Plant Cell Biology PDF - Port Said University

Botany Department Faculty of Science Port Said University Plant Cell Biology Prepared By/ Dr. Raghda Hassan Shahda 2025 1  Cell Biolo...

Botany Department Faculty of Science Port Said University Plant Cell Biology Prepared By/ Dr. Raghda Hassan Shahda 2025 1  Cell Biology What Is a Cell? Cells have all the equipment and expertise necessary to carry out the functions of life. A cell can eat, grow, and move. It can perform necessary maintenance, recycle parts, and dispose of wastes. It can adapt to changes in its environment; and it can even replicate itself. Despite these similarities, all cells are not equal. Some are truly self-sustaining, as with single-celled bacteria or yeast, whereas others live communally, sometimes as part of complex multicellular organisms. Cells also differ in size. Although cells can be quite large — consider a frog's egg, for example — most are too small to see with the naked eye. Indeed, the development of light microscopy was essential to man's discovery of cells. You won't see the familiar schematic drawings of oval-shaped cells. Real cells are three-dimensional, and they exist in a variety of intricate and remarkable shapes. For instance, a single human nerve cell can be over one meter long, extending from your backbone to your big toe. Compare that with the cells that line your small intestine, which have dozens of tiny, fingerlike projections to maximize the surface area across which nutrients can pass. 2 3 History Cells were first seen in 17th-century Europe with the invention of the compound microscope. In 1665, Robert Hooke referred to the building blocks of all living organisms as "cells" (published in Micrographia) after looking at a piece of cork and observing a cell-like structure; however, the cells were dead. They gave no indication to the actual overall components of a cell. A few years later, in 1674, Anton Van Leeuwenhoek was the first to analyze live cells in his examination of algae. All of this preceded the cell theory which states that all living things are made up of cells and that cells are organisms' functional and structural units. This was ultimately concluded by plant scientist Matthias Schleiden and animal scientist Theodor Schwann in 1838, who viewed live cells in plant and animal tissue, respectively. 19 years later, Rudolf Virchow further contributed to the cell theory, adding that all cells come from the division of pre-existing cells. 4 Cell theory Cell theory is a scientific theory first formulated in the mid-nineteenth century by Theodor Schwann and Matthias Jakob Schleiden: 1. All living organisms are composed of one or more cells 2. The cell is the most basic unit of life In 1855, Rudolf Virchow added the third tenet to cell theory. 3. All cells arise only from pre-existing cells The theory was once universally accepted, but now some biologists consider non-cellular entities such as viruses living organisms, and thus disagree with the first tenet. As of 2021: "expert opinion remains divided roughly a third each between yes, no and don’t know". As there is no universally accepted definition of life, discussion still continues. Modern interpretation of Cell theory The generally accepted parts of modern cell theory include: 1. All known living things are made up of one or more cells. 2. All living cells arise from pre-existing cells by division. 3. The cell is the fundamental unit of structure and function in all living organisms. 4. The activity of an organism depends on the total activity of independent cells. 5. Energy flow (metabolism and biochemistry) occurs within cells. 6. Cells contain DNA which is found specifically in the chromosome and RNA found in the cell nucleus and cytoplasm. 7. All cells are basically the same in chemical composition in organisms of similar species. 5 Techniques But how, exactly, do cells accomplish the complex tasks of life? What tools and materials do they need? And what are the key characteristics that define a cell? Cell biology research looks at different ways to culture and manipulate cells outside of a living body to further research in human anatomy and physiology, and to derive medications. The techniques by which cells are studied have evolved. Due to advancements in microscopy, techniques and technology have allowed scientists to hold a better understanding of the structure and function of cells. Many techniques commonly used to study cell biology are listed: 1- Cell culture Cell culture: Utilizes rapidly growing cells on media which allows for a large amount of a specific cell type and an efficient way to study cells. Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis. It is also used in drug screening and development, and large scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins). 6  2- Fluorescence microscopy Fluorescent markers such as Green fluorescent protein (GFP), are used to label a specific component of the cell. Afterwards, a certain light wavelength is used to excite the fluorescent marker which can then be visualized. 3- Phase-contrast microscopy Phase-contrast microscopy uses the optical aspect of light to represent the solid, liquid, and gas-phase changes as brightness differences. It is a technique that reveals the hidden details of transparent specimens without staining them. 7  4- Transmission electron microscopy Involves metal staining and the passing of electrons through the cells, which will be deflected upon interaction with metal. This ultimately forms an image of the components being studied. 5- Cytometry Using a flow cytometer machine, cells or other particles suspended in a liquid stream are passed through a laser light beam in single file fashion, and interaction with the light is measured by an electronic detection apparatus as light scatter and fluorescence intensity. Variables that can be measured by cytometric methods include cell size, cell count, cell morphology (shape and structure), cell cycle phase, DNA content, and the existence or absence of specific proteins on the cell surface or in the cytoplasm. 8  6- Cell fractionation This process requires breaking up the cell using high temperature or sonification followed by centrifugation to separate the parts of the cell allowing for them to be studied separately. a method that separates subcellular components and organelles, so that the structures, functions, and molecular compositions of isolated components may be studied. What Defines a Cell? Cells are considered the basic units of life in part because they come in discrete and easily recognizable packages. That's because all cells are surrounded by a structure called the cell membrane — which, much like the walls of a house, serves as a clear boundary between the cell's internal and external environments. The cell membrane is sometimes also referred to as the plasma membrane. Cell membranes are based on a framework of fat-based molecules called phospholipids, which physically prevent water-loving, or hydrophilic, substances from entering or escaping the cell. These membranes are also studded with proteins that serve various functions. Some of these proteins act as gatekeepers, determining what substances can and cannot cross the membrane. Others function as markers, identifying the cell as part of the same organism or as foreign. Still others work like fasteners, binding cells together so they can function as a unit. Yet other membrane proteins serve as communicators, sending and receiving signals from neighboring cells and the environment — whether friendly or alarming. 9 Within this membrane, a cell's interior environment is water based. Called cytoplasm, this liquid environment is packed full of cellular machinery and structural elements. In fact, the concentrations of proteins inside a cell far outnumber those on the outside — whether the outside is ocean water (as in the case of a single-celled alga) or blood serum (as in the case of a red blood cell). Although cell membranes form natural barriers in watery environments, a cell must nonetheless expend quite a bit of energy to maintain the high concentrations of intracellular constituents necessary for its survival. Indeed, cells may use as much as 30 percent of their energy just to maintain the composition of their cytoplasm 10 Biochemical Composition of cells The cellular body is a dense gel-like mixture crowded with proteins made by ribosomes. Therefore, the term cytoplasm implies the correct constitution of the cellular body rather than the term cytosol. As previously mentioned, a cell's cytoplasm is home to numerous functional and structural elements. These elements exist in the form of molecules and organelles — picture them as the tools, appliances, and inner rooms of the cell. Major classes of intracellular organic molecules include nucleic acids, proteins, carbohydrates, and lipids, all of which are essential to the cell's functions. These macromolecules are assembled from building blocks: monosaccharides, fatty acids, amino acids and nucleotides. Nucleic acids are the molecules that contain and help express a cell's genetic code. There are two major classes of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is the molecule that contains all of the information required to build and maintain the cell; RNA has several roles associated with expression of the information stored in DNA. Of course, nucleic acids alone aren't responsible for the preservation and expression of genetic material: Cells also 11 use proteins to help replicate the genome and accomplish the profound structural changes that underlie cell division. Proteins are a second type of intracellular organic molecule. These substances are made from chains of smaller molecules called amino acids, and they serve a variety of functions in the cell, both catalytic and structural. For example, proteins called enzymes convert cellular molecules (whether proteins, carbohydrates, lipids, or nucleic acids) into other forms that might help a cell meet its energy needs, build support structures, or pump out wastes. Proteins are encoded by the sequence of nucleic acids in the genetic code. 12 Carbohydrates, the starches and sugars in cells, are another important type of organic molecule. Simple carbohydrates are used for the cell's immediate energy demands, whereas complex carbohydrates serve as intracellular energy stores. Complex carbohydrates are also found on a cell's surface, where they play a crucial role in cell recognition. Chains of monosaccharides can also be plugged onto the other molecule classes forming e.g. glycolipids or glycoproteins. Glycoproteins are also an integral part of the bacterial cell wall and the extracellular matrix in tissues of higher organisms. 13 lipids or fat molecules are components of cell membranes — both the plasma membrane and various intracellular membranes. They are also involved in energy storage, Breaking down fatty acids yields even more energy per mass unit than glucose as well as relaying signals within cells and from the bloodstream to a cell's interior. The relative scale of biological molecules and structures Cells can vary between 1 micrometer (μm) and hundreds of micrometers in diameter. Within a cell, a DNA double helix is approximately 10 nanometers (nm) wide, whereas the cellular organelle called a nucleus that encloses this DNA can be approximately 1000 times bigger (about 10 μm). See how cells compare along a relative scale axis with other molecules, tissues, and biological structures (blue arrow at bottom). Note that a micrometer (μm) is also known as a micron. 14 What Are the Different Categories of Cells? Built from these macromolecules at different scales, two kinds of cells can be discriminated. Rather than grouping cells by their size or shape, scientists typically categorize them by how their genetic material is packaged. Types of Cells prokaryote eukaryote prokaryote If the DNA within a cell is not separated from the cytoplasm. All known prokaryotes, such as bacteria and archaea, are single cells. eukaryote If the DNA is partitioned off in its own membrane-bound room called the nucleus, then that cell is a eukaryote. Some eukaryotes, like amoebae, are free-living, single- celled entities. Other eukaryotic cells are part of multicellular organisms. For instance, all plants and animals are made of eukaryotic cells — sometimes even trillions of them. 15 How Did Cells Originate? Researchers hypothesize that all organisms on Earth today originated from a single cell that existed some 3.5 to 3.8 billion years ago. This original cell was likely little more than a sac of small organic molecules and RNA-like material that had both informational and catalytic functions. Over time, the more stable DNA molecule evolved to take over the information storage function. whereas proteins, with a greater variety of structures than nucleic acids, took over the catalytic functions. The absence or presence of a nucleus is important enough to be a defining feature by which cells are categorized as either prokaryotes or eukaryotes. Scientists believe that the appearance of self-contained nuclei and other organelles represents a major advance in the evolution of cells. But where did these structures come from? More than one billion years ago, some cells "ate" by engulfing objects that floated in the liquid environment in which they existed. Then, according to some theories of cellular evolution, one of the early eukaryotic cells engulfed a prokaryote, and together the two cells formed a symbiotic relationship. In particular, the engulfed cell began to function as an organelle within the larger eukaryotic cell that consumed it. Both chloroplasts and mitochondria, which exist in modern eukaryotic cells and still retain their own genomes, are thought to have arisen in this manner. Of course, prokaryotic cells have continued to evolve as well. Different species of bacteria and archaea have adapted to specific environments, and these prokaryotes not only survive but thrive without having their genetic material in its own compartment. For example, certain bacterial species that live in thermal vents along the ocean floor can withstand higher temperatures than any other organisms on Earth. Origin of a eukaryotic cell. A prokaryotic host cell incorporates another prokaryotic cell. Each prokaryote has its own set of DNA molecules (a genome). The genome of the incorporated cell remains separate (curved blue line) from the host cell genome (curved purple line). 16 The incorporated cell may continue to replicate as it exists within the host cell. Over time, during errors of replication or perhaps when the incorporated cell lyses and loses its membrane separation from the host, genetic material becomes separated from the incorporated cell and merges with the host cell genome. Eventually, the host genome becomes a mixture of both genomes, and it ultimately becomes enclosed in an endomembrane, a membrane within the cell that creates a separate compartment. This compartment eventually evolves into a nucleus. Figure: The origin of mitochondria and chloroplasts Mitochondria and chloroplasts likely evolved from engulfed prokaryotes that once lived as independent organisms. At some point, a eukaryotic cell engulfed an aerobic prokaryote, which then formed an endosymbiotic relationship with the host eukaryote, gradually developing into a mitochondrion. Eukaryotic cells containing mitochondria then engulfed photosynthetic prokaryotes, which evolved to become specialized chloroplast. 1.2 Eukaryotic Cells Possess a Nucleus and Membrane-Bound Organelles How do cells accomplish all their functions in such a tiny, crowded package? Eukaryotic cells have evolved ways to partition off different functions to various locations in the cell. In fact, specialized compartments called organelles exist within eukaryotic cells for this purpose. Different organelles play different roles in the cell — for instance, mitochondria generate energy from food molecules; lysosomes break down and recycle organelles and macromolecules; and the endoplasmic reticulum helps build membranes and transport proteins throughout the cell. But what characteristics do all organelles have in common? And why was the development of three particular organelles (the nucleus, the mitochondrion, and the chloroplast) so essential to the evolution of present-day eukaryotes ? 17 What Defines an Organelle? In addition to the nucleus, eukaryotic cells may contain several other types of organelles, which may include mitochondria, chloroplasts, the endoplasmic reticulum, the Golgi apparatus, and lysosomes. Each of these organelles performs a specific function critical to the cell's survival. Moreover, nearly all eukaryotic organelles are separated from the rest of the cellular space by a membrane The membranes that surround eukaryotic organelles are based on lipid bilayers that are similar (but not identical) to the cell's outer membrane. Together, the total area of a cell's internal membranes far exceeds that of its plasma membrane. Like the plasma membrane, organelle membranes function to keep the inside "in" and the outside "out." This partitioning permits different kinds of biochemical reactions to take place in different organelles. Although each organelle performs a specific function in the cell, all of the cell's organelles work together in an integrated fashion to meet the overall needs of the cell. For example, biochemical reactions in a cell's mitochondria transfer energy from fatty acids and pyruvate molecules into an energy-rich molecule called adenosine triphosphate (ATP). Subsequently, the rest of the cell's organelles use this ATP as the source of the energy they need to operate. Because most organelles are surrounded by membranes, they are easy to visualize — with magnification. For instance, researchers can use high resolution electron microscopy to take a snapshot through a thin cross-section or slice of a cell. Other less powerful microscopy techniques coupled with organelle-specific stains have helped researchers see organelle structure more clearly, as well as the distribution of various organelles within cells. However, unlike the rooms in a house, a cell's organelles are not static. Rather, these structures are in constant motion, sometimes moving to a particular place within the cell, sometimes merging with other organelles, and sometimes growing larger or smaller. These dynamic changes in cellular structures can be observed with video microscopic techniques, which provide lower-resolution movies of whole organelles as these structures move within cells. The nucleus 18 Why Is the Nucleus So Important? Of all eukaryotic organelles, the nucleus is perhaps the most critical. In fact, the mere presence of a nucleus is considered one of the defining features of a eukaryotic cell. This structure is so important because it is the site at which the cell's DNA is housed and the process of interpreting it begins. Recall that DNA contains the information required to build cellular proteins. In eukaryotic cells, the membrane that surrounds the nucleus (nuclear envelope) partitions this DNA from the cell's protein synthesis machinery, which is located in the cytoplasm. Tiny pores in the nuclear envelope, called nuclear pores, then selectively permit certain macromolecules to enter and leave the nucleus — including the RNA molecules that carry information from a cellular DNA to protein manufacturing centers in the cytoplasm. This separation of the DNA from the protein synthesis machinery provides eukaryotic cells with more intricate regulatory control over the production of proteins and their RNA intermediates. In contrast, the DNA of prokaryotic cells is distributed loosely around the cytoplasm, along with the protein synthesis machinery. This closeness allows prokaryotic cells to rapidly respond to environmental change by quickly altering the types and amount of proteins they manufacture. Mitochondria and Chloroplasts Why Are Mitochondria and Chloroplasts Special? 19 These specialized structures are enclosed by double membranes, and they are believed to have originated back when all living things on Earth were single-celled organisms. At that time, some larger eukaryotic cells with flexible membranes "ate" by engulfing molecules and smaller cells — and scientists believe that mitochondria and chloroplasts arose as a result of this process. In particular, researchers think that some of these "eater" eukaryotes engulfed smaller prokaryotes, and a symbiotic relationship subsequently developed. Once kidnapped, the "eaten" prokaryotes continued to generate energy and carry out other necessary cellular functions, and the host eukaryotes came to rely on the contribution of the "eaten" cells. Over many generations, the descendants of the eukaryotes developed mechanisms to further support this system, and concurrently, the descendants of the engulfed prokaryotes lost the ability to survive on their own, evolving into present-day mitochondria and chloroplasts. This proposed origin of mitochondria and chloroplasts is known as the endosymbiotic hypothesis. These specialized structures are enclosed by double membranes, and they are believed to have originated back when all living things on Earth were single- celled organisms. At that time, some larger eukaryotic cells with flexible membranes "ate" by engulfing molecules and smaller cells — and scientists believe that mitochondria and chloroplasts arose as a result of this process. In particular, researchers think that some of these "eater" eukaryotes engulfed smaller prokaryotes, and a symbiotic relationship subsequently developed. Once kidnapped, the "eaten" prokaryotes continued to generate energy and carry out other necessary cellular functions, and the host eukaryotes came to rely on the contribution of the "eaten" cells. Over many generations, the descendants of the eukaryotes developed mechanisms to further support this system, and concurrently, the descendants of the engulfed prokaryotes lost the ability to survive on their own, evolving into present-day mitochondria and chloroplasts. This proposed origin of mitochondria and chloroplasts is known as the endosymbiotic hypothesis. In addition to double membranes, mitochondria and chloroplasts also retain small genomes with some resemblance to those found in modern prokaryotes. This finding provides yet additional evidence that these organelles probably originated as self- sufficient single-celled organisms. 20 Today, mitochondria are found in fungi, plants, and animals, and they use oxygen to produce energy in the form of ATP molecules, which cells then employ to drive many processes. Scientists believe that mitochondria evolved from aerobic, or oxygen- consuming, prokaryotes. In comparison, chloroplasts are found in plant cells and some algae, and they convert solar energy into energy-storing sugars such as glucose. Chloroplasts also produce oxygen, which makes them necessary for all life as we know it. Scientists think chloroplasts evolved from photosynthetic prokaryotes similar to modern- day cyanobacteria. 21 Figure: Typical prokaryotic (left) and eukaryotic (right) cells In prokaryotes, the DNA (chromosome) is in contact with the cellular cytoplasm and is not in a housed membrane-bound nucleus. In eukaryotes, however, the DNA takes the form of compact chromosomes separated from the rest of the cell by a nuclear membrane (also called a nuclear envelope). Eukaryotic cells also contain a variety of structures and organelles not present in prokaryotic cells. Throughout the course of evolution, organelles such as mitochondria and chloroplasts (a form of plastid) may have arisen from engulfed prokaryotes. © 1998 Nature Publishing Group Doolittle, W. F. A paradigm gets shifty. Nature 392, 15-16 (1998). All rights reserved. 22 1.3 Cell Function Depends on the Continual Uptake and Conversion of Energy How Do Eukaryotic Cells Handle Energy? Mitochondria enable eukaryotes to make more efficient use of food sources than their prokaryotic counterparts. That's because these organelles greatly expand the amount of membrane used for energy-generating electron transport chains. In addition, mitochondria use a process called oxidative metabolism to convert food into energy, and oxidative metabolism yields more energy per food molecule than non-oxygen-using, or anaerobic, methods. Energywise, cells with mitochondria can therefore afford to be bigger than cells without mitochondria.Within eukaryotic cells, mitochondria function somewhat like batteries, because they convert energy from one form to another: food nutrients to ATP. Accordingly, cells with high metabolic needs can meet their higher energy demands by increasing the number of mitochondria they contain. For example, muscle cells in people who exercise regularly possess more mitochondria than muscle cells in sedentary people. Prokaryotes, on the other hand, don't have mitochondria for energy production, so they must rely on their immediate environment to obtain usable energy. Prokaryotes generally use electron transport chains in their plasma membranes to provide much of their energy. The actual energy donors and acceptors for these electron transport chains are quite variable, reflecting the diverse range of habitats where prokaryotes live. (In aerobic prokaryotes, electrons are transferred to oxygen, much as in the mitochondria.) The challenges associated with energy generation limit the size of prokaryotes. As these cells grow larger in volume, their energy needs increase proportionally. However, as they increase in size, their surface area (and thus their 23 ability to both take in nutrients and transport electron) does not increase to the same degree as their volume. As a result, prokaryotic cells tend to be small so that they can effectively manage the balancing act between energy supply and demand. Figure: The relationship between the radius, surface area, and volume of a cell. Note that as the radius of a cell increases from 1x to 3x (left), the surface area increases from 1x to 9x, and the volume increases from 1x to 27x. Where Do Cells Obtain Their Energy? Cells, like humans, cannot generate energy without locating a source in their environment. However, whereas humans search for substances like fossil fuels to power their homes and businesses, cells seek their energy in the form of food molecules or sunlight. In fact, the Sun is the ultimate source of energy for almost all cells, because photosynthetic prokaryotes, algae, and plant cells harness solar energy and use it to make the complex organic food molecules that other cells rely on for the energy required to sustain growth, metabolism, and reproduction. Cellular nutrients come in many forms, including sugars and fats. In order to provide a cell with energy, these molecules have to pass across the cell membrane, which functions as a barrier — but not an impassable one. Like the exterior walls of a house, the plasma membrane is semi-permeable. In much the same way that doors and windows allow necessities to enter the house, various proteins that span the cell membrane permit specific molecules into the cell, although they may require some energy input to accomplish this task. Cells can incorporate nutrients by phagocytosis. This amoeba, a single-celled organism, acquires energy by engulfing nutrients in the form of a yeast cell (red). Through a process called phagocytosis, the amoeba encloses the yeast cell with its 24 membrane and draws it inside. Specialized plasma membrane proteins in the amoeba (in green) are involved in this act of phagocytosis, and they are later recycled back into the amoeba after the nutrients are engulfed. How Do Cells Turn Nutrients into Usable Energy? cells release the energy stored in their food molecules through a series of oxidation reactions. Oxidation describes a type of chemical reaction in which electrons are transferred from one molecule to another, changing the composition and energy content of both the donor and acceptor molecules. Food molecules act as electron donors. During each oxidation reaction involved in food breakdown, the product of the reaction has a lower energy content than the donor molecule that preceded it in the pathway. At the same time, electron acceptor molecules capture some of the energy lost from the food molecule during each oxidation reaction and store it for later use. Eventually, when the carbon atoms from a complex organic food molecule are fully oxidized at the end of the reaction chain, they are released as waste in the form of carbon dioxide. Figure: The release of energy from sugar 25 Compare the stepwise oxidation (left) with the direct burning of sugar (right). Through a series of small steps, free energy is released from sugar and stored in carrier molecules in the cell (ATP and NADH, not shown). On the right, the direct burning of sugar requires a larger activation energy. In this reaction, the same total free energy is released as in stepwise oxidation, but none is stored in carrier molecules, so most of it will be lost as heat (free energy). This direct burning is therefore very inefficient, as it does not harness energy for later use. Cells do not use the energy from oxidation reactions as soon as it is released. Instead, they convert it into small, energy-rich molecules such as ATP and nicotinamide adenine dinucleotide (NADH), which can be used throughout the cell to power metabolism and construct new cellular components. In addition, workhorse proteins called enzymes use this chemical energy to catalyze, or accelerate, chemical reactions within the cell that would otherwise proceed very slowly. Enzymes do not force a reaction to proceed if it wouldn't do so without the catalyst; rather, they simply lower the energy barrier required for the reaction to begin. Figure: Enzymes allow activation energies to be lowered. Enzymes lower the activation energy necessary to transform a reactant into a product. On the left is a reaction that is not catalyzed by an enzyme (red), and on the right is one that is (green). In the enzyme- catalyzed reaction, an enzyme will bind to a reactant and facilitate its transformation into a product. Consequently, an enzyme-catalyzed reaction pathway has a smaller energy barrier (activation energy) to overcome before the reaction can proceed. What Specific Pathways Do Cells Use? The particular energy pathway that a cell employs depends in large part on whether that cell is a eukaryote or a prokaryote. Eukaryotic cells use three major processes to transform the energy held in the chemical bonds of food molecules into more readily usable forms — often energy- rich carrier molecules. 26 Adenosine 5'-triphosphate, or ATP, is the most abundant energy carrier molecule in cells. This molecule is made of a nitrogen base (adenine), a ribose sugar, and three phosphate groups. The word adenosine refers to the adenine plus the ribose sugar. The bond between the second and third phosphates is a high-energy bond. Figure: An ATP molecule ATP consists of an adenosine base (blue), a ribose sugar (pink) and a phosphate chain. The high-energy phosphate bond in this phosphate chain is the key to ATP's energy storage potential. 27  1- glycolysis The first process in the eukaryotic energy pathway is glycolysis, which literally means "sugar splitting." During glycolysis, single molecules of glucose are split and ultimately converted into two molecules of a substance called pyruvate; because each glucose contains six carbon atoms, each resulting pyruvate contains just three carbons. Glycolysis is actually a series of ten chemical reactions that requires the input of two ATP molecules. This input is used to generate four new ATP molecules, which means that glycolysis results in a net gain of two ATPs. Two NADH molecules are also produced; these molecules serve as electron carriers for other biochemical reactions in the cell. Glycolysis is an ancient, major ATP-producing pathway that occurs in almost all cells, eukaryotes and prokaryotes alike. This process, which is also known as fermentation, takes place in the cytoplasm and does not require oxygen. However, the fate of the pyruvate produced during glycolysis depends upon whether oxygen is present. In the absence of oxygen, the pyruvate cannot be completely oxidized to carbon dioxide, so various intermediate products result. For example, when oxygen levels are low, skeletal muscle cells rely on glycolysis to meet their intense energy requirements. This reliance on glycolysis results in the buildup of an intermediate known as lactic acid, which can cause a person's muscles to feel as if they are "on fire." Similarly, yeast, which is a single-celled eukaryote, produces alcohol (instead of carbon dioxide) in oxygen-deficient settings. 2- Cetric acid cycle when oxygen is available, the pyruvates produced by glycolysis become the input for the next portion of the eukaryotic energy pathway. During this stage, each pyruvate molecule in the cytoplasm enters the mitochondrion, where it is converted into acetyl CoA, a two-carbon energy carrier, and its third carbon combines with oxygen and is released as carbon dioxide. At the same time, an NADH carrier is also generated. Acetyl CoA then enters a pathway called the citric acid cycle, which is the second major energy process used by cells. The eight-step citric acid cycle generates: a. three more NADH molecules and b. two other carrier molecules: FADH2 and GTP. 28  3- Electron transport chain The third major process in the eukaryotic energy pathway involves an electron transport chain, catalyzed by several protein complexes located in the mitochondrional inner membrane. This process, called oxidative phosphorylation, transfers electrons from NADH and FADH2 through the membrane protein complexes, and ultimately to oxygen, where they combine to form water. As electrons travel through the protein complexes in the chain, a gradient of hydrogen ions, or protons, forms across the mitochondrial membrane. Cells harness the energy of this proton gradient to create three additional ATP molecules for every electron that travels along the chain. Overall, the combination of the citric acid cycle and oxidative phosphorylation yields much more energy than fermentation - 15 times as much energy per glucose molecule! Together, these processes that occur inside the mitochondion, the citric acid cycle and oxidative phosphorylation, are referred to as respiration, a term used for processes that couple the uptake of oxygen and the production of carbon dioxide. The electron transport chain in the mitochondrial membrane is not the only one that generates energy in living cells. In plant and other photosynthetic cells, chloroplasts also have an electron transport chain that harvests solar energy. Even though they do not contain mitochondria or chloroplasts, prokaryotes have other kinds of energy- yielding electron transport chains within their plasma membranes that also generate energy. 29 1-4 Photosynthetic Cells Capture Light Energy and Convert It to Chemical Energy Cells get nutrients from their environment, but where do those nutrients come from? Virtually all organic material on Earth has been produced by cells that convert energy from the Sun into energy-containing macromolecules. This process, called photosynthesis, is essential to the global carbon cycle and organisms that conduct photosynthesis represent the lowest level in most food chains. What Is Photosynthesis? Why Is it Important? Cells use carbon dioxide and energy from the Sun to make sugar molecules and oxygen. These sugar molecules are the basis for more complex molecules made by the photosynthetic cell, such as glucose. Then, via respiration processes, cells use oxygen and glucose to synthesize energy- rich carrier molecules, such as ATP, and carbon dioxide is produced as a waste product. Therefore, the synthesis of glucose and its breakdown by cells are opposing processes. Photosynthesis doesn't just drive the carbon cycle — it also creates the oxygen necessary for respiring organisms. Although green plants contribute much of the 30 oxygen in the air we breathe, phytoplankton and cyanobacteria in the world's oceans are thought to produce between one-third and one-half of atmospheric oxygen on Earth. Photosynthetic cells contain special pigments that absorb light energy. Different pigments respond to different wavelengths of visible light. Chlorophyll, the primary pigment used in photosynthesis, reflects green light and absorbs red and blue light most strongly. In plants, photosynthesis takes place in chloroplasts, which contain the chlorophyll. Structure: Chloroplasts are surrounded by a double membrane and contain a third inner membrane, called the thylakoid membrane, that forms long folds within the organelle. In electron micrographs, thylakoid membranes look like stacks of coins, although the compartments they form are connected like a maze of chambers. The green pigment chlorophyll is located within the thylakoid membrane, and the space between the thylakoid and the chloroplast membranes is called the stroma. Chlorophyll A is the major pigment used in photosynthesis, but there are several types of chlorophyll and numerous other pigments that respond to light, including red, brown, and blue pigments. These other pigments may help channel light energy to chlorophyll A or protect the cell from photo-damage. For example, the photosynthetic protists called dinoflagellates, which are responsible for the "red tides" that often prompt warnings against eating shellfish, contain a variety of light-sensitive pigments, including both chlorophyll and the red pigments responsible for their coloration. 31 What Are the Steps of Photosynthesis? Photosynthesis consists of both light-dependent reactions and light-independent reactions. The so-called "light" reactions occur within the chloroplast thylakoids, where the chlorophyll pigments reside. When light energy reaches the pigment molecules, it energizes the electrons within them, and these electrons are shunted to an electron transport chain in the thylakoid membrane. Every step in the electron transport chain then brings each electron to a lower energy state and harnesses its energy by producing ATP and NADPH. Meanwhile, each chlorophyll molecule replaces its lost electron with an electron from water; this process essentially splits water molecules to produce oxygen. 32 Once the light reactions have occurred, the light-independent or "dark" reactions take place in the chloroplast stroma. During this process, also known as carbon fixation, energy from the ATP and NADPH molecules generated by the light reactions drives a chemical pathway that uses the carbon in carbon dioxide to build a three-carbon sugar called glyceraldehyde-3-phosphate (G3P). Cells then use G3P to build a wide variety of other sugars (such as glucose) and organic molecules. Many of these interconversions occur outside the chloroplast, following the transport of G3P from the stroma. The products of these reactions are then transported to other parts of the cell, including the mitochondria, where they are broken down to make more energy carrier molecules to satisfy the metabolic demands of the cell. In plants, some sugar molecules are stored as sucrose or starch. 1.5 Metabolism is the Complete Set of Biochemical Reactions within a Cell A cell's daily operations are accomplished through the biochemical reactions that take place within the cell. Reactions are turned on and off or sped up and slowed down according to the cell's immediate needs and overall functions. At any given time, the numerous pathways involved in building up and breaking down cellular components must be monitored and balanced in a coordinated fashion. To achieve this goal, cells organize reactions into various enzyme-powered pathways. What Do Enzymes Do? Enzymes are protein catalysts that speed biochemical reactions by facilitating the molecular rearrangements that support cell function. Recall that chemical reactions convert substrates into products, often by attaching chemical groups to or breaking off chemical groups from the substrates. Enzymes are flexible proteins that change shape when they bind with substrate molecules. In fact, this binding and shape changing ability is how enzymes manage to increase reaction rates. 33 In many cases, enzymes function by bringing two substrates into close proximity and orienting them for easier electron transfer. Shape or conformational changes can also act as an on/off switch. For example, when inhibitor molecules bind to a site on an enzyme distinct from the substrate site, they can make the enzyme assume an inactive conformation, thereby preventing it from catalyzing a reaction. Conversely, the binding of activator molecules can make an enzyme assume an active conformation, essentially turning it on. What Are Metabolic Pathways? Many of the molecular transformations that occur within cells require multiple steps to accomplish. e.g., Cells split one glucose molecule into two pyruvate molecules by way of a ten- step process called glycolysis. This coordinated series of chemical reactions is an example of a metabolic pathway in which the product of one reaction becomes the substrate for the next reaction. Consequently, the intermediate products of a metabolic pathway may be short-lived. Enzymes can be involved at every step in a reaction pathway. 34 How Do Cells Keep Chemical Reactions in Balance? Cells are expert recyclers. They disassemble large molecules into simpler building blocks and then use those building blocks to create the new components they require. The breaking down of complex organic molecules occurs via catabolic pathways and usually involves the release of energy. Through catabolic pathways, polymers such as proteins, nucleic acids, and polysaccharides are reduced to their constituent parts: amino acids, nucleotides, and sugars, respectively. In contrast, the synthesis of new macromolecules occurs via anabolic pathways that require energy input. Cells must balance their catabolic and anabolic pathways in order to control their levels of critical metabolites (those molecules created by enzymatic activity ) and ensure that sufficient energy is available. For example, if supplies of glucose start to wane, as might happen in the case of starvation, cells will synthesize glucose from other materials or start sending fatty acids into the citric acid cycle to generate ATP. Conversely, in times of plenty, excess glucose is converted into storage forms, such as glycogen, starches, and fats. 35 Not only do cells need to balance catabolic and anabolic pathways, but they must also monitor the needs and surpluses of all their different metabolic pathways. In order to bolster a particular pathway, cells can increase the amount of a necessary (rate-limiting) enzyme or use activators to convert that enzyme into an active conformation. Conversely, to slow down or halt a pathway, cells can decrease the amount of an enzyme or use inhibitors to make the enzyme inactive. Such up- and down-regulation of metabolic pathways is often a response to changes in concentrations of key metabolites in the cell. For example, a cell may take stock of its levels of intermediate metabolites and tune the glycolytic pathway and the synthesis of glucose accordingly. In some instances, the products of a metabolic pathway actually serve as inhibitors of their own synthesis, in a process known as feedback inhibition. For example, the first intermediate in glycolysis, glucose- 6-phosphate, inhibits the very enzyme that produces it, hexokinase. Feedback inhibition When there is enough product at the end of a reaction pathway (red macromolecule), it can inhibit its own synthesis by interacting with enzymes in the synthesis pathway (red arrow). 36  Vacuoles Vacuoles vary in shape and size in relation to the stage of development and the metabolic state of the cell. In meristematic cells, vacuoles are often very small and numerous. In mature cells, There is usually one large vacuole occupying the central part of the protoplast, whereas the cytoplasm and other protoplasmic components are restricted to a parietal position. ERGASTIC SUBSTANCES These are the products of metabolism and represented by reserve or waste products. They include: the visible carbohydrates, cellulose and starch, protein bodies, fats and related substances, mineral matter in the form of crystals. Among the ergastic substances are secondary products, which are not of great importance in plant metabolism. Many of these compounds are not produced by all plants and some by very few. However, these compounds are usually of considerable importance to man as sources of drugs and other purposes. These include tannins, resins, gums, rubber, flavonoids and alkaloids. 37  Carbohydrates These are the most abundant organic compounds of life, such as glucose and fructose. When two monosaccharides combine to form disaccharides, e.g., sucrose. Polysaccharides are formed from the combination of more than two monosaccharides. The principal polysaccharides in the plants are cellulose and starch. Pectic compounds are carbohydrates which often found in association with cellulose and form the primary cementing substances between cell walls. Lignin is also found in conjunction with cellulose in secondary cell walls. It is a very complicated, highly branched polymer that gives rigidity and causes the wood to resist decomposers. How are starch grains formed? Starch grains show obvious concentric layering. The deposition of starch occurs around a point called the hilum. This is referred to: The difference in density and water content in the different successive layers Alternating two different types of starch e.g. amylose and amylopectin. 38  Type of layering depending on the location of the hilum. Excentric Concentric layering layering Type of layering depending on the location of the hilum. Excentric Concentric layering layering 39  Proteins Proteins are the main ingredients of the living protoplasmic bodies, but they also occur as inactive ergastic substances. Ergastic protein occurs in the cell as a reserve material in amorphous or crystal-like form. In many seeds, there are special type of protein grains known as the Aleurone grains, e.g., in the seeds of castor oil plant (Ricinus conumunis). Each aleurone grain is enclosed within a membrane and contains inclusions of globoids and crystalloids of protein, which are embedded in simple proteins. One or more globoid or crystalloid is present in the aleurone grain. Crystals Crystals are the end products of the metabolic processes of the cell. These crystals are of differing chemical composition and are found in many kinds of cells and plants. The inorganic deposits in plants consist mostly of calcium salts and of anhydrides of silica. 40  CALCIUM CRYSTALS Calcium Calcium oxalates carbonate Solita raphi druse Cysto ry des s lith 41  Unit 2: How Do Cells Decode Genetic Information into Functional Proteins? How Do Cells Decode Genetic Information into Functional Proteins? It is a beautiful system made complex by many levels of control, on-off switches, feedback, and fine-tuning. Segments of DNA are transcribed into RNA, and this RNA is then translated into proteins. The resulting proteins then fold into their three-dimensional configurations and combine with other proteins, or are decorated with sugars or fats to create finely-crafted tools for carrying out specific cellular functions. Protein functions range from structural supports and motors to catalysts of biochemical reactions and monitors of the cell's internal and external environments. 2.1 Information Transfer in Cells Requires Many Proteins and Nucleic Acids How Is Genetic Information Passed on in Dividing Cells? When a cell divides, it creates one copy of its genetic information — in the form of DNA molecules — for each of the two resulting daughter cells. The accuracy of these copies determines the health and inherited features of the nascent cells, so it is essential that the process of DNA replication be as accurate as possible. One factor that helps ensure precise replication is the double-helical structure of DNA itself.. In particular, the two strands of the DNA double helix are made up of combinations of molecules called nucleotides. DNA is constructed from just four different nucleotides — adenine (A), thymine (T), cytosine (C), and guanine (G) — each of which is named for the nitrogenous base it contains. Moreover, the nucleotides that form one strand of the DNA double helix always bond with the nucleotides in the other strand according to a pattern known as complementary base-pairing — 42 specifically, A always pairs with T, and C always pairs with G (Figure 2). Thus, during cell division, the paired strands unravel and each strand serves as the template for synthesis of a new complementary strand. In most multicellular organisms, every cell carries the same DNA, but this genetic information is used in varying ways by different types of cells. In other words, what a cell "does" within an organism dictates which of its genes are expressed. Nerve cells, for example, synthesize an abundance of chemicals called neurotransmitters, which they use to send messages to other cells. What Are the Initial Steps in Accessing Genetic Information? Transcription is the first step in decoding a cell's genetic information. During transcription, enzymes called RNA polymerases build RNA molecules that are complementary to a portion of one strand of the DNA double helix 43 RNA molecules differ from DNA molecules in several important ways: They are single stranded rather than double stranded; their sugar component is a ribose rather than a deoxyribose; and they include uracil (U) nucleotides rather than thymine (T) nucleotides. Also, because they are single strands, RNA molecules don't form helices; rather, they fold into complex structures that are stabilized by internal complementary base-pairing. 44 Three general classes of RNA molecules are involved in expressing the genes encoded within a cell's DNA. Messenger RNA (mRNA) molecules carry the coding sequences for protein synthesis and are called transcripts; ribosomal RNA (rRNA) molecules form the core of a cell's ribosomes (the structures in which protein synthesis takes place); and transfer RNA (tRNA) molecules carry amino acids to the ribosomes during protein synthesis. mRNA is the most variable class of RNA, and there are literally thousands of different mRNA molecules present in a cell at any given time. Some mRNA molecules are abundant, numbering in the hundreds or thousands, as is often true of transcripts encoding structural proteins. Other mRNAs are quite rare, with perhaps only a single copy present, as is sometimes the case for transcripts that encode signaling proteins. mRNAs also vary in how long-lived they are. In eukaryotes, transcripts for structural proteins may remain intact for over ten hours, whereas transcripts for signaling proteins may be degraded in less than ten minutes. Cells can be characterized by the spectrum of mRNA molecules present within them; this spectrum is called the transcriptome. Whereas each cell in a multicellular organism carries the same DNA or genome, its transcriptome varies widely according to cell type and function. For instance, the insulin-producing cells of the pancreas contain transcripts for insulin, but bone cells do not. Even though bone cells carry the gene for 45  insulin, this gene is not transcribed. Therefore, the transcriptome functions as a kind of catalog of all of the genes that are being expressed in a cell at a particular point in time. What Is the Function of Ribosomes? Ribosomes are the sites in a cell in which protein synthesis takes place. Cells have many ribosomes. the number depends on the activity of a particular cell in synthesizing proteins. For example, rapidly growing cells usually have a large number of ribosomes What Is the Function of Ribosomes? Ribosomes are complexes of rRNA molecules and proteins. Sometimes, ribosomes are visible as clusters, called polyribosomes. In eukaryotes, some of the ribosomes are attached to internal membranes, where they synthesize the proteins that will later reside in those membranes, or are destined for secretion. Although only a few rRNA molecules are present in each ribosome, these molecules make up about half of the ribosomal mass. The remaining mass consists of a number of proteins — nearly 60 in prokaryotic cells and over 80 in eukaryotic cells. Function of rRNA:Within the ribosome, the rRNA molecules direct the catalytic steps of protein synthesis — the stitching together of amino acids to make a protein molecule. In fact, rRNA is sometimes called a ribozyme or catalytic RNA to reflect this function. Eukaryotic and prokaryotic ribosomes: They are different from each other and these differences are exploited by antibiotics, which are designed to inhibit the prokaryotic ribosomes of infectious bacteria without affecting eukaryotic ribosomes, thereby not interfering with the cells of the sick host. 46 How Does the Whole Process Result in New Proteins? After the transcription of DNA to mRNA is complete, translation — or the reading of these mRNAs to make proteins — begins. Recall that mRNA molecules are single stranded, and the order of their bases — A, U, C, and G — is complementary to that in specific portions of the cell's DNA. Each mRNA dictates the order in which amino acids should be added to a growing protein as it is synthesized. In fact, every amino acid is represented by a three-nucleotide sequence or codon along the mRNA molecule. For example, AGC is the mRNA codon for the amino acid serine, and UAA is a signal to stop translating a protein — also called the stop codon. A ribosome is composed of two subunits: large and small. During translation, ribosomal subunits assemble together like a sandwich on the strand of mRNA, where they proceed to attract tRNA molecules tethered to amino acids (circles). 47 A long chain of amino acids emerges as the ribosome decodes the mRNA sequence into a polypeptide, or a new protein. tRNA are responsible for matching amino acids with the appropriate codons in mRNA. Each tRNA molecule has two distinct ends: one of which binds to a specific amino acid, and the other which binds to the corresponding mRNA codon. During translation, these tRNAs carry amino acids to the ribosome and join with their complementary codons. Then, the assembled amino acids are joined together as the ribosome, with its resident rRNAs, moves along the mRNA molecule in a ratchet-like motion. The resulting protein chains can be hundreds of amino acids in length, and synthesizing these molecules requires a huge amount of chemical energy. 48  In prokaryotic cells, transcription (DNA to mRNA) and translation (mRNA to protein) are so closely linked that translation usually begins before transcription is complete. In eukaryotic cells, however, the two processes are separated in both space and time: mRNAs are synthesized in the nucleus, and proteins are later made in the cytoplasm. 49  Figure: (1) Translation begins when a ribosome (gray) docks on a start codon (red) of an mRNA molecule in the cytoplasm. (2) Next, tRNA molecules attached to amino acids (spheres) dock at the corresponding triplet codon sequence on the mRNA molecule. (3) (3, 4, and 5) This process repeats over and over, with multiple tRNAs docking and connecting successive amino acids into a growing chain that elongates out of the top of the ribosome. (6) When the ribosome encounters a stop codon, it falls off the mRNA molecule and releases the protein for use in the cell. 50 2.2 DNA Is Extensively Compacted with Proteins Chromosomes Cells package their DNA to protect it and regulate which genes are accessed and when. DNA packaging helps conserve space in cells. approximately two meters of human DNA can fit into a cell that is only a few micrometers wide. But how, exactly, is DNA compacted to fit within eukaryotic and prokaryotic cells? And what mechanisms do cells use to access this highly compacted genetic material? What Are Chromosomes? Cellular DNA always forms a complex with various protein partners that help package it into such a tiny space. This DNA-protein complex is called chromatin, wherein the mass of protein and nucleic acid is nearly equal. Within cells, chromatin usually folds into characteristic formations called chromosomes. Each chromosome contains a single double-stranded piece of DNA along with the packaging proteins. During cell division,, they become more tightly packed, and their condensed form can be visualized with a light microscope. This condensed form is approximately 10,000 times shorter than the linear DNA strand would be if it was devoid of proteins and pulled taut. However, when eukaryotic cells are not dividing — a stage called interphase — the chromatin within their chromosomes is less tightly packed. This looser configuration is important because it permits transcription to take place. In contrast to eukaryotes, the DNA in prokaryotic cells is generally present in a single circular chromosome that is located in the cytoplasm. Prokaryotic chromosomes are less condensed than their eukaryotic counterparts and don't have easily identified features when viewed under a light microscope. 51 How Are Eukaryotic Chromosomes Structured? Eukaryotic chromosomes consist of repeated units of chromatin called nucleosomes Nucleosomes are made up of double-stranded DNA that has complexed with small proteins called histones. The core particle of each nucleosome consists of eight histones. Histones carry positive charges and bind negatively charged DNA in a specific conformation. In particular, a segment of the DNA double helix wraps around each histone core particle a little less than twice. The exact length of the DNA segment associated with each histone core varies from species to species, but most such segments are approximately 150 base pairs in length. Furthermore, each histone molecule within the core particle has one end that sticks out from the particle. These ends are called N-terminal tails, and they play an important role in higher-order chromatin structure and gene expression. 52 Why Is Complex Packing Critical for Eukaryotic Chromosomes? Although nucleosomes may look like extended "beads on a string" under an electron microscope, they appear differently in living cells. In such cells, nucleosomes stack up against one another in organized arrays with multiple levels of packing. The first level of packing is thought to produce a fiber about 30 nanometers (nm) wide. These 30 nm fibers then form a series of loops, which fold back on themselves for additional compacting. 53 1- The multiple levels of packing that exist within eukaryotic chromosomes not only permit a large amount of DNA to occupy a very small space, but they also serve several functional roles. For example, the looping of nucleosome-containing fibers brings specific regions of chromatin together, thereby influencing gene expression. In fact, the organized packing of DNA is malleable and appears to be highly regulated in cells. 2- Chromatin packing also offers an additional mechanism for controlling gene expression. Specifically, cells can control access to their DNA by modifying the structure of their chromatin. Highly compacted chromatin simply isn't accessible to the enzymes involved in DNA transcription, replication, or repair. Thus, regions of chromatin where active transcription is taking place 54 (called euchromatin) are less condensed than regions where transcription is inactive or is being actively inhibited or repressed (called heterochromatin). 3- The dynamic nature of chromatin is regulated by enzymes. For example, chromatin can be loosened by changing the position of the DNA strands within a nucleosome. This loosening occurs because of chromatin remodeling enzymes, which function to slide nucleosomes along the DNA strand so that other enzymes can access the strand. This process is closely regulated and allows specific genes to be accessed in response to metabolic signals within the cell. 4- Another way cells control gene expression is by modifying their histones with small chemical groups, such as methyl and acetyl groups in the N-terminal tails that extend from the core particle. Different enzymes catalyze each kind of N- terminal modification. Scientists occasionally refer to the complex pattern of histone modification in cells as a "histone code." Some of these modifications increase gene expression, whereas others decrease it. How Are Chromosomes Organized in the Nucleus? chromosomes have functional and nonrandom arrangements. chromosomes occupy characteristic regions of the nucleus, which they termed chromosome territories. The spatial localization of these territories is thought to be important for gene expression. In fact, with the advent of gene-specific probes, researchers are beginning to understand how the arrangement of chromosome territories can bring particular genes closer together. A second major observation related to chromosome territories is that the position of chromosomes relative to one another differs from cell to cell. Such differences reflect variation in gene expression patterns. 55 2.3 Differential Control of Transcription and Translation Underlies Changes in Cell Function Each somatic cell in the body generally contains the same DNA. (A few exceptions include red blood cells, which contain no DNA in their mature state, and some immune system cells that rearrange their DNA while producing antibodies). In general, the genes that determine whether you have green eyes or brown hair, or how fast you metabolize food are the same in eye cells and liver cells, even though these organs function quite differently. If each cell has the same DNA, how is it that cells differ in their structure and function? Why do cells in the eye differ so dramatically from cells in the liver? Genes encode proteins and proteins dictate cell function. Therefore, the thousands of genes expressed in a particular cell determine what that cell can do. Moreover, each step in the flow of information from DNA to RNA to protein provides the cell with a potential control point for self-regulating its functions by adjusting the amount and type of proteins it manufactures. At any given time, the amount of a particular protein in a cell reflects the balance between that protein's synthetic and degradative biochemical pathways. On the synthetic side of this balance, recall that protein production starts at transcription (DNA to RNA) and continues with translation (RNA to protein). Thus, control of these processes plays a critical role in determining what proteins are present in a cell and in what amounts. In addition, the way in which a cell processes its RNA transcripts and newly made proteins also greatly influences protein levels. 56 Figure: Prokaryotic transcription and translation occur simultaneously in the cytoplasm, and regulation occurs at the level of transcription. In eukaryotes, transcription and translation are physically separated, and gene expression is regulated at many different levels. Prokaryotic Gene Regulation The DNA of prokaryotes is organized into a circular chromosome that resides in the cell’s cytoplasm. Proteins that are needed for a specific function, or that are involved in the same biochemical pathway, are often encoded together in blocks called operons. For example, all five of the genes needed to make the amino acid tryptophan in the bacterium E. coli are located next to each other in the trp operon. The genes in an operon are transcribed into a single mRNA molecule. This allows the genes to be controlled as a unit: either all are expressed, or none is expressed. Each operon needs only one regulatory region, including: a promoter, where RNA polymerase binds, and an operator, where other regulatory proteins bind. 57 In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of operons. 1. Activators are proteins that increase the transcription of a gene. 2. Repressors are proteins that suppress transcription of a gene. 3. Inducers are molecules that bind to repressors and inactivate them. Below are two examples of how these molecules regulate different operons. The trp Operon The lac Operon The trp Operon: A Repressor Operon Like all cells, bacteria need amino acids to survive. Tryptophan is one amino acid that the bacterium E. coli can either ingest from the environment or synthesize. When E. coli needs to synthesize tryptophan, it must express a set of five proteins that are encoded by five genes. These five genes are located next to each other in the tryptophan (trp) operon. When tryptophan is present in the environment, E. coli does not need to synthesize it, and the trp operon is switched off. When tryptophan availability is low, the trp operon is turned on so that the genes are transcribed, the proteins are made, and tryptophan can be synthesized. A DNA sequence called the operator is located between the promoter and the first trp gene. The operator contains the DNA code to which the repressor protein can bind. The repressor protein is regulated by levels of tryptophan in the cell. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor. This causes the repressor to change shape and bind to the trp operator. Binding of the tryptophan–repressor complex at the operator physically blocks the RNA polymerase from binding, and transcribing the 58 downstream genes. Thus, when the cell has enough tryptophan, it is preventing from making more. When tryptophan is not present in the cell, the repressor has no tryptophan to bind to it. The repressor is not activated and it does not bind to the operator. Therefore, RNA polymerase can transcribe the operon and make the enzymes to synthesize tryptophan. Thus, when the cell does not have enough tryptophan, it synthesizes it. Figure: The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. The lac Operon: An Inducer Operon The lac operon in E. coli has more complex regulation, involving both a repressor and an activator. E. coli uses glucose for food, but is able to use other sugars, such as lactose, when glucose concentrations are low. Three proteins are needed to break down lactose; they are encoded by the three genes of the lac operon. 59 When lactose is not present, the proteins to digest lactose are not needed. Therefore, a repressor binds to the operator and prevents RNA polymerase from transcribing the operon. When lactose is present, lactose binds to the repressor and removes it from the operator. RNA polymerase is now free to transcribe the genes necessary to digest lactose Figure: Transcription of the lac operon only occurs when lactose is present. Lactose binds to the repressor and removes it from the operator. However, the story is more complex than this. Since E. coli prefers to use glucose for food, the lac operon is only expressed at low levels even when the repressor is removed. 60 But what happens when ONLY lactose is present? Now the bacterium needs to ramp up production of the lactose-digesting proteins. It does so by using an activator protein called catabolite activator protein (CAP). When glucose levels drop, cyclic adenosine mono phosphate (cAMP) begins to accumulate in the cell. cAMP binds to CAP and the complex binds to the lac operon promoter. This increases the binding ability of RNA polymerase to the promoter and ramps up transcription of the genes. When there is no glucose, the CAP activator increases transcription of the lac operon. However, if no lactose is present, the operon is not activated. Figure: When glucose levels drop: 61  1. cyclic AMP (cAMP) begins to accumulate in the cell. 2. cAMP binds to CAP and the complex binds to the lac operon promoter. 3. This increases the binding ability of RNA polymerase to the promoter and ramps up transcription of the genes. In summary, for the lac operon to be fully activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent, and lactose is present will the lac operon be transcribed maximally. This makes sense for the cell, because it would be energetically wasteful to create the proteins to process lactose if glucose was plentiful or lactose was not available. Summary of signals that induce or repress transcription of the lac operon. Eukaryotic Gene Regulation How Is Gene Expression Regulated? Eukaryotic transcripts are also more complex than prokaryotic transcripts. For instance, the primary transcripts synthesized by RNA polymerase contain sequences that will not be part of the mature RNA. These intervening sequences are called introns, and they are removed before the mature mRNA leaves the nucleus. The remaining regions of the transcript, which include the protein-coding regions, are called exons, and they are spliced together to produce the mature mRNA. Eukaryotic transcripts are also modified at their ends, which affects their stability and translation. 62 In eukaryotes, control of gene expression is more complex and can happen at many different levels. Eukaryotic genes are not organized into operons, so each gene must be regulated independently. In addition, eukaryotic cells have many more genes than prokaryotic cells. Regulation of gene expression can happen at any of the stages as DNA is transcribed into mRNA and mRNA is translated into protein. For convenience, regulation is divided into five levels: epigenetic transcriptional post-transcriptional translational post-translational 63 Figure: Regulation of gene expression in eukaryotes can occur at five different levels. Here, the Central Dogma is diagrammed with arrows showing where each type of eukaryotic regulation of gene expression interrupts it. 1-Epigenetic Control of Gene Expression The first level of control of gene expression is epigenetic (“around genetics”) regulation. Epigenetics is a relatively new, but growing, field of biology. Epigenetic control involves: changes to genes that do not alter the nucleotide sequence of the DNA and are not permanent. Instead, these changes alter the chromosomal structure so that genes can be turned on or off. This level of control occurs through heritable chemical modifications of the DNA and/or chromosomal proteins. One example of chemical modifications of DNA is the addition of methyl groups to the DNA, in a process called methylation, In general, methylation suppresses transcription. Interestingly, methylation patterns can be passed on as cells divide. Thus, parents may be able to pass on the tendency of a gene to be expressed in their offspring. Other heritable chemical modifications of DNA may also occur. 64 Modification of Histone Proteins is an Example of Epigenetic Control Histone proteins can move along the DNA and change the structure of the molecule. If a gene is to be transcribed, the nucleosomes surrounding that region of DNA can slide down the DNA to open that specific chromosomal region and allow access for RNA polymerase and other proteins, called transcription factors, to bind to the promoter region and initiate transcription. If a gene is to remain turned off, or silenced, the histone proteins and DNA have different modifications that signal a closed chromosomal configuration. In this closed configuration, the RNA polymerase and transcription factors do not have access to the DNA and transcription cannot occur. How the histone proteins move is dependent on signals found on the histone proteins. These signals are “tags” – in the form of phosphate, methyl, or acetyl groups – that open or close a chromosomal region. These tags are not permanent, but may be added or removed as needed. Since DNA negatively charged, changes in the charge of the histone will change how tightly wound the DNA molecule will be. When unmodified, the histone proteins have a large positive charge; by adding chemical modifications like acetyl groups, the charge becomes less positive. 65 2- Transcriptional Control of Gene Expression Transcriptional regulation is control of whether or not an mRNA is transcribed from a gene in a particular cell. Like prokaryotic cells, the transcription of genes in eukaryotes requires an RNA polymerase to bind to a promoter to initiate transcription. In eukaryotes, RNA polymerase requires other proteins, or transcription factors, to facilitate transcription initiation. Transcription factors are proteins that bind to the promoter sequence and other regulatory sequences to control the transcription of the target gene. RNA polymerase by itself cannot initiate transcription in eukaryotic cells. Transcription factors must bind to the promoter region first and recruit RNA polymerase to the site for transcription to begin. 66 In eukaryotic genes, the promoter region is immediately upstream of the coding sequence. This region can range from a few to hundreds of nucleotides long. The length of the promoter is gene-specific and can differ dramatically between genes. The longer the promoter, the more available space for proteins to bind. Consequently, the level of control of gene expression can differ quite dramatically between genes. The purpose of the promoter is to bind transcription factors that control the initiation of transcription. Within the promoter region, just upstream of the transcriptional start site, resides the TATA box. This box is simply a repeat of thymine and adenine dinucleotides (literally, TATA repeats). Transcription factors bind to the TATA box, assembling an initiation complex. Once this complex is assembled, RNA polymerase binds to its upstream sequence and becomes phosphorylated. This releases part of the protein 67 from the DNA, activates the transcription initiation complex, and places RNA polymerase in the correct orientation to begin transcription. Enhancers and Repressors In some eukaryotic genes, there are regions that help increase transcription. These regions, called enhancers, Enhancers are not necessarily close to the genes; they can be located thousands of nucleotides away. They can be found upstream, within the coding region, or downstream of a gene. Enhancers are binding sites for activators. When an enhancer is far away from a gene, the DNA folds such that the enhancer is brought into proximity with the promoter, allowing interaction between the activators and the transcription initiation complex. Like prokaryotic cells, eukaryotic cells also have mechanisms to prevent transcription. Transcriptional repressors can bind to promoter or enhancer regions and block transcription. Both activators and repressors respond to external stimuli to determine which genes need to be expressed. 68  3- Post-transcriptional Control of Gene Expression Post-transcriptional regulation occurs after the mRNA is transcribed but before translation begins. This regulation can occur at the level of mRNA processing, transport from the nucleus to the cytoplasm, or binding to ribosomes. A- Alternative RNA splicing In eukaryotic cells the RNA primary transcript often contains introns, which are removed prior to translation. Alternative RNA splicing is a mechanism that allows different combinations of introns, and sometimes exons, to be removed from the primary transcript. This allows different protein products to be produced from one gene. Alternative splicing can act as a mechanism of gene regulation. Differential splicing is used to produce different protein products in different cells or at different times within the same cell. Alternative splicing is now understood to be a common mechanism of gene regulation in eukaryotes; up to 70 percent of genes in humans are expressed as multiple proteins through alternative splicing. 69 Before a RNA can be translated, introns must be removed by splicing. Pre-mRNA can be alternatively spliced to create different proteins. B- Control of RNA Stability in the cytoplasm The longer an mRNA exists in the cytoplasm, the more time it has to be translated, and the more protein is made. Many factors contribute to mRNA stability, including the length of its poly-A tail. Proteins, called RNA-binding proteins (RBPs) can bind to the regions of the RNA just upstream or downstream of the protein-coding region. These regions in the RNA that are not translated into protein are called the untranslated regions, or UTRs. The region just before the protein-coding region is called the 5′ UTR, whereas the region after the coding region is called the 3′ UTR. The binding of RBPs to these regions can increase or decrease the stability of an RNA molecule, depending on the specific RBP that binds. The protein-coding region of mRNA is flanked by 5′ and 3′ untranslated regions (UTRs). RNA-binding proteins at the 5′ or 3′ UTR influence the stability of the RNA molecule. 4- Translational Control of Gene Expression 70 Translation can also be regulated at the level of binding of the mRNA to the ribosome. Once the mRNA bound to the ribosome, the speed and level of translation can still be controlled. An example of translational control occurs in proteins that are destined to end up in the endoplasmic reticulum (ER). The first few amino acids of these proteins are a tag called a signal sequence. As soon as these amino acids are translated, a signal recognition particle (SRP) binds to the signal sequence and stops translation while the mRNA-ribosome complex is shuttled to the ER. Once they arrive, the signal recognition particle SRP is removed and translation resumes. 5- Post-translational Control of Gene Expression This type of control involves modifying the protein after it is made, in such as way as to affect its activity. 1- Enzyme inhibition One example of post-translational regulation is enzyme inhibition. When an enzyme is no longer needed, it is inhibited by a competitive or allosteric inhibitor, which prevents it from binding to its substrate. The inhibition is reversible, so that the enzyme can be reactivated later. This is more efficient than degrading the enzyme when it is not needed and then making more when it is needed again. 71 Competitive and allosteric inhibitor 72 2- The activity and/or stability of proteins can also be regulated by adding functional groups, such as methyl, phosphate, or acetyl groups. Sometimes these modifications can regulate where a protein is found in the cell—for example, in the nucleus, the cytoplasm, or attached to the plasma membrane. The addition of an ubiquitin group to a protein marks that protein for degradation. Ubiquitin acts like a flag indicating that the protein’s lifespan is complete. Tagged proteins are moved to a proteasome, an organelle that degrades proteins. One way to control gene expression, therefore, is to alter the longevity of the protein. Proteins with ubiquitin tags are marked for degradation within the proteasome. 73  3-THE CELL CYCLE & MITOSIS A human, as well as every sexually reproducing organism, begins life as a fertilized egg or zygote. Trillions of cell divisions subsequently occur in a controlled manner to produce a complex, multicellular human. Single-celled organisms use cell division as their method of reproduction. 3.1 DNA Organization and the Cell Cycle The cell cycle is an orderly sequence of events that describes the stages of a cell’s life from the division of a single parent cell to the production of two genetically identical new daughter cells. The mechanisms involved in the cell cycle are highly regulated. 3.1.1 Genomic DNA Before discussing the steps a cell must undertake to replicate, we need a deeper understanding of the structure and function of a cell’s genetic information. A cell’s DNA, packaged as double-stranded DNA molecules, is called its genome. In prokaryotes, the genome is composed of a single circular double-stranded DNA molecule. The region in the cell containing this genetic material is called a nucleoid. Some prokaryotes also have smaller loops of non- essential DNA called plasmids. Bacteria can exchange these plasmids with other bacteria, sometimes receiving beneficial new genes that the recipient can add to their chromosomal DNA. Antibiotic resistance is one trait that often spreads through a bacterial colony through plasmid exchange. 74 In eukaryotic cells, the genome consists of several double-stranded linear DNA molecules. Each species has a characteristic number of chromosomes in the nuclei of its cells. Human body cells have 46 chromosomes, while human gametes (sperm or eggs) have 23 chromosomes each. A typical body cell, or somatic cell, contains two matched sets of chromosomes, a configuration known as diploid. The letter n is used to represent a single set of chromosomes; therefore, a diploid organism is designated 2n. Human cells that contain one set of chromosomes are called gametes, or sex cells; these are eggs and sperm, and are designated 1n, or haploid. Matched pairs of chromosomes in a diploid organism are called homologous (“same knowledge”) chromosomes. Homologous chromosomes are the same length and have specific nucleotide segments called genes in exactly the same location, or locus. Genes are the functional units of chromosomes and determine specific characteristics by coding for specific proteins. Traits are the variations of those characteristics. For example, hair color is a characteristic with traits that are blonde, brown, or black. Each copy of a homologous pair of chromosomes originates from a different parent; therefore, the genes themselves are not identical. 75 The variation of individuals within a species is due to the specific combination of the genes inherited from both parents. Even a slightly altered sequence of nucleotides within a gene can result in an alternative trait. A karyotype of human chromosomes, showing their distinct sizes and banding patterns. In this image, the chromosomes were exposed to fluorescent stains to highlight chromosomes in different colors. 3.1.2 Eukaryotic Chromosomal Structure and Compaction If the DNA from all 46 chromosomes in a human cell nucleus was laid out end to end, it would measure approximately two meters; however, its diameter would be only 2 nm. Considering that the size of a typical human cell is about 10 µm (100,000 cells lined up to equal one meter), DNA must be tightly packaged to fit in the cell’s nucleus. At the same time, it must also be readily accessible for the genes to be expressed. During some stages of the cell cycle, the long strands of DNA are condensed into compact chromosomes. There are a number of ways that chromosomes are compacted. 76 DNA replicates in the S phase of interphase. After replication, the chromosomes are composed of two linked sister chromatids. When fully compact, the pairs of identically packed chromosomes are bound to each other by cohesin proteins. The connection between the sister chromatids is closest in a region called the centromere. The conjoined sister chromatids, with a diameter of about 1 µm, are visible under a light microscope. The centromeric region is highly condensed and thus will appear as a constricted area. 77 78  3.2 | The Cell Cycle The cell cycle is an ordered series of events involving cell growth and cell division that produces two new daughter cells. Somatic cells on the path to cell division proceed through a series of precisely timed and carefully regulated stages of growth, DNA replication, and division that produces two genetically identical cells. In other words, a typical 2n somatic cell will divide into two 2n somatic cells that are genetically identical. This is a form of asexual reproduction. we see two major phases: interphase and the M phase. During interphase, the cell undergoes three distinct periods: G1, S, and G2. During G1, S, and G2, the cell grows, DNA is replicated, and the cell grows some more. During the M phase, the cell undergoes two distinct periods: mitosis (also called karyokinesis) and cytokinesis, the division of the cytoplasm. Figure: cell cycle 79 3.2.1 Interphase During interphase, the cell undergoes normal growth processes while also preparing for cell division. In order for a cell to move from interphase into the mitotic phase, many internal and external conditions must be met. The three aspects or stages of interphase are called G1, S, and G2. G1 Phase (First Gap) The first stage of interphase is called the G1 phase (first gap) because, from a microscopic aspect, little change is visible. However, during the G1 stage, the cell is quite active at the biochemical level. The cell is accumulating the building blocks of chromosomal DNA and the associated proteins as well as accumulating sufficient energy reserves to complete the task of replicating each chromosome in the nucleus. S Phase (Synthesis of DNA) Throughout interphase, nuclear DNA remains in a semi-condensed chromatin configuration. In the S phase, DNA replication can proceed to form identical pairs of DNA molecules—sister chromatids—that are firmly attached to the centromeric region. The centrosome is duplicated during the S phase. The two centrosomes will give rise to the mitotic spindle, the apparatus that orchestrates the movement of chromosomes during mitosis. At the center of each animal cell, the centrosomes of animal cells are associated with a pair of rod-like objects, the centrioles, which are at right angles to each other. Centrioles help organize cell division. Centrioles are not present in the centrosomes of other eukaryotic species, such as plants and most fungi. G2 Phase (Second Gap) In the G2 phase, the cell replenishes its energy stores and synthesizes proteins necessary for chromosome manipulation. Some cell organelles are duplicated, and the cytoskeleton is dismantled to provide resources for the mitotic phase. There may be additional cell growth during G2. The final preparations for the mitotic phase must be completed before the cell is able to enter the first stage of mitosis. 80  3.2.2 The Mitotic Phase The mitotic phase is a multistep process during which the duplicated chromosomes are aligned, separated, and moved into two new, identical daughter cells. M phase is divided into mitosis and cytokinesis. Mitosis Mitosis is divided into a series of phases—Prophase, Prometaphase, Metaphase, Anaphase, and Telophase—that result in the division of the cell nucleus. 81 82 Cytokinesis Cytokinesis, or “cell motion,” is the second main stage of the mitotic phase during which cell division is completed via the physical separation of the cytoplasmic components into two daughter cells. Division is not complete until the cell components have been apportioned and completely separated into the two daughter cells. Although the stages of mitosis are similar for most eukaryotes, the process of cytokinesis is quite different for eukaryotes that have cell walls, such as plant cells. In cells such as animal cells that lack cell walls, cytokinesis actually begins at the midpoint of Anaphase. A contractile ring composed of actin filaments forms just inside the plasma membrane at the former Metaphase plate. The actin filaments pull the equator of the cell inward, forming a constriction. This constriction or fissure is called the cleavage furrow. The furrow deepens as the actin ring contracts, and eventually the membrane is cleaved in two. In plant cells, a new cell wall must form between the daughter cells. During interphase, the Golgi apparatus accumulates enzymes, structural proteins, and glucose molecules prior to breaking into vesicles and dispersing throughout the dividing cell. During telophase, these Golgi vesicles are transported on microtubules to form a phragmoplast (a vesicular structure) at the metaphase plate. There, the vesicles fuse and coalesce from the center toward the cell walls; this structure is called a cell plate. As more vesicles fuse, the cell plate enlarges until it merges with the cell walls at the periphery of the cell. Enzymes use the glucose that has accumulated between the membrane layers to build a new cell wall made of cellulose. Remember that cellulose is a structural polymer of glucose. The Golgi membranes become parts of the plasma membrane on either side of the new cell wall. 83  3.2.3 G0 Phase (A variation of the cell cycle) Not all cells adhere to the classic cell cycle pattern in which a newly formed daughter cell immediately enters the preparatory phases of interphase, closely followed by the mitotic phase. Cells in G0 phase (“G zero”) are not actively preparing to divide. The cell is in a quiescent (inactive) stage that occurs when cells exit the cell cycle. Some cells enter G0 temporarily until an external signal triggers the onset of G1. Other cells that never or rarely divide, such as mature cardiac muscle and nerve cells, remain in G0 permanently. Although cells in G0 are inactive in the sense that they are not actively preparing for and undergoing cell division, they can be very active in other ways. Many cells in the adult body for instance, are permanently in G0 while fulfilling their specialized functions. 84  3.3.2 Regulation of the Cell Cycle at Internal Checkpoints It is essential that the daughter cells that have been produced be exact duplicates of the parent cell. Mistakes in the duplication or distribution of the chromosomes lead to mutations that may be passed forward to every new cell produced from an abnormal cell. To prevent a compromised cell from continuing to divide, there are internal control mechanisms that operate at three main cell cycle checkpoints. A checkpoint is one of several points in the eukaryotic cell cycle at which the progression of a cell to the next stage in the cycle can be halted until conditions are favorable. These checkpoints occur: 1. near the end of G1, 2. at the G2/M transition, and 3. during metaphase. 1- The G1 Checkpoint The G1 checkpoint determines whether all conditions are favorable for cell division to proceed. The G1 checkpoint, also called the restriction point (in yeast), is a point at which the cell irreversibly commits to the cell division process. External influences, such as growth factors, play a large role in carrying the cell past the G1 checkpoint. In addition to adequate reserves and cell size, there is a check for genomic DNA damage at the G1 checkpoint. A cell that does not meet all the requirements will not be allowed to progress into the S phase. The cell can halt th