called aquaporins. Osmosis does not require external energy input. Similarly, passive transport of charged substances depends on both the concentration gradient and the membrane potential, with the electrochemical gradient determining the direction of movement. Passive transport always occurs from high to low concentration. Simple diffusion is when solutes move across the membrane along their concentration gradient. The rate of diffusion is in uenced by factors like concentration difference, electric eld across the membrane, hydrostatic pressure gradient, permeability of the membrane, and temperature. Facilitated diffusion is another form of passive transport that does not require energy. It involves membrane proteins, either protein channels or transporters, to help move substances across the membrane. Ion channels: Facilitated diffusion can occur through ion channels, which are protein structures that connect the intracellular and extracellular spaces and are lled with water. These ion channels are selective, meaning they allow only speci c ions to pass through based on factors like the diameter and shape of the channel, the arrangement of charged amino acids lining the channel, and the type of ion (such as anions or cations). The main function of ion channels is to temporarily increase the membrane's permeability to speci c inorganic ions. Ion channels can either be open, allowing ions to pass freely, or closed, permitting ions to pass through periodically. The opening and closing of these channels are controlled by external stimuli, such as temperature, electrochemical gradient, mechanical stimuli, and concentration gradient. The concentration of the opening stimulus in uences how many channels are open at any given time, making this a highly ef cient form of transport. fi fl fi fi fi fl Transporters: Transporters, also known as carrier proteins, are responsible for moving small water-soluble organic molecules and some inorganic ions across cell membranes. These transporters are highly selective, typically transporting only one type of solute. They function by opening on one side of the cell membrane at a time, but never on both sides simultaneously, ensuring that the transport process is controlled and ef cient. Facilitated diffusion Facilitated diffusion through a carrier protein involves the protein transitioning through different conformational states. For glucose transport, the carrier alternates between an **outward open state**, where binding sites are exposed to the outside of the membrane; a **closed state**, where the binding sites are inaccessible from both sides; and an **inward open state**, where binding sites are exposed to the cell's interior. This process depends on the concentration gradient, the rate of interaction between the carrier protein and the transported substance, the speed of conformational changes in the protein, and hormonal regulation. For example, **insulin** enhances glucose transport into adipose and muscle tissues, as well as amino acid transport in the liver. Similarly, **glucocorticoids** increase amino acid transport into liver cells. Coupled transport Coupled transport is a form of carrier-mediated facilitated diffusion in which the transporter protein has binding sites for two different substances. The direction of transport depends on the relationship between the movement of these substances. In **symport**, both substances are transported in the same direction across the membrane. In **antiport**, the substances move in opposite directions. This coordinated movement allows the ef cient transfer of molecules or ions across the cell membrane. fi Active transport Active transport moves substances against their concentration gradients, requiring energy input to supply the cell with essential molecules like amino acids, sugars, sodium, and potassium ions while maintaining proper osmotic pressure. There are two types of active transport: **primary** and **secondary**. In **primary active transport**, energy (e.g., from ATP hydrolysis) directly powers the transport process, as seen in the sodium-potassium (Na+/ K+) pump, which uses ATP to export Na+ out of the cell and import K+, maintaining their concentration gradients. **Secondary active transport** relies on the electrochemical gradient of one substance, such as Na+, to drive the co-transport of another molecule, like glucose, against its gradient. For example, glucose transport uses the Na+ gradient to actively import glucose into the cell. Bulk transport Bulk transport is a process used to move large molecules, such as amino acids and proteins, across the cell membrane. Since these molecules cannot pass directly through the membrane, their transport requires temporary disruption of the membrane and is facilitated by vesicles. Bulk transport includes two main types: **endocytosis** and **exocytosis**. In **endocytosis**, substances like bacteria, viruses, or cell fragments are engulfed by the cell membrane, which forms a vesicle to enclose and internalize the material. Conversely, **exocytosis** involves the removal of waste or the secretion of substances, such as hormones, where a vesicle containing the material fuses with the cell membrane to release its contents outside the cell. Transmembrane transport Endocytosis, a type of bulk transport, can be categorized into three types: **phagocytosis**, **pinocytosis**, and **receptor-mediated endocytosis**. Each type involves distinct processes for internalizing substances into the cell. **Phagocytosis** involves the uptake of macromolecules or bacteria, forming a vesicle called a phagosome. This phagosome fuses with a primary lysosome to create a secondary lysosome, facilitating substance breakdown. **Pinocytosis** involves the uptake of uids and dissolved substances, forming vesicles called pinosomes. Similar to phagocytosis, these pinosomes fuse with primary lysosomes to form secondary lysosomes for substance processing. **Receptor-mediated endocytosis** begins with the binding of speci c molecules (ligands) to receptors located on the cell membrane, forming a specialized pit. An endosome is then formed and progresses from an early to a late stage, ultimately delivering its contents to speci c cellular compartments or lysosomes for further processing. Lysosomal degradation The breakdown of macromolecules occurs in **lysosomes**, specialized organelles containing a variety of speci c enzymes known as **hydrolases**. These enzymes are responsible for hydrolyzing different types of biomolecules, including proteins, lipids, nucleic acids, and carbohydrates, ensuring proper cellular recycling and waste disposal. fi fi fl Protein degradation In a healthy organism, **3-5% of proteins are degraded**, while in diseased states, protein breakdown increases signi cantly. Protein metabolism is under **strict regulation** to maintain cellular function. In healthy cells, proteins are degraded if they have reached the end of their lifespan, have an improper structure, are damaged, or exist in excessive amounts. In eukaryotic cells, protein degradation occurs through two main pathways: **lysosomal proteolysis** and **proteasomal proteolysis**. Lysosomal proteolysis is **non-selective** and involves the degradation of exogenous proteins and old endogenous proteins, such as structural proteins. Proteasomal proteolysis is **selective** and relies on **ubiquitination**, a process where proteins marked with ubiquitin are targeted for degradation by the proteasome. Ubiquination The **ubiquitin system** is essential for protein degradation and involves several key components: **ubiquitin**, **ubiquitin-activating enzyme (E1)**, **ubiquitin-conjugating enzyme (E2)**, **ubiquitin ligase (E3)**, **proteasome**, and **deubiquitinase (DUB)**. Ubiquitin, a **globular protein** made of 76 amino acids, plays a crucial role by attaching to the protein targeted for degradation. This allows the **proteasome** to recognize and degrade the protein. Ubiquitin consists of **alpha-helical segments** (shown in blue) and **beta-sheets** (shown in green), which form its structure. The ubiquitination process ensures that only the appropriate proteins are broken down by the proteasome, maintaining proper cellular function. fi Proteasomes Proteasomes are found in all eukaryotic cells and play a crucial role in breaking down proteins that are tagged with ubiquitin. They are located in both the **cytoplasm** and the **nucleus** of the cell. The number of proteasomes in a cell can vary depending on the cell’s need for protein degradation, with an average of about 30,000 proteasomes per eukaryotic cell. These proteasomes are large, high-molecular-weight enzyme complexes that perform several functions. They **bind to** the target protein, **unfold** it, and bring it into the central part of the proteasome (a cylindrical structure). Once inside, the protein is **cut into small peptides**, and these peptides are then released from the ends of the cylinder. This process requires **energy from ATP hydrolysis** to function. Structurally, the proteasome consists of 28 protease units in a cylindrical shape, with **active sites** directed inward. The ends of the cylinder are closed by large protein complexes that act as **plugs**. Organelles degradation Dying organelles, such as mitochondria, endoplasmic reticulum membranes, nuclei, and peroxisomes, send signals that trigger the formation of autophagosome membranes. Autophagosomes then enclose the damaged organelles, isolating them from the rest of the cytosol. After joining with a primary lysosome, the autophagosome forms a secondary lysosome called an **autolysosome**, where the organelles are degraded. Speci c types of degradation include **nucleophagy**, which targets the nucleus, especially in cases of DNA damage or improper chromosome separation during cell division. This process leads to the formation of micronuclei, containing chromosome fragments or whole chromosomes. **Mitophagy** targets damaged mitochondria, often triggered by **hypoxia** (oxygen deprivation). **Lysophagy** involves the degradation of lysosomes, marked by increased permeability of their membranes and the appearance of lysosomal proteins in the cytoplasm. **Ribophagy**, initiated by a demand for nitrogen and amino acids like arginine and leucine, results in the breakdown of ribosomes. Lastly, **proteaphagy** involves the degradation of proteasomes, where receptors bind to autophagosome proteins to facilitate the process. 3rd presentation: Cell signaling Homeostasis: Organisms maintain a relatively stable internal environment despite external changes through a process called **homeostasis** ("homeo" meaning similar and "stasis" meaning state). When homeostasis is disrupted, the organism attempts to restore balance. If successful, stability is regained; if unsuccessful, the disruption can lead to disease. Key components of homeostasis include **extracellular uid (ECF)**, which links the external environment and cells, and **intracellular uid (ICF)**, located inside cells. Both uids remain relatively stable, but their dynamic steady state involves constant substance movement between them. This steady state is not equilibrium because concentrations of substances differ between ECF and ICF, creating a purposeful imbalance. In multicellular organisms, homeostasis requires coordination at various levels, including cells within tissues, tissues within organs, and systems within the whole organism. To maintain balance, cells cooperate by exchanging information through **intercellular signaling**. fl fl Intercellular signaling Intercellular signaling is crucial for essential cellular processes such as survival, division, differentiation, and death. Coordination between cells relies on the transmission of signals, which can be either **electrical**, involving changes in the cell's membrane potential, or **chemical**, involving molecules secreted into the extracellular space. Cells that respond to these signals are known as **target cells**. Communication between cells can be classi ed based on the distance the signal travels: **local communication**, which includes **juxtacrine**, **paracrine**, and **autocrine** signaling, and **distant communication**, which involves **endocrine (hormonal)** and **neuronal** signaling. These mechanisms ensure precise coordination of cellular activities across the body. Types of cell communication: Juxtacrine communication involves direct contact between cells, allowing the transfer of signaling molecules. This can occur through **gap junctions**, which enable direct exchange of molecules between adjacent cells. Alternatively, a signaling molecule located on the surface of one cell's membrane can bind to a receptor on the membrane of a neighboring cell. Cellular receptors, which are specialized proteins, play a central role in this process by receiving, transforming, and transmitting signals from the external environment to intracellular effectors, enabling an appropriate cellular response. Paracrine communication involves molecules released by a cell into the extracellular uid, which act on nearby neighboring cells. In contrast, autocrine communication occurs when molecules secreted by a cell act on the same cell that released them. Endocrine communication operates through the endocrine system, where hormones— chemical compounds secreted into the bloodstream—are distributed throughout the body via the circulatory system. Although hormones contact most cells in the body, only speci c cells with appropriate receptors, known as target cells, respond to the hormonal signal. fl Neuronal (synaptic) communication in the nervous system involves a combination of electrical and chemical signals to transmit information over long distances. An electrical signal travels along the neuron until it reaches its endpoint, where it is converted into a chemical signal. Neurons communicate with each other at specialized junctions called synapses. The chemicals released by neurons during this process are referred to as neurocrine molecules. Neurocrine molecules include neurotransmitters, which diffuse across the synaptic cleft to exert a rapid effect, and neurohormones, which enter the bloodstream and are distributed throughout the body, resulting in a slower but widespread effect. Cells communication principles Cells respond selectively to signaling molecules because only those with the appropriate receptors can detect and react to a speci c signal. The cellular response to a signal depends on the cell's functional specialization and the type of receptor involved, not the signaling molecule itself. For example, a single signaling molecule can interact with different receptors, inducing various changes in different target cells. Additionally, the same signaling molecule may trigger multiple effects within a single target cell. Acetylcholine illustrates this concept, as it can cause a wide range of reactions depending on the cell type and receptor it interacts with. Intercellular communication Signaling molecules, known as ligands, bind to receptors to deliver information to target cells. Ligands are classi ed based on their interaction with receptors: **agonists** stimulate the receptor, while **antagonists** inhibit it. When a ligand binds to a receptor, it activates the receptor, which then triggers one or more intracellular signaling molecules. These molecules ultimately generate a nal response, such as protein modi cation or synthesis. Ligands are further divided into two categories: those that are small and hydrophobic can pass through the cell membrane and require intracellular receptors, while large or hydrophilic ligands cannot cross the membrane and require cell-surface receptors. fi fi fi Types of receptors: Types of Cellular Receptors Receptor Classi cation - Cell-Surface Receptors** are divided into three main categories: * **Ion Channel-Linked Receptors** * **G Protein-Coupled Receptors** * **Enzyme-Linked Receptors** **Intracellular Receptors**: - Located in **cytoplasm and nucleus** - Bind to **small, hydrophobic molecules** that can diffuse through cell membranes * Examples include **steroid hormones**, **thyroid hormones**, **vitamin D**, and **retinoic acid** **Nuclear Receptor Structure**: - Composed of key structural elements: * **Ligand Binding Domain (LBD)** * **DNA Binding Domain (DBD)** * **Hinge Region** controlling nuclear movement * **Transcription-Activating Domain** **Receptor Binding and Activation**: - Bind to **Hormone Response Element (HRE)** sequences of 15 nucleotides - Can exist as **monomeric or dimeric** structures - Dimers can be **homodimers or heterodimers** **Receptor Families**: - Include receptors for: * Lipophilic hormones * Active vitamin A * Active vitamin D3 * Unidenti ed "orphan" receptors fi fi Steroid hormones The steroid hormone cortisol (involved in stress response) activates a target gene transcription regulator Cell surface receptors Cell-surface receptors are proteins located on/within the membrane of the target cell. The ligands are large and hydrophilic molecules that cannot diffuse through the cell membrane. All cell-surface receptor proteins bind to an extracellular signal molecule and transduce its message into one or more intracellular signaling molecules that alter the cell’s behavior Ion channel linked receptors When a ligand binds to a receptor, it causes a structural change in the receptor protein, often part of an ion channel. This change alters the membrane potential and affects the permeability of the cell membrane to speci c ions like Na⁺, K⁺, Ca²⁺, or Cl⁻. Ions then ow through the open channel according to their concentration gradients. Most neurotransmitter receptors work this way, producing a rapid response. The binding of the ligand triggers a sequence: the receptor changes shape, the membrane depolarizes or hyperpolarizes, and an action potential may be generated as the channel opens to allow ion ow. The type of ion channel involved depends on the stimulus: voltage-gated, ligand-gated by extracellular or intracellular ligands, or mechanically gated. Ion channels are selective, allowing only speci c ions to pass—like sodium, potassium, or calcium. Voltage-gated ion channels, for example, respond to changes in membrane potential. They stay closed when the cell is at rest, open during depolarization, temporarily inactivate (refractory period), and close again after repolarization. Ligand-gated ion channels activated by neurotransmitters translate chemical signals into electrical ones. This process leads to changes in membrane potential, depolarization, and the potential generation of an action potential. fi G protein coupled receptors G protein-coupled receptors (GPCRs) are receptors that combine with ligands to transmit a signal to secondary signaling molecules. Such signal transmission is usually much slower and more complex than via ion-channel-coupled receptors. GPCR effects last longer. GPCRs constitute the largest group of receptors 7TM proteins G proteins G proteins are specialized proteins bound to guanosine-5′-diphosphate (GDP) and located on the cytoplasmic side of the cell membrane. They consist of three subunits: alpha (α), beta (β), and gamma (γ). Different types of G proteins are associated with speci c receptors and target proteins, acting as intermediaries to transmit signals from modi ed 7-transmembrane (7TM) receptors to effector proteins. The α subunit determines the speci city of signal transduction, and G proteins are classi ed based on this subunit into stimulating (Gs) or inhibitory (Gi). In their resting state, both 7TM receptors and G proteins are inactive. When a ligand binds to the 7TM receptor, it triggers a conformational change in both the receptor (on the extracellular side) and the G protein (on the cytoplasmic side). In the inactive state, GDP is bound to the α subunit. Activation occurs when GDP is replaced by guanosine-5′-triphosphate (GTP) on the α subunit, splitting the G protein into two active components: the α subunit bound to GTP and the G βγ complex. These active components interact with target proteins in the plasma membrane to propagate the signal. fi Second messenger molecules: The primary targets of G proteins are typically proteins found in the cell membrane, such as adenylyl cyclase and phospholipase C. These target proteins play a crucial role in signal transduction by catalyzing the production of second messenger molecules within the cell. Adenylyl cyclase generates cyclic adenosine-3′,5′-monophosphate (cAMP), a key second messenger involved in various cellular processes. Phospholipase C produces two important molecules: inositol trisphosphate (IP3), which in uences calcium release, and diacylglycerol (DAG), which activates protein kinase C. These second messengers help amplify and transmit signals inside the cell, allowing for an appropriate physiological response. Protein kinase: Protein kinases are enzymes that add phosphate groups to speci c proteins, a process known as phosphorylation. This phosphorylation causes a change in the conformation of the target protein, which can alter its activity, modify its ability to bind to other proteins, or even cause it to relocate within the cell. Around 30% of proteins are regulated through this mechanism. Protein kinases play a central role in many cellular processes, particularly in cell signaling and metabolic pathways, making them essential for regulating various cellular functions. fl Adenylyl cyclase Ligands such as adrenaline, acetylcholine, and glucagon activate adenylyl cyclase, an enzyme involved in signal transduction. The process begins when the ligand binds to a 7- transmembrane (7TM) receptor on the cell membrane. This binding activates the G protein, leading to the α subunit of the G protein associating with adenylyl cyclase. Once activated, adenylyl cyclase synthesizes cyclic adenosine-3′,5′-monophosphate (cAMP), a crucial second messenger that regulates various cellular processes. Cyclic AMP The activity of cyclic adenosine-3′,5′-monophosphate (cAMP) begins with the activation of protein kinase A (PKA). PKA, in turn, activates various proteins responsible for speci c cellular responses. For example, in muscle cells, PKA activates proteins involved in glycogen breakdown, providing energy for cellular activities. In endocrine cells of the hypothalamus, PKA regulates gene expression, in uencing hormone production. cAMP also plays a role in other cellular processes, making it a versatile second messenger in signal transduction pathways. fl Phospholipase C Ligands such as acetylcholine, vasopressin, and thrombin activate phospholipase C through a series of steps. First, the ligand binds to the 7-transmembrane (7TM) receptor, which activates a G protein. The α subunit of the G protein then binds to phospholipase C, activating it. This leads to the breakdown of phosphatidylinositol, a component of the inner cell membrane, producing two second messengers: inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 diffuses into the cytosol, where it binds to calcium channels on the endoplasmic reticulum (ER) membrane, causing calcium ions (Ca²⁺) to ow into the cytosol. Meanwhile, DAG stays membrane-bound and, together with Ca²⁺ ions, activates protein kinase C (PKC) in the cytosol. Active PKC then moves to the inner side of the cell membrane, where it phosphorylates various target proteins, contributing to the regulation of multiple cellular processes. Enzyme linked receptors Enzyme-linked receptors are a type of receptor that either act as enzymes themselves or bind to proteins that function as enzymes. These receptors typically have a single transmembrane segment that spans the lipid bilayer as an α-helix. On the outside of the cell membrane, this segment binds to a ligand, while on the inside, it contains a catalytic center or enzyme- binding site. Enzyme-linked receptors are sensitive to low ligand concentrations and produce slower responses, often taking hours to unfold. Their effects typically require multiple steps of intracellular signaling, usually resulting in changes to gene expression. Enzyme-linked receptors are categorized into different types based on their function, including tyrosine kinase, serine-threonine kinase, and guanylate cyclase receptors. Tyrosine kinase Growth factors such as FGF, EGF, PDGF, and VEGF are ligands that activate tyrosine kinases, which are important in cell signaling. When a ligand binds to the receptor on the cell membrane, it triggers a series of events. First, two receptor molecules come together to form a dimer. This dimerization activates the kinase activity of the receptor, leading to the phosphorylation of tyrosine residues on both receptors. This mutual phosphorylation creates binding sites for other signaling proteins. These signaling proteins then bind to the phosphorylated receptors and become phosphorylated themselves, allowing the signal to be transmitted and ampli ed inside the cell. This process is crucial for regulating various cellular responses, including growth, differentiation, and metabolism. Ras protein Most tyrosine kinases activate Ras proteins, which play a key role in cell signaling. Ras is a monomeric G protein, meaning it consists of a single protein unit, and it acts as a GTPase. It is attached to the cell membrane by a lipid. Ras exists in two states: an inactive state when bound to GDP and an active state when bound to GTP. When a tyrosine kinase receptor is activated, an adaptor protein binds to a speci c phosphorylated tyrosine residue on the receptor. This adaptor protein then interacts with a protein called GEF (guanine nucleotide exchange factor), forming a Ras-GEF complex. The Ras-GEF complex causes the Ras protein to exchange GDP for GTP, which activates the Ras protein and allows it to initiate downstream signaling pathways in the cell. fi fi The phosphorylation cascade The activated Ras protein triggers a series of events known as a phosphorylation cascade, involving three serine-threonine kinases that carry the signal forward. Each kinase in this cascade is phosphorylated and activated by enzymes called kinase kinases. The nal kinase in the cascade is MAP kinase (mitogen-activated protein kinase), which plays a crucial role in further signaling. MAP kinase phosphorylates additional target proteins, continuing the signaling process and helping regulate important cellular activities such as growth, differentiation, and response to stress. This cascade ampli es the initial signal and ensures proper cellular responses. PI3K/Akt pathway Growth and survival signaling, such as that triggered by IGF (Insulin-like Growth Factor), follows a series of steps to promote cell survival and growth. First, a tyrosine kinase is activated. This leads to the activation of PI3K (phosphoinositide 3-kinase), which then phosphorylates a lipid called phosphatidylinositol. This phosphorylated lipid binds to protein kinase B (also called PKB or Akt). Akt is then phosphorylated by two enzymes, protein kinase 1 and protein kinase 2, which activates it. Once activated, Akt is released from the cell membrane and moves into the cell. In its active form, Akt participates in the phosphorylation of various other proteins inside the cell, which helps regulate processes like cell growth and survival. Cell signal response Extracellular signals can lead to both fast and slow responses in cells. Rapid changes, such as motility (movement), secretion, and metabolism, occur quickly because they involve changes in the activity of existing proteins. On the other hand, processes like growth, differentiation, and cell division are slower because they require the synthesis of new proteins and changes in gene expression. These slower responses involve more complex cellular processes, such as turning on or off speci c genes, which take more time to produce noticeable effects. Signal transduction Signal transduction is the process through which a cell communicates with another cell. It begins when a signaling cell releases a ligand, which is an extracellular signaling molecule, such as a hormone or neurotransmitter. The ligand binds to a speci c receptor on the membrane of the target cell. This binding triggers the production of intracellular signaling molecules within the target cell. These molecules then initiate a series of reactions, often called a signaling cascade, which ultimately leads to the transmission of the signal to an effector protein. The effector protein then carries out the nal response in the cell, such as a change in cell behavior. fi fi Molecular relay race The signaling process inside a cell is often compared to "a molecular relay race," where information is passed from one signaling molecule to another in a chain of events. Each signaling molecule activates the next one, continuing this relay until a speci c outcome is achieved. This process can lead to various cellular changes, such as the activation or inactivation of enzymes involved in metabolism, the rearrangement of the cytoskeleton to alter cell shape, or the activation or repression of certain genes, leading to changes in gene expression. These steps are essential for controlling the cell's responses to signals from its environment. Molecular switches: Many signaling molecules function as molecular switches, meaning they can be turned on or off in response to a signal. When a signaling molecule is activated, it switches from an inactive to an active state, allowing it to either activate or inhibit other proteins in the signaling pathway. These proteins stay active until another process turns them off. Molecular switches can be divided into two main types: 1) proteins whose activity is regulated by adding or removing phosphate groups, a process called phosphorylation (turning the protein on) or dephosphorylation (turning it off), and 2) proteins whose activity depends on the exchange of GDP (guanosine-5′-diphosphate) for GTP (guanosine-5′-triphosphate), with the binding of GTP activating the protein and GDP inactivating it. Fourth presentation: Cell cycle and regulation Types of cells: In eukaryotic organisms, cells can be **diploid** or **haploid**. **Diploid cells** (2n) contain two sets of chromosomes, such as **somatic cells** (the body's cells). In humans, somatic cells have 46 chromosomes (2n=46). **Haploid cells** (1n) have a single set of chromosomes, such as **gametes** (eggs and sperm). Human gametes contain 23 chromosomes (n=23). Eukaryotic organisms undergo two types of cell division: **mitosis**, where the daughter cells receive the same number of chromosomes as the parent cell, and **meiosis**, where the daughter cells receive half the number of chromosomes, which is essential for the formation of gametes. The cell cycle: The **cell cycle** is a sequence of events that occur in a eukaryotic cell leading to its division. The primary goal of the cell cycle is to **accurately duplicate DNA** in the chromosomes and then **segregate** it into genetically identical daughter cells. This ensures that each new cell receives a complete copy of the entire genome. The length of the cell cycle can vary widely depending on the type of cell, but it is crucial for maintaining proper cell function and genetic integrity during cell division. Cell cycle: The cell cycle, which varies in duration depending on the cell type, consists of several key phases: interphase, the mitotic (or meiotic) phase, and cytokinesis. Interphase makes up about 95% of the cycle and includes the G1 phase ( rst gap), where the cell grows; the S phase (synthesis), where DNA is replicated; and the G2 phase (second gap), where the cell continues to grow and prepare for division. The mitotic phase involves the M phase, where mitosis (or meiosis) occurs, followed by cytokinesis, which is the division of the cytoplasm. This process ensures the cell's growth and division into two daughter cells. fi Interphase Interphase is a crucial part of the cell cycle, consisting of the G1, S, and G2 phases, each with speci c roles in preparing the cell for division. During the G1 phase, the cell synthesizes structural and enzymatic proteins, increases the number of mitochondria and lysosomes, and grows in mass and volume. By the end of G1, regulatory proteins are synthesized to transition the cell into the S phase. In the S phase, nuclear DNA is replicated, histone proteins are synthesized, and centrosomes are duplicated. At this point, each chromosome consists of two identical sister chromatids, linked by cohesin protein complexes. The G2 phase involves synthesizing mitotic spindle proteins (like α- and β-tubulin), non-histone proteins needed for chromatin condensation, and proteins and lipids for rebuilding the cell membrane. The G1 and G2 phases are critical for allowing the cell to grow and replicate its cytoplasmic organelles, ensuring it doubles its mass before division. Without these phases, cells would shrink after each division during the M phase. fi Cell cycle exit The cell cycle can include a temporary or permanent exit into the G0 phase, also known as the resting phase, when conditions for division are not favorable (e.g., lack of nutrients or oxygen). The G0 phase can last from days to months, and some cells, like nerve cells, skeletal muscle, and kidney and liver parenchyma, remain in this phase permanently. However, certain cells, such as hepatocytes and lymphocytes, can re-enter the cell cycle from G0 to divide again. The M phase, or mitotic phase, includes **karyokinesis** (nuclear division) and **cytokinesis** (cytoplasmic division). Karyokinesis is divided into prophase, prometaphase, metaphase, anaphase, and telophase, while cytokinesis starts in anaphase and concludes in telophase. Additionally, the centrosome cycle is synchronized with the cell cycle. During the G1 and S phases, centrosomes duplicate; in the G2 phase, they mature and connect; and in the M phase, daughter centrosomes separate and form a bipolar mitotic spindle with an astrosphere at each pole to assist in cell division. During **prophase**, chromosomes condense with the help of protein complexes called condensins, centrosomes move to opposite poles of the cell, and the mitotic spindle begins to form. Kinetochores, which are protein structures, also develop on the chromosomes. The mitotic spindle is assembled as new microtubules grow in random directions from both centrosomes. Some microtubules from one centrosome connect with those from the other, stabilizing the microtubules, preventing their depolymerization, and giving the spindle its bipolar shape. In **prometaphase**, the nuclear membrane disintegrates, allowing the microtubules of the mitotic spindle to attach to the kinetochores of the chromosomes. Each chromosome connects to spindle microtubules from opposite poles, generating tension in the kinetochores and ensuring proper alignment for subsequent phases of mitosis. During **metaphase**, chromosomes reach their maximum condensation and align along the equatorial plane of the mitotic spindle, forming the metaphase plate. In **anaphase**, the cohesin proteins holding sister chromatids together are broken, allowing the centromeres to separate. The sister chromatids, now individual chromosomes, move toward opposite poles of the cell. At the same time, cell organelles are divided into two similarly-sized groups and move alongside the chromosomes. By the end of anaphase, the chromosomes are evenly distributed at each spindle pole. **Telophase** follows, marked by the disappearance of the mitotic spindle, decondensation of chromosomes, and reformation of the nuclear envelope and nucleolus. Additionally, a contractile ring forms, initiating cytokinesis, the process of dividing the cytoplasm. **Cytokinesis** occurs during anaphase and telophase and involves the physical division of the cytoplasm between two daughter cells. A contractile ring made of actin and myosin micro laments forms in the equatorial plane of the cell. The contraction of this ring creates a cleavage furrow, which is positioned by the mitotic spindle, leading to the division of the cytoplasm. New cell membranes are then constructed in the daughter cells to complete the process. In humans, **sexual reproduction** involves the diploid form as the dominant life stage, with haploid cells limited to gametes. **Meiosis**, which only occurs in germ cells, involves two successive divisions of the diploid nucleus, ultimately producing four haploid cells. The rst division, meiosis I, is a reduction division that halves the chromosome number, while the second division, meiosis II, is a compensatory division that ensures each haploid cell receives a complete set of chromosomes. fi  M phase meiosis The **M phase of meiosis** consists of two divisions: **meiosis I** and **meiosis II**, each with four stages—prophase, metaphase, anaphase, and telophase. **Prophase I**, the longest and most complex stage of meiosis, is divided into ve sub-stages: leptotene, zygotene, pachytene, diplotene, and diakinesis. In the **leptotene** stage, chromatin condenses to form chromosomes, each composed of two sister chromatids. Cell organelles migrate to the periphery of the cell. During **zygotene**, maternal and paternal homologous chromosomes of the same shape and size pair up to form bivalents (or tetrads) consisting of four chromatids. A protein structure called the synaptonemal complex connects the homologous chromosomes during this pairing process, known as conjugation or synapsis. Simultaneously, the centrioles divide and move toward opposite poles of the cell, preparing for subsequent stages of meiosis. The **synaptonemal complex** is a protein structure that forms during meiosis to hold homologous chromosomes together. Sister chromatids of each homologous chromosome are connected by cohesins, and these cohesins associate with axial cores. When homologous chromosomes pair up to form bivalents, their axial cores are held together by transverse laments, forming the synaptonemal complex. In **prophase I**, the **pachytene** stage involves further chromosome condensation and histone synthesis. At this stage, chiasmata form between non-sister chromatids of homologous chromosomes, and homologous recombination occurs through a process called crossing-over, where DNA segments are exchanged to shuf e genetic material. During **diplotene**, crossing-over is completed, and homologous chromosomes begin to separate in the middle, though chromatids remain connected at the chiasmata. Finally, in **diakinesis**, chromosome condensation concludes, and chiasmata move toward the ends of the chromosomes. The nucleolus and nuclear membrane disassemble, signaling the end of prophase I and the transition to the next phase of meiosis. fi Crossing over **Crossing-over**, or homologous recombination, is a critical process during meiosis in which alleles are physically exchanged between non-sister chromatids of homologous chromosomes within a bivalent. The synaptonemal complex stabilizes bivalents and facilitates this DNA exchange. Each sister chromatid of one homologous chromosome can form chiasmata with one or two chromatids of the other chromosome, and multiple chiasmata typically occur within a single bivalent, allowing for multiple recombination events. In **metaphase I**, bivalents align along the equatorial plane of the cell. Mitotic spindle bers attach to only one chromatid of each homologous chromosome, and only one kinetochore is active. During **anaphase I**, spindle bers shorten, separating the homologous chromosomes and pulling them toward opposite poles of the cell. This ensures the chromosomes migrate to opposite poles. In **telophase I**, two daughter cells are formed, each with a haploid (1n) set of chromosomes, representing a reduction in chromosome number. The chromosomes undergo partial decondensation, and the nuclear envelope and nucleolus are reassembled. Finally, **cytokinesis** divides the cytoplasm, completing the formation of two daughter cells. fi Meiosis II **Meiosis II** occurs immediately after meiosis I and is not preceded by an S phase. This phase separates sister chromatids within the two daughter cells formed during meiosis I, resulting in four haploid gametes. Meiosis II is similar to mitosis, except each dividing cell has only one set of homologous chromosomes. In **prophase II**, chromatin condenses, and chromosomes become visible again. The nuclear envelope and nucleolus disappear, and the mitotic spindle re-forms, oriented perpendicular to the rst division. Each chromosome consists of two sister chromatids connected by a centromere. During **metaphase II**, chromosomes align along the equatorial plane, forming the metaphase plate, with spindle bers attaching to the centromeres of both sister chromatids. In **anaphase II**, the centromeres divide, and spindle bers shorten, separating sister chromatids and pulling them to opposite poles of the cell. Each chromatid becomes an individual daughter chromosome. Finally, in **telophase II**, four haploid nuclei are formed, each with a (1n) set of chromosomes. Chromosomes decondense into chromatin bers, and the nuclear envelope and nucleolus are re-formed. Cytokinesis follows, completing the formation of four haploid gametes. fi fi fi Cell cycle control system The **cell cycle control system** ensures that each cell progresses through the cell cycle in a controlled and regulated manner. **Checkpoints** are critical stages during the cell cycle where both internal and external conditions are assessed to determine whether the cell should continue dividing. Key checkpoints occur between the G1 and S phase (the **G1 checkpoint**), during the S phase (the **S checkpoint**), between the G2 and M phase (the **G2 checkpoint**), and between metaphase and anaphase of M phase (the **spindle checkpoint**). **Regulators**, which include both external and internal signals, control the activation or inhibition of pathways that affect processes like DNA replication, mitosis, and cytokinesis, ensuring proper progression through the cell cycle. **Mitogens** are speci c proteins or peptides that trigger a cell to start or increase the rate of cell division. They bind to receptors on the cell membrane, initiating a cascade of events that ultimately transmit the signal to the cell’s nucleus, promoting cell division. External regulators A **mitogen** is a small protein or peptide that triggers a cell to begin cell division or increase the rate of division (mitosis). Mitogens act as speci c ligands that bind to receptors on the cell membrane, initiating a cascade of signaling processes that transmit the signal to the cell nucleus. The effect of a mitogen depends on the activation of protein kinases, which play a key role in starting mitosis. This action typically occurs in the **G0 phase**, allowing the cell to transition into the **G1 phase** of the cell cycle. The signaling pathways that drive this process are linked to two important pathways: the **mitogen-activated protein kinase (MAPK)** pathway and the **phosphatidylinositol 3-kinase (PI-3K)** pathway, both of which help regulate the progression of cell division. fi Internal regulators **Internal cell cycle regulators** are essential for controlling the progression of the cell cycle. **Cyclins** are proteins that bind to speci c **cyclin-dependent kinases (Cdks)**, forming a complex that drives the cell cycle forward. The activity of these cyclin-Cdk complexes is crucial for the cell’s transition through various phases of the cycle. Another important regulator is the **anaphase-promoting complex (APC/C complex)**, also known as the **cyclosome**, which helps regulate the separation of chromosomes during cell division. In addition, **cyclin-dependent kinase inhibitor proteins (CdKIs)** act as inhibitors that block the activity of cyclin-Cdk complexes, preventing the cell from progressing through the cycle when necessary, such as in response to DNA damage or other stresses. These internal regulators work together to ensure proper control and timing of the cell cycle. Cyclins The concentration of **cyclins** remains low throughout most of the cell cycle but uctuates depending on the phase. As the cell cycle progresses, the levels of speci c cyclins rise and fall, and these cyclins activate **cyclin-dependent kinases (Cdks)**. Once activated, the cyclin-Cdk complexes carry out their function, after which the cyclins are degraded by **ubiquitination** in the proteasome. There are four main types of cyclins in humans: **G1- phase cyclins (D-cyclins)**, **G1/S-phase transition cyclins (E-cyclins)**, **S-phase cyclins (A-cyclins)**, and **M-phase cyclins (B-cyclin)**. **Cdks**, which are a family of proteins, maintain relatively constant levels throughout the cell cycle, but their enzymatic activity relies on their association with speci c cyclins. The activity of the cyclin-Cdk complex varies at different stages of the cell cycle, with increased concentrations of the respective cyclins leading to the formation of active cyclin-Cdk complexes. These complexes play a key role in regulating speci c phases of the cycle. While the levels of **Cdks** remain constant, their activity and protein targets change depending on the cyclin they are paired with, ensuring proper control of the cell cycle. fi Activation of cyclin Cdk complexes Initially, **cyclin-Cdk complexes** are inactive due to the attachment of two phosphate groups to the **Cdk** by an inhibitory kinase called **Wee1**. The **Cdc25** phosphatase activates the cyclin-Cdk complex by removing these deactivating phosphate groups. The activity of these complexes is tightly regulated by the synthesis and degradation of cyclins, which ensures the proper timing and control of **Cdk** activity throughout the cell cycle. This regulation is essential for the smooth progression of the cell cycle and ensures that cells only move forward at the right times. G1 phase During the **G1 phase**, several key events occur to regulate the cell cycle. First, the **S- Cdk** and **M-Cdk** complexes are inactivated. There is an increase in the synthesis of **cyclin D**, which then associates with **Cdk4** and **Cdk6** kinases to form **G1-Cdk complexes**. The activity of these complexes is highest in the middle of the G1 phase. The **G1-Cdk complexes** transfer phosphate groups from ATP to speci c substrates, primarily phosphorylating the **Rb protein (pRb)**. This phosphorylation helps control the cell's progression. Later in G1, **cyclin E** forms a complex with **Cdk2** to create the **G1/S-Cdk complex**, which prepares the cell to move into the S phase and begin DNA replication. S phase At the end of the **G1 phase**, the **S-Cdk complex** is formed, which consists of **cyclin A** and **Cdk2 kinase**. In the **S phase**, this complex plays a crucial role by triggering the activity of the **pre-replication complex**, ensuring that DNA replication occurs only once during the cell cycle. It also prevents a second round of DNA replication from happening. Additionally, the S-Cdk complex is essential for the transition from the S phase to the **G2 phase**, allowing the cell to proceed with preparation for cell division.  G2 phase In the **G2 phase**, **cyclin A** binds to **Cdk1**, forming a complex that helps the cell transition into the **M phase**. Meanwhile, **cyclin B** binds to **Cdk1** to form the **M-Cdk complex**, but this complex is inactive during the G2 phase. Once activated, the **cyclin B- Cdk1 complex** (also known as **M-phase Promoting Factor, or MPF**) plays a key role in controlling the events of **mitosis**. It regulates the progression of the cell through the stages of mitosis, ensuring that cell division occurs correctly. M phase The **MPF complex** (also known as **M-Cdk**) activates proteins that play key roles in **mitosis**. One of the most important actions is the disintegration of the **nuclear envelope**, which marks the start of early **M phase**. Additionally, MPF activation causes **chromosome condensation** and other essential processes for mitosis. The activation of the MPF complex occurs when **Cdc25 phosphatase** removes inhibitory phosphate groups. Once activated, MPF complexes also trigger the activation of additional MPF complexes, creating a positive-feedback loop that ampli es the process and ensures the cell moves ef ciently through mitosis. Activation of the MPF complex The entry of the cell into the **M phase** is controlled by the activity of the **MPF complex**, also known as the **M-Cdk complex**. This complex is crucial for initiating the events of mitosis. Its activation ensures that the cell transitions from the **G2 phase** into the **M phase**, where it will begin the processes necessary for cell division, such as chromosome condensation and the breakdown of the nuclear envelope. Therefore, the activity of the MPF complex is essential for the proper progression of the cell cycle into mitosis. fi fi APC/C The **MPF complex** activates the **APC/C complex** (anaphase-promoting complex/ cyclosome), which plays a key role in mitosis. The APC/C triggers the breakdown of the inactive **securin-separase complex**, leading to the degradation of securin and the release of **separase**, an enzyme that breaks down **cohesins**. Cohesins are proteins that hold sister chromatids together, so their destruction by separase allows the sister chromatids to separate and move to opposite poles of the cell. This marks the end of the M phase and enables the new daughter cells to enter the **G1 phase**. Additionally, the APC/C degrades the MPF complex by adding a small protein called **ubiquitin (Ub)** to **cyclin**. The ubiquitin-tagged cyclin is then separated from the cyclin-dependent kinase and sent to the **proteasome** for degradation. This process ensures that cyclin levels decrease, allowing the cell cycle to progress correctly. Protein p53 When DNA damage occurs, the concentration of the **p53** protein increases, and it becomes activated. Activated p53 stimulates the transcription of the **p21** gene, which leads to the production of the **p21 protein**. The p21 protein then binds to the **G1/S-Cdk** and **S-Cdk complexes**, inhibiting their activity. This blockage prevents the cell from progressing through the **G1 phase** into the **S phase**, effectively arresting the cell cycle in the **G1 phase**. This mechanism helps to stop the division of damaged cells, allowing time for repair or, if necessary, triggering cell death. Cyclin dependent kinase inhibitors Cdk inhibitors (CDKIs) are classi ed into two families based on their protein structure: **CIP/ KIP** and **INK4**. The **CIP/KIP** family, which includes proteins like **p21**, **p27**, and **p57**, binds to and inhibits the activity of **Cdk1**, **Cdk2**, **Cdk4**, and **Cdk6** complexes. On the other hand, the **INK4** family, represented by proteins such as **p15**, **p16**, **p18**, and **p19**, speci cally binds to and inhibits the activity of complexes containing only **Cdk4** and **Cdk6**. These inhibitors regulate the progression of the cell cycle by controlling the activity of cyclin-dependent kinases (Cdks), which are crucial for cell division. KIP p27 inhibitor The **p27** protein, a member of the KIP family, plays a crucial role in determining whether a cell enters a new cell cycle or enters the resting phase (G0). If the cell is to start a new cycle, the concentration of p27 decreases, while an increase in p27 concentration pushes the cell into the G0 phase. **p27** binds to and inactivates **E-Cdk2** and **A-Cdk2** complexes, preventing them from driving the cell cycle forward. In response to DNA damage, the level of p27 is regulated by the tumor suppressor protein **p53**, which controls p27 production at the transcriptional level. Apoptosis Apoptosis, or programmed cell death, is an energy-dependent process that involves the activation of speci c genes. It typically affects individual cells and plays a crucial role in regulating cell numbers and types within tissues. Apoptosis removes damaged, infected, or mutated cells, helping maintain tissue homeostasis. This process ensures the proper functioning of tissues by eliminating unwanted or harmful cells, contributing to the overall balance and health of the organism. fi fi fi Necrosis Necrosis is a type of cell death that typically affects entire groups of cells and does not require energy. It is a passive and random process that occurs due to external factors, such as physical or chemical damage. As a defensive response, necrosis triggers an in ammatory reaction, which involves cell swelling, loss of membrane integrity, and the leakage of cell contents into the surrounding extracellular space. This process often leads to tissue damage and is different from apoptosis, which is a controlled and programmed form of cell death. Lecture 7: DNA replication, transcription and translation DNA replication is called semiconservative because each original strand of the double helix serves as a template for creating a new, complementary strand. DNA synthesis can only occur in the 5’ to 3’ direction. As new nucleotides are added, they are linked together by a phosphodiester bond. This process is enzymatic, meaning it involves various protein complexes working together. Additionally, DNA replication requires energy, which is provided by the hydrolysis of high-energy phosphate bonds. Replication proteins: Several key proteins are involved in DNA replication. Helicase, speci cally the MCM 2-7 complex, unwinds the DNA double helix. Topoisomerases help prevent DNA from becoming too twisted during unwinding. Replication Protein A (RPA) binds to single-stranded DNA, protecting it from damage. Proliferating Cell Nuclear Antigen (PCNA) acts as a sliding clamp, helping DNA polymerase stay attached to the template strand. Replication Factor C (RFC) helps load PCNA onto the DNA. DNA polymerases (α, β, γ, δ, and ε) are responsible for adding nucleotides to the growing strand. RNase H and other endonucleases remove RNA primers, while FEN1 exonuclease removes any remaining RNA fragments. Finally, DNA ligase joins the newly synthesized DNA fragments to complete the process. Helicase: Several key proteins are involved in DNA replication. Helicase, speci cally the MCM 2-7 complex, unwinds the DNA double helix. Topoisomerases help prevent DNA from becoming too twisted during unwinding. Replication Protein A (RPA) binds to single-stranded DNA, protecting it from damage. Proliferating Cell Nuclear Antigen (PCNA) acts as a sliding clamp, helping DNA polymerase stay attached to the template strand. Replication Factor C (RFC) helps load PCNA onto the DNA. DNA polymerases (α, β, γ, δ, and ε) are responsible for adding nucleotides to the growing strand. RNase H and other endonucleases remove RNA primers, while FEN1 exonuclease removes any remaining RNA fragments. Finally, DNA ligase joins the newly synthesized DNA fragments to complete the process. Topoisomerases During DNA replication, helicase unwinds the DNA strands, which generates torsional stress, or twisting tension, in the DNA. If this tension becomes too high, it can stop further unwinding of the DNA. Topoisomerases help relieve this tension by changing the structure of the DNA molecule. There are two types of topoisomerases: topoisomerase I, which makes temporary single-strand cuts to relieve tension, and topoisomerase II, which makes double-strand cuts. Both work to reduce the twisting stress and allow the DNA to continue unwinding for replication. RPA When helicase separates the complementary DNA strands during replication, single-stranded DNA fragments are created. These fragments can form short double-stranded regions that obstruct the action of DNA polymerase. The RPA (Replication Protein A) protein helps resolve this issue by binding to the single-stranded DNA. This prevents the formation of unwanted hydrogen bonds between the strands, ensuring that DNA polymerase can properly synthesize the new DNA strand without interference. PCNA The Proliferating Cell Nuclear Antigen (PCNA) is a ring-shaped homotrimer composed of three subunits that encircle the DNA strand. PCNA is loaded onto the DNA with the help of Replication Factor C (RFC). Acting as a scaffold, PCNA recruits proteins essential for DNA replication, repair, chromatin remodeling, and epigenetic regulation. During replication, PCNA moves along the DNA strand and serves two critical functions: it prevents DNA polymerase from dissociating from the template strand, ensuring processivity, and it releases DNA polymerase after the synthesis of each Okazaki fragment on the lagging strand. RFC Replication Factor C (RFC), also known as the "ring-applying protein," is composed of ve distinct subunits. In the presence of ATP, RFC opens the PCNA homotrimer, positions the DNA strand into the center of the ring, and subsequently closes the ring around the DNA. For the leading strand, this process occurs only once during replication. However, on the lagging strand, due to the discontinuous synthesis of Okazaki fragments, RFC must repeatedly place the PCNA protein onto the DNA strand to facilitate replication. DNA polymerase DNA polymerases are essential enzymes in DNA replication, with several key characteristics. They require a DNA template to function and cannot initiate complementary strand synthesis independently, necessitating an RNA primer. DNA polymerases synthesize DNA in the 5’ → 3’ direction and have 3’ → 5’ exonuclease activity for proofreading. Polymerase α, with primase and polymerase activity, synthesizes an RNA primer (10 nucleotides) and adds a short DNA sequence (20–30 nucleotides), which is then extended by polymerases δ and ε. Polymerase δ, composed of four subunits, is primarily responsible for leading strand synthesis, while polymerase ε, also made of four subunits, mainly replicates the lagging strand. Both δ and ε exhibit 5’ → 3’ polymerase and 3’ → 5’ exonuclease activities. During replication, DNA polymerases operate in two modes: polymerization (P) for synthesis and editing (E) for error correction. If an incorrect nucleotide is incorporated, the polymerase shifts to exonuclease activity, removes the error, and resumes polymerization to ensure high- delity DNA replication. Ribonucleases RNases are enzymes belonging to the hydrolase class that degrade RNA molecules by hydrolyzing phosphodiester bonds. This process breaks RNA into shorter chains or single nucleotides. RNases are categorized based on their action: endoribonucleases cleave RNA molecules internally, dividing them in the middle of the chain, while exoribonucleases remove nucleotides sequentially from either the 3' or 5' end of the RNA strand. These enzymes play critical roles in RNA processing, maturation, and degradation, ensuring proper RNA turnover and regulation within the cell. Replication proteins RNAse H, an endoribonuclease, removes the iRNA primer from the DNA strand, leaving only the last ribonucleotide behind. FEN1 (Flap Endonuclease 1), an exoribonuclease, removes this nal ribonucleotide. DNA ligase then seals the DNA strand by forming phosphodiester bonds, using ATP, to join deoxyribonucleotides, including the Okazaki fragments on the lagging strand. DNA replication faces key challenges: the need to unwind the DNA double helix, the inability of DNA polymerase to initiate new strand synthesis independently, and the restriction of nucleotide addition to the 5’→3’ direction. New strand synthesis begins with an RNA primer synthesized by the primase unit of polymerase α, which creates a short 10- nucleotide iRNA strand. Polymerase α extends this with 20–30 deoxynucleotides of iDNA, after which polymerase δ or ε continues DNA strand