Lecture 2 - Proteins and Nucleic Acids (2) PDF

Proteins  The Many Functions of Proteins - immensely diverse in structure and function 1. Enzyme catalysis 2. Defense 3. Transport 4. Support 5. Motion 6. Regulation The Many Functions of Proteins The Many Functions of Proteins Amino Acids Are the Building Blocks of Proteins Although p...

Proteins  The Many Functions of Proteins - immensely diverse in structure and function 1. Enzyme catalysis 2. Defense 3. Transport 4. Support 5. Motion 6. Regulation The Many Functions of Proteins The Many Functions of Proteins Amino Acids Are the Building Blocks of Proteins Although proteins are complex and versatile molecules, they are all polymers of only 20 amino acids An amino acid is a molecule containing an amino group (NH2), a carboxyl group (—COOH), and a hydrogen atom, all bonded to a central carbon atom: Each amino acid has unique chemical properties determined by the nature of the side group (indicated by R) covalently bonded to the central carbon atom. 1. Nonpolar amino acids, such as leucine, often have R groups that contain —CH2 or —CH3. 2. Polar uncharged amino acids, such as threonine, have R groups that contain oxygen (or only —H). 3. Ionizable amino acids, such as glutamic acid, have R groups that contain acids or bases. 4. Aromatic amino acids, such as phenylalanine, have R groups that contain an organic (carbon) ring with alternating single and double bonds. 5. Special-function amino acids have unique individual properties; methionine often is the first amino acid in a chain of amino acids, proline causes kinks in chains, and cysteine links chains together. Proteins Are Polymers of Amino Acids The amino and carboxyl groups on a pair of amino acids can undergo a condensation reaction, losing a molecule of water and forming a covalent bond- peptide bond A protein is composed of one or more long chains, or polypeptides, composed of amino acids linked by peptide bonds. Although many different amino acids occur in nature, only 20 commonly occur in proteins. Overview of Protein Structure Proteins consist of long amino acid chains folded into complex shapes. The structure of proteins is traditionally discussed in terms of four levels of structure, as primary, secondary, tertiary, and quaternary Primary Structure. The specific amino acid sequence of a protein is its primary structure. Secondary Structure. The folding of the amino acid chain by hydrogen bonding into these characteristic coils and pleats is called a protein’s secondary structure. Tertiary Structure. The final folded shape of a globular protein, which positions the various motifs and folds nonpolar side groups into the interior, is called a protein’s tertiary structure. Quaternary Structure. When two or more polypeptide chains associate to form a functional protein, the individual chains are referred to as subunits of the protein. https://www.youtube.com/watch?v=wvTv8TqWC48  17 Nucleic acids Nucleotides – building blocks of nucleic acids Each nucleotide consists of three components: a five-carbon sugar (ribose in RNA and deoxyribose in DNA); a phosphate (—PO4) group; and an organic nitrogen containing base Two types of organic bases occur in nucleotides. The first type, purines, are large, double-ring molecules found in both DNA and RNA; they are adenine (A) and guanine (G). The second type, pyrimidines, are smaller, single-ring molecules; they include cytosine (C, in both DNA and RNA), thymine (T, in DNA only), and uracil (U, in RNA only).  Nucleotides – building blocks of nucleic acids When a nucleic acid polymer forms, the phosphate group of one nucleotide binds to the hydroxyl group of another, releasing water and forming a phosphodiester bond. A nucleic acid, then, is simply a chain of five- carbon sugars linked together by phosphodiester bonds with an organic base protruding from each sugar.  DNA The bases that participate in base-pairing are said to be complementary to each other. Primary DNA structure – sequence Secondary DNA structure - Double helix 2 nm Anotation: 5’CATT…AGT3’ Prokaryotes – 3х106 bp Eukaryotes – 1,7 nm 3х109 bp Possible combinations – 4n (n – number of nucleotides); 1,1 nm for 10 000 nucl. there are 410 000 combinations  23 The double helix is supported by two types of bonds between the bases : - hydrophobic bonds - hydrogen bonds “stacking” Small turn Large turn  Denaturation and renaturation of DNA The separation of the two DNA strands after breaking the weak bonds /heat or chemical agents is called denaturation/. The restoration of the double helix is referred to as renaturation.  Tertiary DNA structure: 26 - cyclic (bacteria, - linear (eukaryotic nucleus, karyotype) mitochondria and plastids) Interphase nucleus Metaphase chromosome  28 Functions of DNA – hereditary information Genetic code - триплетен 42=16; 43=64 - изроден - специфичен - еднопосочен - универсален Gene – sequence of DNA encoding one protein of RNA molecule ATP. Adenosine triphosphate (ATP) contains adenine, a five carbon sugar, and three phosphate groups. This molecule serves to transfer energy rather than store genetic information. Ribonucleic acids (RNA) Contain ribonucleotides Ribose Deoxyribose in RNA in DNA Uracil Thymine In RNA In DNA Primary RNA structure – sequence complementary to specific section of DNA DNA Matrix RNA synthesis (Transcription) RNA Secondary structures – short double-stranded sections Types of RNA and their functions: 1. Messenger RNA (mRNA) – encodes proteins 2. Ribosomal RNA (rRNA) – participates in ribosomes and translation 3. Transport RNA (tRNA) – transfers amino acids and participates in translation In cytoplasm 4. Small nuclear RNAs (snRNA) – role in modifications of mRNA 5. Small nucleolar RNAs (snoRNAs) – role in modifications of rRNA In nucleus 1) Messenger RNA (mRNA) – 2% of cellular RNA Eukaryotes Prokaryotes Transport RNA (tRNA) - 16% of cellular RNA Bound AA 5’ Ribosomal RNA (rRNA) - 82% of cellular RNA Ribosomal proteins rRNA  Ribosomes Prokaryotes Eukaryotes https://www.youtube.com/watch?v=o_-6JXLYS-k Cell cycle and division Chromosomes Prokaryote cell division In prokaryotes, division is called simple, but is essentially equivalent to mitosis in eukaryotic cells. The genetic material in bacteria is localized in a chromosome (a double-stranded ring DNA molecule) located in a nucleoid nuclear zone and attached to the bacterial plasmalemma by folding, not called a mesosome. After replication, the two daughter specimens attached to the plasmalemma are separated by lateral growth of the latter, and subsequent pinching. As a result of the simple division of prokaryotes, the two daughter cells receive the same amount of genetic material identical to the mother cell. Small circular DNA molecules called plasmids are also found in bacteria. They contain genetic information different from that on the bacterial chromosome - e.g. it can determine resistance to certain types of antibiotics. Some of the plasmids replicate independently of the bacterial genome and the amount of plasmid DNA in the two daughter cells is not the same. Evolutionarily, simple division in prokaryotes is considered to be the oldest. No dividing figures are observed. As the organization of cells becomes more complicated, so does the mechanism of their proliferation.  Cell cycle and mitotic cycle Cell cycle – the time from appearance of a cell to its division or death CC = MC CC ≠ MC CC > MC автосинтетична хетеросинтетична хетеросинтетична пролиферативен интерфаза (G1,S, G2) интерфаза(G0) интерфаза (G0) стимул автосинтетична интерфаза(G1) death Daughter Daughter cells cells The cell life cycle is a series of processes through which cell growth and division take place. In different cell types, this cycle is of different duration. Two main components form the life cycle of cells - interphase and mitosis. The interphase has three periods. G1 - also called presynaptic. It begins after the end of mitosis. It is characterized by rapid growth of the cytoplasm, preparation for DNA synthesis, synthesis of enzymes. During the G1 period, many cancer cells are removed. This period is the longest in duration. S - in this period DNA and histones are synthesized, DNA is replicated. G2 - this is the post-synthetic period and is the shortest. The dividing spindle is formed and energy accumulates for the course of mitosis. A decrease in protein synthesis is characteristic. There is an additional period called G0, in which some cells enter after leaving G1. Once in this period, the cells are at rest and can remain in it for a long time (years), after which they are included again in the cell cycle. Possible reasons why cells enter G0 are: a signal for differentiation, the onset of aging, cell death and others. Cell division is their main property. About 1012 cell divisions take place in 24 hours. Proper control of the cell cycle and elimination of errors is carried out in several control points. The first is checkpoint G1 - if the cell is the right size and the DNA is not damaged - the cell cycle can continue and move to the S period. At the end of the latter, a new control is performed to determine if there is damage to the DNA. In the period between G2 and the interphase, it is checked whether the DNA has replicated correctly. During the metaphase, the fourth checkpoint is performed, followed by the anaphase one (during the anaphase), where the correct arrangement of the chromosomes and the formation of the dividing spindle are monitored. The last checkpoint - the sixth is in the telophase period, where the correct distribution of chromosomes is monitored. If a deviation from the norm is found at any of these control points, the cell cycle stops. Under natural conditions, there are always cells in animal and plant organs that are outside the mitotic cycle and do not go through G1 S G2 M periods. Such cells are in the G0-period - they are at rest. Some of them can stay for such a long time without changing their morphology, retain their ability to divide, turn into stem cells - for example in the blood-forming tissue. More often, the loss of division is accompanied by specialization and differentiation of cells. Such cells that have left the cycle may, in special cases, re-enter it (for example, most of the liver cells are in the G0 period, but when part of the liver is removed, they begin to divide again). In other cases, leaving the cycle, the cells differentiate irreversibly and lose their ability to divide forever. This is seen in embryonic nerve cells, which differentiate and remain in this state for the rest of the body's life. In multicellular adult organisms, most of the cells are in the G0 period. The higher the specialization of the cells, the weaker their ability to divide.After mitosis, the cell has two options - either to re-enter the process of cell division, or to begin to differentiate. The period from the formation of a cell to its division into two daughter cells or to its death is called the life cycle. The life cycle coincides with the mitotic one, when the cell enters a preparation for a new division after its appearance. The length of the life cycle of cells depends on their type and environmental conditions. It is between 8 minutes and a year and a half. The life cycle differs for different cell types. In prokaryotic cells and unicellular organisms, the life cycle is short. Each division produces two new cells. The cells of multicellular organisms divide in different ways. Embryonic, epithelial and connective tissue cells divide continuously. Nerve cells, transverse striated and cardiac muscle cells do not divide. There are also cells that do not divide under normal conditions, but when injured, when tissue repair is required, they begin to divide. There are similar types of cells in plant organisms. Types of cell division Types видове of делене division при in еукариотни eukaryotesклетки amitosis mitosis meiosis амитоза митоза мейоза AMITOSIS An archaic form of simple division in eukaryotes No dividing figures are formed Chromatin remains in the interphase (non-compact) state The nucleus divides by folding the nuclear envelope There is often an uneven distribution of genetic material in daughter cells Occurs at: o Dinuclear unicellular with micro- and macronucleus. The micronucleus divides mitotically and the macronucleus divides amitotically (physiological amitosis) o Cells that have completed their functions and are not needed by the body (eg amnion cells) o Cells placed in highly unfavorable living conditions (degenerative amitosis) / regenerative – liver o Some tumors, such as Ehrlich ascites tumor The result is multinucleated cells as well as cells with fragmented nuclei without a uniform distribution of chromatin. Amitosis The cell divides unevenly - different amounts of nuclear material and cytoplasm enter the daughter cells Mitosis The nuclear material spirals and compact structures - chromosomes - are formed A complex dividing apparatus is formed - a "spindle", through which the chromosomes are divided equally between the daughter cells. Conditionally, mitosis is divided into 2 processes: Karyokinesis - division of the nucleus Cytokinesis - separation of the cytoplasm Karyokinesis occurs in 5 phases:  Prophase  Prometaphase  Metaphase  Anaphase  Telophase Периоди на митозата ИНТЕРФАЗА ЦИТОКИНЕЗА ПРОФАЗА ТЕЛОФАЗА ПРОМЕТАФАЗА АНАФАЗА МЕТАФАЗА МИТОЗА Mitosis is a division in which the resulting daughter cells are completely identical to the mother cell. Mitosis is a process consisting of six phases. Prophase - characteristic of this process is the condensation of chromosomes, the disappearance of the nucleus, fragmentation of the Golgi apparatus and EPR and stopping the processes of endo- and exocytosis. The poles of the dividing spindle are formed. Prometaphase - fragmentation of the nuclear envelope begins. The chromosomes attach to the dividing spindle. Metaphase - chromosomes are arranged at the equator of the dividing spindle. A metaphase plate forms. Anaphase - metaphase chromosomes are divided into chromatids, which shorten, condense and move to the poles of the dividing spindle. The latter are separated from each other. Telophase - daughter chromosomes are located at the poles of the dividing spindle, where they despiralize. The appearance of nuclei, formation of nuclear pores and others is characteristic. Cytokinesis - the last phase of mitosis. The cytoplasm is pinched between the two nuclei and the mother cell divides into two daughters, which are rounded. Mitosis is a universal form of proliferation of eukaryotic cells. In it, cell- significant processes take place, through which the chromosomes divide evenly in the two daughter cells. Biologists conditionally divide mitosis into four phases, namely: prophase, metaphase, anaphase, and telophase. In fact, mitosis is a long process in which each phase merges with the next. Prophase - the first stage of mitosis. It occupies about 60% of the total time required for mitosis. The most notable events that take place with the mother cell are:- two pairs of centrioles are formed, which separate from each other and located at opposite poles of the cell. Between them are formed protein filaments, called filaments of the dividing spindle, through which the chromosomes move;·Interphase chromosomes (chromatin filaments) become mitotic - they begin to spiral, fold, shorten and thicken and become short rods;· The nucleus and nuclear envelope gradually disintegrate;· The chromosomes move to the equator of the dividing spindle, catching their strands with their centromeres. The centromere of each chromosome is connected to a separate filament of the dividing spindle. With this the prophase passes into metaphase. Metaphase - the filaments of the dividing spindle pull the chromosomes and they are located fixed to the equatorial region of the dividing spindle. Anaphase:· The two sister chromatids on each chromosome are separated in the centromere region;· The filaments of the dividing spindle begin to shorten, thus pulling the chromatids. Now each chromatid is one chromosome;· Both groups of chromosomes move through the cytoplasm to the opposite ends of the cell - the poles. Telophase:· Once the individual chromosomes have reached the opposite poles of the cell, the dividing spindle disappears;· A nuclear membrane is formed around each group of chromosomes, and the chromosomes take on their filamentous shape again - they despiralize;· Centrioles double, i.e. two centrioles are formed in each daughter cell;· Nucleus and nuclear envelope appear. After the telophase of karyokinesis (the division of the nucleus), the cytoplasm - cytokinesis - divides. It begins during the telophase. In an animal cell, the cell membrane is pinched and formed along the equator of the dividing spindle - indentation. It increases and as a result the cell membrane divides, thus differentiating two daughter cells. In plants, the cell wall is relatively rigid, which prevents plant cells from dividing by pinching. In the middle of the dividing plant cell, a fragmoplast is formed from the membrane vacuoles formed by the Golgi apparatus. Just before cytokinesis occurs, they move to the area where the fragmoplast is about to form. The membrane vacuoles then fuse into a single membrane that divides the cells. The fragmoplastexpands until it separates the two daughter cells. Each of the newly formed cells then forms a cell wall on the adjacent side of the fragmoplast. Центрозомен цикъл S/G2 Early Prophase Metaphase prophase Polar microtubules Astral microtubules Formation of metaphase chromosomes The first level of chromatin organization is called the nucleosomal level - DNA is like a necklace. The diameter of the DNA strand is 11 nm. At this level, sections of DNA that are free of nucleosomal structure are observed. These regions must be recognized by various regulatory proteins that initiate the process of DNA replication or transcription. Second level of organization In the second level of organization, 11 nm of filament is converted into 30 nm of fibril, which is formed due to the interaction of neighboring H1. Beads are obtained, but each contains 6, 7 beads together, ie the nucleosomes are collected and they determine a diameter of 30 nm. At this level we are talking about a solenoid. The reduction in DNA length that results from the formation of a solenoid is six times the compression in the nucleosome. Tertiary and quaternary level of organization At the tertiary level of organization - from 30 nm thread by bending can be obtained fibrils with different length sections. These are loop-like structures with a size of 20 - 80,000 nucleotides. Each loop structure represents a domain, ie a functionally active unit in the chromatin structure. From the 3rd to the 4th level there are several levels of structuring in the space of these loop- like structures. The axial part that supports the loops begins to twist and loop- like structures converge again. H1 plays a major role, the more phosphorylated it is, the more compact the DNA. Mitotic spindle The mitotic spindle is composed of 3 different functional types of microtubules. All three types of microtubules with "-" ends are directed to the centrioles (the poles of the spindle). Microtubules that point at the "+" end to the cell periphery are referred to as astral microtubules. In the new daughter cells, they will form the cytocenter. Two types of microtubules are observed in the direction from the poles to the equator of the spindle. Some are referred to as overlapping (polar) and bind by cross-linking proteins in the equatorial region, and those associated with the kinetochore plates of chromosomes are called kinetochore microtubules.. Main events during mitosis Mitosis (IF: green – microtubules; blue – chromosomes) Mitosis – phase-contrast microscopy Mitosis in animal cells Mitosis in plant cells Mitosis If the mother cell has 6 chromosomes (DNA molecules), then they double in phase. The double chromosomes are arranged at the equator (metaphase) and separated (anaphase). After division, the daughter cells again have 6 chromosomes (DNA molecules) similar to the mother cell. Митоза http://www.youtube.com/watch?v=lf9rcqifx34&featur e=player_embedded#! http://www.youtube.com/watch?v=rgLJrvoX_qo&feat ure=related http://www.youtube.com/watch?v=vw1qGtVR5lI&NR =1 http://www.biology.arizona.edu/Cell_Bio/tutorials/cell _cycle/MitosisFlash.html http://www.youtube.com/watch?v=m73i1Zk8EA0&fe ature=related Meiosis Benefits: - New gene combinations - New chromosome combinations between maternal and paternal DNA - Overcome of mutations through addition of “healthy” allele 2n combination >>> 2n combinations n – number of chromosomes  Meiosis division of germ cells If the mother cell has 6 chromosomes, then as a result of the two divisions of meiosis (M1 and M2) their number in the four daughter cells is reduced by half. Each daughter cell (gamete) contains 3 chromosomes. Meiosis - DNA replication - first division (reduction of chromosomes), separation of homologous chromosomes - second division (reduction of DNA), separation of sister chromatids  Meiosis During the prophase of M1 of meiosis (pachytene stage) crossover is performed - (crossing of chromosomes). Хромозоми тип лампови четки (диплотен)  - Meiotic chromosomes - Transcriptionally active Организация на хромозомите тип лампови четки (mRNA) in prophase - the biggest known chromosomes - are observed in oocytes of amphibians хиазма - are formed during prophase I примка of meiosis хромомер - active - synthesizes of mRNA needed to build the backup egg proteins - (metaphase chromosomes are inactive in terms of RNA synthesis) хроматинова - contain 4 molecules (threads) примка DNA сестрински хроматиди хромомер Meiosis http://www.youtube.com/watch?feature=end screen&v=kVMb4Js99tA&NR=1 Polyten chromosomes - in insect larvae - interphase chromosomes- differences from the usual metaphase chromosomes: - repeatedly replicated (thicker, 210 = 1024) - not condensed (longer) - haploid number (n) - are in somatic conjugation - active (RNA synthesis) Polyten chromosomes discs Interdisc spaces puffs  FLOW OF INFORMATION Replication Transcription Translation DNA RNA PROTEIN Reverse transcription  RNA replication 1. Basic features Complementary А-Т, G-C Parent ds DNA Semi-conservative mechanism: -template strand -new strand Replication rate: - prokaryota - 500 bp/s - eukaryota – 50 bp/s 2. Mechanism - Opening of the double helix Helikase – eliminates hydrogen bonds and “stacking” bonds using ATP /without enzyme this is achievable by heating to 90°С – denaturation/ - Preventing renaturation of single-stranded regions of DNA strand SSB (single-stranded binding) белтъци ss DNA region with short ds sequences monomere SSB proteins SSB protein binding keeps ss DNA annealed and available for ensymes - Enzymes relieving tension in the unwind DNA and preventing tangling DNA topoisomerases І and ІІ Relieving tension caused by unwinding of the helix (superspiralization), DNA helix needs to make a turn DNA polymerase Topoisomerase І – performs single-stranded nicks Topoisomerase ІІ – makes double-stranded nicks - A new DNA molecule synthesis 3’ end 5’ end Template DNA 5’ New DNA 3’ end 3’ Pyrophosphate New dNTP added 5’ end  New nucleotides are added to the new strand by DNA polymerase III New dNTP New dNTP New strand “thumb” New Old strand strand cavity pyrophosphate 5’-3’ polymerization “fingers” template “palm” DNA polymerase III: - Adds dNTPs to 3’ OH end - Requires the presence of template - Catalyzes the growing of the strand in 5’ to 3’ direction’ - has 3’ 5’ correction exonuclease activity  Characteristics of DNA Poly III and antiparallel of DNA strands result in different pattern of new DNA strand synthesis 5’ 3’ 5’ Replication fork 3’  DNA primase synthesizes RNA primers required for synthesis of Okkazaki fragments RNA primer new RNA primer template DNA Poly forms new fragment of Okkazaki using the primer DNA Poly completes Okkazaki fragment RNA primer is degraded (enzyme) And replaced by DNA DNA ligase forms the absent Phosphodiester bond  DNA replication in prokaryotes 5’3’ 3’ 5’  Replication complex in prokrayotes SSB белтъци Replication fork in eukaryotes Differences – the lagging strand uses two polymerases -  and δ. DNA Poly /primase complex forms RNA primers and short pieces of DNA, Which are then extended by DNA Poly δ. ХелиHelicase contains 6 different subunits. Transcription of TNA Transcription DNA RNA - Process of transcription of information DNA in DNA to RNA - Complementarity А-U, G-C, uses NTP’s, not dNTPs) - Products: rRNAК, mRNA, tRNA other types of RNA  Enzymes – RNA polymerases - Use NTPs - Work in 5’ 3’ direction - May start the synthesis without primer (more mistakes – 1 per 104 nucleotides) Basic characteristics and differences in transcription between prokrayotes and eukaryotes 1.RNA polymerases prokaryotes eukaryotes One type synthesizes - RNA polymerase І (А) – rRNA all RNAs - RNA polymerase ІІ (В) – mRNA - RNA polymerase ІІІ (С) – small RNAs Differences between prokaryotes and eukaryotes 2. Number of proteins encoded by single mRNA molecule Prokaryote mRNA Coding Non-coding sequence sequence protein  protein  protein  Eukaryotic mRNA Coding Non-coding sequence sequence protein  Differences between prokaryotes and eukaryotes 3. Modifications of RNA Eukaryotes cytoplasm Prokaryotes intrones exones DNA DNA transcritpion Pre-RNA transcript TRAANSCRIPTION mRNA translation protein SPLICING cap mRNA EXPORT mRNA TRANSLATION protein Stages of transcription: 1. Initiation – starts from specific places – promotors promotors determine: - frequency of transcription - which strand shall be transcribed - supported by initiating factors 2. Elongation - elongation of the strand complementary 3. Termination - termination of synthesis after reaching “stop” signal and release of the new RNA molecule 1. Transcription in prokaryotes σ factor RNA promotor DNA Synthesis is terminated σ factor recognizes the promotor RNA polymerase When reaching And places RNA Poly on DNA Terminating signal – AT rich sequences RNA Poly has helicase activity and opens DNA without ATP Fast synthesis of RNA RNA synthesis starts, and after approx. 10 nucleotides are added, Release of the σ factor РНК σ factor is released Results in conformation РНК Changes in RNA Poly – Frontal parts are closed and the Other part forms a channel for exit of the RNA molecule конформационни промени в РНК полимеразата – предните “челюсти” се затварят, а в задната част се оформя канал през който излиза синтезираната молекула РНК 2. Mechanism of transcription in eukaryotes (RNA Poly ІІ) Basic transcription factors – sufficient for transcription over “naked” DNA molecule ТАТА biox РНК 3. Processing of RNA (eukaryotes) Eukaryotic mRNA Coding Non-coding sequence sequence 5’ cap RNA Poly Polyadenylating factors Splicing factor Factors for Cap formation RNA  А) Formation of cap 5’-end of primary transcript Methylguanozyne Functions of cap: - Identifies RNA as messenger /the other RNA polymerases 5’- 5’ produce transcripts without cap трифосфатен мост - protective - Export from the nucleus - Role in translation  B) Splicing – deletion of non-coding sequences (intrones) and ligation of coding sequences (exons) of the primary transcript Splicing is performed in spliceosomes intron – part of the gene sequence that is transcribed to RNA, but is cut through splicing and not present in mature RNA exon – part of the gene sequence that is present in mature mRNA, tRNA and rRNA Translation of RNA Translation RNA PROTEIN - Translation of the information into proteins - Genetic code - triplets (43 = 64) - alternative - universal - codon – triplet of mRNA coding one AA start  Players: - mRNA - tRNA - ribosomes - translation factors Transport RNAs Bound AA (Phe) AA end 5’end loop tRNA loop anticodon Loop of anticodon anticodon codon anticodon mRNA  Transport RNAs carry their respective AA (binding is performed during synthesis of proteins in cytosol) Macro energetic bond Amino acid /tryptophan/ тРНК Binding of amino acid with tRNA Aminoacyl /tryptophanyl/ tRNA synthetase  Ribosomes Prokaryotes Eukaryotes белтъка Zones in the ribosome participating in translation Phases of translation – initiation, elongation and termination 1. Initiation in eukaryotes ADP Initiation factors GTP separate Initiating tRNA Large ribosomal subunit binds Small ribosomal subunit Bound to initiating tRNA mRNA Adding of new initiation factors Next AA-tRNA is bound GTP to RNA In A zone ATP Initiating tRNA is moving First peptide bond is Along mRNA in search of formed ADP The first AUG codon GTP  Initiation in prokaryotes 5’ 3’ mRNA AGGAGGU AUG AGGAGGU AUG UCCUCCA UCCUCCA Small ribosomal subunit protein 1 protein 2 Differences with eukaryotes: - mRNA has no cap - small subunit recognizes small complementary sequences of mRNA - one mRNA encodes several proteins 2. Elongation Steps 1, 2 and 3 repeat with new AA-tRNA (№5) Growing polypeptide chain 1 1 New AA-tRNA 2 2 3 Factor of elongation: Catalyze the process (use ATP) And monitor the pairing between codons and anticodons 3. Termination TERMINATION Stop codon Binding of releasing factor to А-zone “Stop” codons – UAA, UAG, UGA  Process of translation is multiplied in polyzomes mRNA Stop Start codon codon poly А Binding protein Growing polypeptide chain Cell signaling Intercellular signaling: — Communication between cells. Intracellular signaling: — Signaling chains within the cell, responding to extracellular and intracellular stimuli.  Extracellular messengers: Cells send out signals in the form of specific messenger molecules that the target cell transmits into a biochemical reaction. Signaling cells can simultaneously influence many cells by messenger molecules so as to enable a temporally coordinated reaction in an organism. Gap junctions: Communication between bordering cells is possible via direct contact in the form of “gap junctions.” Gap junctions are channels that connect two neighboring cells to allow a direct exchange of metabolites and signaling molecules between the cells. Cell–cell interaction via cell-surface proteins: Another form of direct communication between cells occurs with the help of surface proteins. In this process, a cell-surface protein of one cell binds a specific complementary protein on another cell. As a consequence of the complex formation, an intracellular signal chain is activated which initiates specific biochemical reactions in the participating cells. Electrical signaling: A further intercellular communication mechanism relies on electrical processes. The conduction of electrical impulses by nerve cells is based on changes in the membrane potential. SYNAPTIC JUNCTIONS Steps of Intercellular Signaling 1) Trigger signal induces release of stored messenger or stimulates its biosynthesis 2) Transport to target cell 3) Receipt of signal by the target cell 4) Conversion of signal into intracellular signal chain in the target cell. There are two principal ways by which target cells can process incoming signals: Cell-surface receptors receive the signal (e.g., a messenger substance) at the outside of the cell, become activated, and initiate signaling events in the interior of the cell. In such signaling pathways, the membrane-bound receptor transduces the signal at the cell membrane so that it is not necessary for the signal to actually enter the cell. The messenger enters into the target cell and binds and activates the receptor localized in the cytosol or nucleus. Hormone signaling is mainly regulated via: Hormones can be: — External trigger signals Amino acids and amino acid — Feedback loops derivatives — Degradation Derivatives of fatty acids — Modification Peptides — Amount of receptors Steroids — Activity of receptor. Proteins Retinoids Nucleotides Small inorganic molecules, for example, NO Endocrine, Paracrine, and Autocrine Signaling Endocrine signaling: — Production of hormone in endocrine cells — Transport of hormone to target cell via circulation. Paracrine signaling: — Hormone reaches target cell by diffusion — Close neighborhood of signaling cell and target cell. Autocrine signaling: — Hormone-producing cell and target cell are of the same cell type. Second messengers and other regulatory proteins in signal transduction Second messengers may be rapidly formed from precursors by enzymatic reactions. Second messengers may be rapidly released from intracellular stores Second messengers may be rapidly inactivated or stored in specific compartments. Second messengers may activate different effector proteins Second messengers allow the amplification of signals Transmembrane Intracellular receptors: receptors: — Receive signals — Are nuclear- at the extracellular and/or cytoplasm- side and transmit localized the signal into the — Function as cytosol ligand-controlled — Structural parts: transcriptional Extracellular activators. domain Transmembrane domain Cytosolic domain. G protein-coupled receptors: https://www.youtube.com/watch?v=gPyo7k9E_-w Intracelullar receptors: https://www.youtube.com/watch?v=Ul0ecXrRwN4&t=397s Chemical foundations of cells. Inorganic and small organic molecules. Sugars. Lipids. Assoc. Prof. Georgi Georgiev, PhD Department of Cell and Developmental Biology, Faculty of Biology, Sofia University Cells come in variety of shapes and sizes Cells are Prokaryotic and Eukaryotic Property Prokaryotic cells Eukaryotic cells Size 0.2 - 2 μm to 10μm 10-100 μm Cell border complex Cell membrane + cell wall of carbohydrates Cytoplasm with cytoskeleton and proteins Cell membrane Cell surface coat – cellulose cell wall (plants), chitin (fungi), glycocalyx (animal cells) Cellular organelles No membrane organelles, “nucleoid”, Endomembrane system, cytoskeleton of proteins, ribosomes 70S (30S+50S) nucleus, ribosomes 80 S (40S + 60S) Genetic apparatus 1 cyclic DNA molecule + plasmids (non- DNA + proteins – chromatine, chromosomes, chromosome DNA) plasmids in yeasts; DNA in mitochondria and plastids Metabolism Aerobic and anaerobic Aerobic, rarely anaeorobic Motility Static or motile using flagella Various cell movements (actin, myosin, dynein; Cilia, flagella Reproduction No mitosis, but precise division of the genetic Amitosis, mitosis and meiosis information Differentiation and Absent or very simple due to their simple In most cases – facilitates the life of the muclticellular specialization structure. organisms. It is due to loss and/or appearance of new genes, and not change of the gene expression. Chemical composition of the cells. Chemical elements. Water. Small organic molecules Chemical composition No substantial difference in the variety of elements composing the non-living nature and the organisms. The difference is in their quantities.  Groups of elements composing the organisms: More than 95% of the weight of an organism consists of oxygen, Macroelements - 1Н, 12С, 14N, 16О; composing 98-99% of the mass of organisms hydrogen, carbon, and nitrogen, all of С which form strong С С covalent bonds С С С С С С С С С with one another. С Microelements – P, S, Na, K, Ca, Cl, Mg, Fe; Composing between 0,01 to 1,0% Ultramicroelements – Mn, Co, Cu, Zn, Mo, etc. composing under 0,01%  Types of chemical bonds Hydrogen bond Hydrophobic bonds Ionic bond Van der Vaals bonds Ionic bonds form when electrons transfer from one atom to another, and the resulting oppositely charged ions attract one another. Properties of non-covalent bonds - Weak - Multiple  Inorganic substances Water is the cradle of life + _ The chemistry of life is the chemistry of water (H2O). The central oxygen atom in water attracts the electrons it shares with the two hydrogen atoms. This charge separation makes water a polar molecule. A hydrogen bond is formed between the partial positive charge of a hydrogen atom in one molecule and the partial negative charge of another atom, either in another molecule or in a different portion of the same molecule. Water is cohesive and adhesive, has a great capacity for storing heat, is a good solvent for other polar molecules, and tends to exclude nonpolar molecules. The H+ concentration in a solution is expressed by the pH scale, in which pH equals the negative logarithm of the H+ concentration. Salts, acids and bases – all in the form of ions - maintain pH - maintain osmotic pressure - role in transformation and flow of energy - role in protein modifications - cofactors of various enzymes - represent signals Small organic molecules – carbon molecules with molecular mass between 100 and 1000 D Four families of small organic molecules: Малки органични Биополимери и молекули производни прости захари полизахариди мастни киселини мазнини, липиди аминокиселини белтъци нуклеотиди нуклеинови киселини Образуват метаболитния фонд на клетките  Carbohydrates (sugars) trioses pentoses hexoses 1. Monosacharides – (CH2O)n, n = 3 to 8 aldoses The carbohydrates are a loosely defined group of Cyclic forms molecules that contain H OH H carbon, hydrogen, and O oxygen in the molar ratio 1:2:1. Their C + OH C C O C empirical formula H (which lists the atoms in H ketoses the molecule with subscripts to indicate how many there are of each) is (CH2O)n, where n is the number of carbon atoms. Because they contain many carbon- hydrogen (C—H) bonds, which release energy when they are broken, Isomers carbohydrates are well suited for energy storage. galactose glucose manose Glucose Ribose Derivatives of simple sugars - amino sugars - esters P Products of carbon metabolism Glucosamine - chitin 2 Galactosamine – cartilage OH (glucose-6-phosphate) - disaccharides fructose glucose sucrose /glucose + fructose/ (table sugar) in sugarcane lactose /glucose + galactose/ in milk sucrose Glycosidic bond The glycoside bond is flexible   The human digestive enzymes are able to degrade - glycoside bond (starch), but not  bond /cellulose/. Two glucose molecules can bind in 11 different ways Oligo- and polysaccharides glycogen Glycogen may reach up to 10% of the liver’s weight According to types of monomers: - homopolysaccharides and heteropolysaccharides According to functions: - energy storage – glycogen (animal cells), starch (plant cells) and dextran (yeasts and bacteria) - structural – cellulose (50% of the organic carbon), chitin (protective covers) and agar (cell wall of sea algae) Lipids – molecules dissolved in organic solvents Fatty acids unsaturated saturated СООН Most fatty acids in the cell possess an even number of (СН2)n carbon atoms – usually 14, 16, 18 or 20. СН3 n – 12 до 24 Simple lipids – esters of fatty acids and alcohols -Triglycerides (neutral lipids) esters of glycerol and fatty acids Steroids Steroid hormones of the adrenals, gonads cholesterol testosterone Complex lipids – in addition to alcohol and fatty acids contain other components - Phospholipids Hydrophilic head - contains glycerol, phosphor acid and additional group Hydrophobic tail (fatty acid residues) - Sphingolipids /contain sphyngosine/, glycolipids /contain sugar component/