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Cells and Genomes 21:54 What do I need to know? What are the universal features common to all life on our planet? The diversity of cells Universal Features of Cells on Earth All living cells on earth store their hereditary information in DNA. All cells replicate their hereditary information by templated polymerization: Templated polymerization is the way in which information is copied throughout the living world. Each nucleotide consists of two parts: a base (either A, C, T, or G), and a sugar with a phosphate attached to it. Each sugar is linked to the next via the phosphate group, creating a long polymer chain. For a single, isolated strand of DNA, monomers can be added in any order because they all attach in the same way Individual sugar-phosphate groups are asymmetric, giving the strand a polarity or directionality. Transcription: Transcription: where segments of the DNA sequence are used as templates for the synthesis of shorter molecules of the closely related polymer RNA. In RNA, the backbone is formed of ribose instead of deoxyribose. Uracil replaces thymine. DNA is fixed, but RNA transcripts are mass-produced and disposable. mRNA: guides the synthesis of proteins according to the genetic instructions stored in the DNA. Proteins: Proteins are long unbranched polymer chains, formed by stringing together monomeric building blocks. Proteins form most of the cell’s mass. Amino acids are the monomers of proteins; there are 4 types. Polypeptides are protein molecules. Each polypeptide is created by joining amino acids in a particular sequence that folds into a precise 3D form with reactive sites on its surface. A groove in the surface of the polypeptide, the enzyme lysozyme, forms a catalytic site. Polysaccharide molecule: a polymer chain of sugar monomers; binds to the catalytic site of lysozyme and is broken apart, as a result of a covalent bond-breaking reaction catalyzed by the amino acids lining the groove. Proteins are the molecules that put the cell’s genetic information into action. Translation www.notesolution.com The info in the sequence of a messenger RNA molecule is read out in groups of three nucleotides at a time: each triplet (codon) specifies a single amino acid in a corresponding protein. The code is read out by a special class of small RNA molecules, the tRNAs. Each type of tRNA becomes attached at one end to a specific amino acid, and displays at its other end a specific sequence of three nucleotides – an anticodon – that enables it to recognize, through base pairing, a particular codon or subset of codons in mRNA. Ribosomal RNA: any one of specific RNA molecules that form part of the structure of a ribosome and participate in the synthesis of proteins. One protein = one gene Individual segments of the entire DNA sequence are transcribed into separate mRNA molecules, with each segment coding for a different protein. Each DNA segment represents one gene. Gene: the segment of DNA sequence corresponding to a single protein or set of alternative protein variants. The Plasma Membrane The plasma membrane acts as a selective barrier that enables the cell to concentrate nutrients gathered from its environment and retain the products it synthesizes for its own use, while excreting its waste products. The hydrophobic tails of the predominant membrane molecules in all cells are hydrocarbon polymers (-CH2-CH2-CH2-). A living cell can exist with fewer than 500 genes. The Diversity of Genomes and the Tree of Life Prokaryotes DNA, RNA, and protein are composed of just six elements: H, C, N, O, S, P. Mutation: alteration of the nucleotide sequence. Most bacteria and Archaea have 1000 – 6000 genes. New Genes are Generated from Preexisting Genes 1) Intragenic mutation: an existing gene can be modified by changes in its DNA sequence, through various types of error that occur mainly in the process of DNA replication. www.notesolution.com 2) Gene duplication: an existing gene can be duplicated so as to create a pair of initially identical genes within a single cell; these two genes may then diverge in the course of evolution. 3) Segment shuffling: two or more existing genes can be broken and rejoined to make a hybrid gene consisting of DNA segments that originally belonged to separate genes. 4) Horizontal (intercellular) transfer: a piece of DNA can be transferred from the genome of one cell to that of another - even to that of another species. This process is in contrast with the usual vertical transfer of genetic information from parent to progeny. Orthologs: genes that are related by descent (genes in two separate species that derive from the same ancestral gene in the last common ancestor of those two species). Paralogs: genes that have resulted from a gene duplication event within a single genome and are likely to have diverged in their function. Homologs: genes that are related by descent in either way. More than 200 gene families are common to all three branches of the tree of life. Genetic Information in Eukaryotes Mitochondria: create most of the cell’s energy in the form of ATP. Genetic Redundancy: the presence of two or more similar genes with overlapping functions. www.notesolution.com Cell Chemistry and Biosynthesis 21:54 The Chemical Components of a Cell Acid: a substance that releases protons to form hydronium ions when they dissolve in water. Base: a base accepts protons so as to lower the concentration of hydronium ions, and thereby raise the concentration of hydroxyl ions. Cells contain four major families of small organic molecules: the sugars, the fatty acids, the amino acids, and the nucleotides. Monosaccharide: a simple sugar with the formula (CH2O)n, where n is usually 3, 4, 5, 6, 7, or 8. Sugars, and the molecules made from them, are also called carbohydrates because of this simple formula. Isomers: sets of molecules with the same chemical formula but different structures. Optical Isomers: the subset of such molecules that are mirror-image pairs. Condensation reaction: where a molecule of water is expelled as the bond is formed. Hydrolysis: the reverse of a condensation reaction. A molecule of water is consumed. Lipids: comprise a loosely defined collection of biological molecules that are insoluble in water, while being soluble in fat and organic solvents such as benzene. The most important function of fatty acids in cells is in the construction of cell membranes. These thin sheets enclose all cells and surround their internal organelles. Amino acids all possess a carboxylic acid group and an amino group, both linked to a single carbon atom called the α-carbon. Amino acids make proteins, which are polymers of amino acids joined head to tail in a long chain that is then folded into a 3D structure unique to each type of protein. Nucleotides A nucleotide is a molecule made up of a nitrogen ring compound linked to a five-carbon sugar, which in turn carries one or more phosphate groups. The five-carbon sugar can be either ribose or deoxyribose. Nucleotides can act as short term carriers of chemical energy. The most fundamental role of nucleotides in the cell is in the storage and retrieval of biological information. ATP: transfers energy in hundreds of different cell reactions. ATP is formed through reactions that are driven by the energy released by the oxidative breakdown of foodstuffs. www.notesolution.com Cell Chemistry and Biosynthesis 21:54 Phosphodiester bond: a bond which covalently links nucleotide subunits in long polymer chains. Two main types of nucleic acids: Ribonucleic acids, or RNA: normally contain the bases A, G, U, and C. Deoxyribonucleic acid: contains A, G, C, and T. Three types of macromolecules: polysaccharide, protein, nucleic acid. Nucleotides have many other functions 1) They carry chemical energy in their easily hydrolyzed phosphoanhydride bonds. 2) They combine with other groups to form coenzymes. 3) They are used as specific signaling molecules in the cell. www.notesolution.com Chapter 3 21:54 Proteins A protein molecule is made from a long chain of amino acids, each linked to its neighbor through a covalent peptide bond. Also known as polypeptides. Polypeptide backbone: the repeating sequence of atoms along the core of the polypeptide chain. Attached to this repetitive chain are those portions of the amino acids that are not involved in making a peptide bond and that give each amino acid its unique properties: the 20 different amino acid side chains. The two ends of a polypeptide chain are chemically different: The end carrying the free amino group (NH2 or NH3+) is the amino terminus, and that carrying the free carboxyl group (COO- or COOH) is the carboxyl terminus. The amino acid sequence of a protein is always presented in the N to C direction, reading from left to right. An important factor governing the folding of any protein is the distribution of its polar (hydrophilic) and non-polar (hydrophobic) amino acids. Polar amino acids buried within the protein are usually hydrogen-bonded to other polar amino acids or to the polypeptide backbone. A typical amino acid. Basic Amino Side Chains: having a positive charge. These groups are very basic because it’s positive charge is stabilized by resonance. Acidic Amino Side Chains: having a negative charge. Proteins Fold into a Conformation of Lowest Energy Large proteins usually consist of several distinct protein domains The α Helix and the β Sheet are Common Folding Patterns α Helix is a folding pattern that was first found in the protein α-keratin. The β sheet was discovered in the protein fibroin, the major constituent of silk. www.notesolution.com Chapter 3 21:54 These two patterns are common because they result from hydrogen-bonding between the N-H and C=O groups in the polypeptide backbone without involving the side chains of the amino acids. β-sheets can form either from neighboring polypeptide chains that are parallel or from a polypeptide chain that folds back and forth upon itself, with each section of the chain running antiparallel. Both types of β-sheets produce a very rigid structure held together by H-bonds that connect the peptide bonds in neighboring chains. An α helix is generated when a single polypeptide chain twists around on itself to form a rigid cylinder. A hydrogen bond forms between every fourth peptide bond, linking the C=O of one peptide bond to the N-H of another. A complete turn of the helix occurs every 3.6 amino acids. In other proteins, α helices wrap around each other to form a particularly stable structure, known as thecoiled-coil. This structure forms when the two or three α helices have most of their non- polar (hydrophobic) side chains on one side, so that they can twist around each other with these side chains facing inward. Four Levels of Organization in the Structure of a Protein Primary Structure: the amino acid sequence. Secondary Structure: stretches of polypeptide chain that form α helices and β-sheets. Tertiary Structure: the full 3D organization of a polypeptide chain. Quaternary Structure: when a particular protein molecule is formed as a complex of more than one polypeptide chain. Protein Subunit: each polypeptide chain in a large protein molecule. Disulfide bonds are the most common cross-linkage in proteins. These bonds form as cells prepare newly synthesized proteins for export. www.notesolution.com Chapter 4 21:54 DNA, Chromosomes, and Genomes Chromosome: each chromosome consists of a single, enormously long linear DNA molecule associated with proteins that fold and pack the fine DNA thread into a more compact structure. The complex of DNA and protein is called chromatin. Exon: the coding sequences. A nucleic acid sequence that is represented in the mature form of an RNA molecule. Intron: the intervening (noncoding) sequences. A DNA region within a gene that does not translate into a protein. The majority of human genes thus consists of a long string of alternating exons and introns, with most of the gene consisting of introns. Cell Cycle Interphase: chromosomes are replicated. Cells spend most of their time in interphase. Mitosis: chromosomes become highly condensed and then are separated and distributed to the two daughter nuclei. Each DNA molecule that forms a linear chromosome must contain a centromere, two telomeres, and replication origins. Centromere: allows one copy of each duplicated and condensed chromosome to be pulled into each daughter cell when a cell divides. Telomere: the end of a chromosome. Telomeres contain repeated nucleotide sequences that enable the ends of chromosomes to be efficiently replicated. The proteins that bind to the DNA to form eukaryotic chromosomes are traditionally divided into two general classes: the histones and the nonhistone chromosomal proteins. Nucleosome: composed of an octameric core of histone proteins around which the DNA double helix is wrapped. Histones are responsible for the first and most basic level of chromosome packing, the nucleosome. When interphase nuclei are broken open very gently and their contents examined under the electron microscope, most of the chromatin is in the form of a fiber with a diameter of about 30 nm. www.notesolution.com Chapter 4 21:54 Uncondensed chromatin: “beads on string”. The string is the DNA, the beads are the chromatin. The histone octamer forms a protein core around which the double-stranded DNA is wound. Each nucleosome core particle is separated from the next by a region of linker DNA. All four of the histones that make up the core of the nucleosome are relatively small proteins. When DNA is linked around the histone octamer, G-C is preferred on the outside minor groove while T-A is preferred on the inside minor grooves. www.notesolution.com DNA Replication, Repair, and Recombination 21:54 DNA Replication Mechanisms DNA Polymerase: enzyme that synthesizes DNA by joining nucleotides together using a DNA template as a guide. The polymerase “reads” an intact DNA strand as a template and uses it to synthesize the new strand. This process copies a piece of DNA. The newly polymerized molecule is complementary to the template strand and identical to the template’s original partner strand. DNA polymerase is NOT able to create a new strand. DNA Primase: enzyme that synthesizes a short strand of RNA on a DNA template, producing a primer for DNA synthesis. The replication fork is asymmetrical. The fundamental reaction by which DNA is synthesized is the addition of a deoxyribonucleotide to the 3’ end of a polynucleotide chain. Because errors made in RNA are not passed on to the next generation, the RNA polymerase enzymes involved in gene transcription do not need such an efficient exonucleolytic proofreading mechanism. This is why RNA polymerases are able to start new polynucleotide chains without a primer. DNA replication only occurs in the 5’ to 3’ direction because of the need for accuracy. It is only possible to correct a mismatched base if it has been added to the 3’ end of a DNA chain. For the leading strand, a special primer is needed only at the start of replication: once a replication fork is established, the DNA polymerase is continuously presented with a base-paired chain end on which to add new nucleotides. RNA Primer: a short stretch of RNA synthesized on a DNA template. It is required by DNA polymerases to start their DNA synthesis. Because an RNA primer contains a properly base-paired nucleotide with a 3’-OH group at one end, it can be elongated by the DNA polymerase at this end to begin an Okazaki fragment. The synthesis of each Okazaki fragment ends when this DNA polymerase runs into the RNA primer attached to the 5’ end of the previous fragment. RNA is used rather than DNA for priming because any enzyme that starts chains anew cannot be efficient at self-correction. DNA helicases and single-strand DNA-binding proteins are needed to help open up the double helix ahead of the replication fork because DNA cannot unwind itself unless it is in near boiling temperatures. DNA Helicase: an enzyme that is involved in opening the DNA helix into its single strands for DNA replication. www.notesolution.com DNA Replication, Repair, and Recombination 21:54 Since both DNA strands have opposite polarities, the helicase could move in either direction along each strand. Both types of DNA helicase exist. A helicase moving 5’ to 3’ along the lagging-strand template appears to have the predominant role. Single-strand DNA-binding (SSB) Proteins: bind tightly and cooperatively to exposed single- stranded DNA without covering the bases, which therefore remain available for templating. SSB proteins are unable to open a long DNA helix directly, but they aid helicases by stabilizing the unwound, single-stranded conformation. In addition, their cooperative binding coats and straightens out the regions of single-stranded DNA on the lagging-strand template, thereby preventing the formation of the short hairpin helices that readily form in single-strand DNA. Sliding Clamp: this clamp keeps the polymerase firmly on the DNA when it is moving, but releases it as soon as the polymerase runs into a double-stranded region of DNA. The Initiation and Completion of DNA Replication in Chromosomes DNA Synthesis Begins at Replication Origins The process of DNA replication is begun by special initiator proteins that bind to double-stranded DNA and pry the two strands apart, breaking the H-bonds between the bases. The positions at which the DNA helix is first opened are called replication origins. Since an A-T pair is held together by fewer hydrogens than a C-G pair, DNA rich in A-T pairs is relatively easy to pull apart, and regions of DNA enriched in A-T pairs are typically found at replication origins. Prokaryotic Chromosomes Typically have a Single Origin of DNA Replication In E.coli, DNA replication begins at a single origin of replication and the two replication forks assembled there proceed in opposite directions until they meet up roughly halfway around the chromosome. Eukaryotic Chromosomes Contain Multiple Origins of Replication 1) Replication origins tend to be activated in clusters, called replication units, of perhaps 20 – 80 origins. 2) New replication units seem to be activated at different times during the cell cycle until all of the DNA is replicated. www.notesolution.com DNA Replication, Repair, and Recombination 21:54 3) Within a replication unit, individual origins are spaced at intervals of 30000 – 250000 nucleotide pairs from one another. 4) As in bacteria, replication forks are formed in pairs and create a replication bubble as they move in opposite directions away from a common point of origin, stopping only when they collide head-on with a replication form moving in the opposite direction (or when they reach a chromosome end). In eukaryotes, DNA replication takes place during only one part of the cell cycle. DNA replication in most eukaryotic cells only occurs during a specific part of the cell division cycle, called the DNA synthesis phase or S phase. By its end, each chromosome has been replicated to produce two complete copies, which remain joined together at their centromeres until the M phase (M for mitosis). Replication origins are not all activated simultaneously; the DNA in each replication unit is replicated during only a small part of the total S-phase interval. The origin in which replication origins are activated depends, in part, on the chromatin structure in which the origins reside. The regions of the genome whose chromatin is least condensed are replicated first. A Strand-Directed Mismatch Repair System Removes Replication Errors that Escape from the Replication Machine Strand-Directed Mismatch Repair System: a system for recognizing and repairing erroneous insertion, deletion, and misincorporation of bases that can arise during DNA replication and recombination. To be effective, such a proofreading system must be able to distinguish and remove the mismatched nucleotide only on the newly synthesized strand, where the replication error occurred. Nicks in DNA provide the signal that directs the mismatch proofreading system to the appropriate strand. This idea requires that the newly synthesized DNA on the leading strand be transiently nicked, but it is uncertain how this occurs. DNA Topoisomerases Prevent DNA Tangling During Replication DNA Topoisomerase: a reversible nuclease that adds itself covalently to a DNA backbone phosphate, thereby breaking a phosphodiester bond in a DNA strand. This reaction is reversible, and the phosphodiester bond re-forms as the protein leaves. Topoisomerase I: produces a transient single-stranded bread (or nick); this break in the phosphodiester backbone allows the two sections of DNA helix on either side of the nick to www.notesolution.com DNA Replication, Repair, and Recombination 21:54 rotate freely relative to each other, using the phosphodiester bond in the strand opposite the nick as a swivel point. Any tension in the DNA helix will drive this rotation in the direction that relieves the tension. Topoisomerase II: forms a covalent linkage to both strands of the DNA helix at the same time, making a transient double-strand nick in the helix. These enzymes are activated by sites on chromosomes where two double helices cross over each other. Topoisomerase II then binds to this crossing site, and breaks one double helix reversibly to create a DNA “gate”. It then causes the second, nearby double helix to pass through this break, and it then reseals the break and dissociates from the DNA. Telomerase Replicates the Ends of Chromosomes Since there is no place to produce the RNA primer needed to start the last Okazaki fragment at the very tip of a linear DNA molecule, telomerase is needed. Telomere DNA sequences are recognized by sequence specific DNA binding proteins that attract an enzyme, called telomerase, that replenishes these sequences each time a cell divides. Telomerase recognizes the tip of an existing telomere DNA repeat sequence and elongates it in the 5’-3’ direction, using an RNA template that is a component of the enzyme itself to synthesize new copies of the repeat. This mechanism is aided by a nuclease that chews back the 5’ end, ensuring that the 3’ DNA end at each telomere is always longer than the 5’ end with which it is paired, leaving a protruding single-stranded end. DNA Repair DNA Damage Can be Removed by More Than One Pathway Pathway 1: called base excision repair, involves a battery of enzymes called DNA glycosylases, each of which can recognize a specific type of altered base in DNA and catalyze its hydrolytic removal. There are at least 6 types of these enzymes; including those that remove deaminated C’s, deaminated A’s, different types of alkylated or oxidized bases, bases with opened rings, and bases in which a C=C bond has been accidentally converted to a C-C bond. These enzymes flip over the bases to inspect for damage. AP Endonuclease: an enzyme used in pathway 1 which cuts the phosphodiester backbone after which the damage is removed and the resulting gap is repaired. Pathway 2: called nucleotide excision repair. This mechanism can repair the damage caused by almost any large change in the structure of the DNA double helix. www.notesolution.com DNA Replication, Repair, and Recombination 21:54 This mechanism goes after damage by sunlight creating dimers, etc. In this pathway, a large multienzyme complex scans the DNA for a distortion in the helix, rather than for a specific base change. Once it finds the lesion, it cleaves the phosphodiester backbone of the abnormal strand on both sides of the distortion, and a DNA helicase peels away a single-strand oligonucleotide containing the lesion. The large gap produced in the DNA helix is then repaired by DNA polymerase and ligase. Direct Chemical Reversal of DNA Damage: this is a strategy employed for the rapid removal of certain highly mutagenic or cytotoxic lesions. Cells have a way of directing DNA repair to the DNA sequences that are most urgently needed. They do this by linking RNA polymerase, the enzyme that transcribes DNA into RNA as the first step in gene expression, to the repair of DNA damage. Transcription-coupled repair: works with base excision, nucleotide excision, and other repair machinery to direct repair immediately to the cell’s most important DNA sequences, namely those being expressed when the damage occurs. The Chemistry of the DNA Bases Facilitates Damage Detection The genetic code was initially carried out by A, C, G, and U. The question of why the U in RNA was replaced in DNA by T is answered by the fact that the repair system would find it difficult to distinguish a deaminated C from a naturally occurring U. Double-stranded Breaks are Efficiently Repaired An especially dangerous type of DNA damage occurs when both strands of the double helix are broke, leaving no intact template strand to enable accurate repair. If these lesions were left unrepaired, they would quickly lead to the breakdown of chromosomes into smaller fragments and to the loss of genes when the cell divides. Non-homologous End Joining: the broken ends are simply brought together and rejoined by DNA ligation, generally with the loss of one or more nucleotides at the site of joining. A much more accurate type of double-strand break repair occurs in newly replicated DNA. Here, the DNA is repaired using the sister chromatid as a template. This reaction is an example of homologous recombination. Eukaryotic cells delay progression of the cell cycle until DNA repair is complete. www.notesolution.com DNA Replication, Repair, and Recombination 21:54 www.notesolution.com How Cells Read the Genome: From DNA to Protein 21:54 From DNA to RNA RNA differs from DNA chemically in two respects: 1) the nucleotides in RNA are ribonucleotides – they contain the sugar ribose rather than deoxyribose. 2) Although, like DNA, RNA contains A, G, and C, it contains U instead of T. Transcription differs from DNA replication in several crucial ways: Unlike a newly formed DNA strand, the RNA strand does not remain H-bonded to the DNA template strand. Instead, just behind the region where the ribonucleotides are being added, the RNA chain is displaced and the DNA helix re-forms. Thus, the RNA molecules produced by transcription are released from the DNA template as single strands. RNAs are also much shorter than DNAs because they are copied from only a limited region of the DNA. RNA Polymerases: the enzymes that perform transcription. RNA polymerases catalyze the formation of the phosphodiester bonds that link the nucleotides together to form a linear chain. Differences between RNA Polymerase and DNA Polymerase 1) RNA polymerase catalyzes the linkage of ribonucleotides, not deoxyribonucleotides. 2) Unlike the DNA polymerases involved in DNA replication, RNA polymerases can start an RNA chain without a primer. This difference exists because transcription need not be as accurate as DNA replication. 3) Unlike DNA, RNA does not permanently store genetic information in cells. 4) The consequences of an error in RNA transcription are much less significant than that in DNA replication. If an incorrect ribonucleotides is added to the growing RNA chain, the polymerase can back up, and the active site of the enzyme can perform an excision reaction that resembles the reverse of the polymerization reaction, except that water instead of pyrophosphate is used and a nucleoside monophosphate is released. Cells Produce Several Types of RNA www.notesolution.com How Cells Read the Genome: From DNA to Protein 21:54 Messenger RNA (mRNA): the RNA molecules that are copied from these genes and which ultimately direct the synthesis of proteins. rRNA: ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis. tRNA: transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids. snRNAs: small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNA. snoRNAs: small nucleolar RNAs. Used to process and chemically modify rRNAs. scaRNAs: small cajal RNAs, used to modify snoRNAs and snRNAs. miRNAs: micro RNAs, regulates gene expression typically by blocking translation of selective mRNAs. siRNAs: small interfering RNAs, turn off gene expression by directing degradation of selective mRNAs and the establishment of compact chromatin structures. Other non-coding RNAs: function in diverse cell processes, including telomere synthesis, X- chromosome inactivation, and the transport of proteins into the ER. Signals Encoded in DNA Tell RNA Polymerase Where to Start and Stop To transcribe a gene accurately, RNA polymerase must recognize where on the genome to start and where to finish. The initiation of transcription is an especially important step in gene expression because it is the main point at which the cell regulates which proteins are to be produced and at what rate. A detachable subunit called sigma factor associates with the core enzyme and assists it in reading the signals in the DNA that tell it where to begin transcribing. Together, sigma factor and core enzyme are known as the RNA polymerase holoenzyme; this complex adheres only weakly to bacterial DNA when the two collide, and a holoenzyme typically slides rapidly along the long DNA molecule until it dissociates again. When the polymerase holoenzyme slides into a region on the DNA double helix called a promoter, a special sequence of nucleotides indicating the starting point for RNA synthesis, the polymerase binds tightly to this DNA. Terminator: the nucleotide sequence where the polymerase halts and releases both the newly made RNA chain and the DNA template. Transcription Start and Stop Signals are Heterogeneous in Nucleotide Sequence www.notesolution.com How Cells Read the Genome: From DNA to Protein 21:54 Consensus Nucleotide Sequence: derived by comparing many sequences with the same basic function and tallying up the most common nucleotide found at each position. It therefore serves as a summary or average of a large number of individual nucleotide sequences. Transcription Initiation in Eukaryotes Requires many Proteins In contrast to bacteria, which contain a single type of RNA polymerase, eukaryotic nuclei have three: RNA polymerase I, RNA polymerase II, and RNA polymerase III. These different polymerases transcribe different types of genes. RNA polymerases I and III transcribe the genes encoding tRNA, rRNA, and various small RNAs. RNA polymerase II transcribes most genes, including all those that encode proteins. Differences between RNA polymerase II and bacterial RNA polymerase: While bacterial RNA polymerase requires only a single additional protein (sigma factor) for transcription initiation to occur in vitro, eukaryotic RNA polymerases require many additional proteins, collectively called the general transcription factors. Eukaryotic transcription initiation must deal with the packing of DNA into nucleosomes and higher-order forms of chromatin structure, features absent from bacterial chromosomes. RNA Polymerase II Requires General Transcription Factors General Transcription Factors help to position eukaryotic RNA polymerase correctly at the promoter, aid in pulling apart the two strands of DNA to allow transcription to begin, and release RNA polymerase from the promoter into the elongation mode once transcription has begun. The proteins are general because they are needed at nearly all promoters used by RNA polymerase II; consisting of a set of interacting proteins, they are designated as TFII (for transcription factor for polymerase II), and are denoted arbitrarily as TFIIB, TFIID, and so on. The assembly process begins when the general transcription factor TFIID binds to a short double-helical DNA sequence primarily composed of T and A nucleotides. This sequence described above is known as a TATA box, and the subunit of TFIID that recognizes it is called TBP (for TATA-binding protein). The TATA box is typically located 25 nucleotides upstream from the transcription start site. The binding of TFIID causes a large distortion in the DNA of the TATA box. C-Terminal Domain: the addition of phosphate groups the “tail” of the RNA polymerase. TFIID: TBP subunit recognizes TATA box www.notesolution.com How Cells Read the Genome: From DNA to Protein 21:54 TAF subunits recognizes other DNA sequences near the transcription start point; regulates DNA- binding by TBP. TFIIB: recognizes BRE element in promoters; accurately positions RNA polymerase at the start site of transcription. TFIIF: stabilizes RNA polymerase interaction with TBP and TFIIB; helps attract TFIIE and TFIIH. TFIIE: attracts and regulates TFIIH. TFIIH: unwinds DNA at the transcription start point, phosphorylates Ser5 of the RNA polymerase CTD; releases RNA polymerase from the promoter. Polymerase II Also Requires Activator, Mediator, and Chromatin-Modifying Proteins Transcriptional Activators: gene regulatory proteins. These proteins must bind to specific sequences in DNA and help to attract RNA polymerase II to the start point of transcription. Their presence on DNA is required for transcription initiation in a eukaryotic cell. Mediator: needed for eukaryotic transcription initiation in vivo, which allows the activator proteins to communicate properly with the polymerase II and with the general transcription factors. Transcription initiation in a eukaryotic cell typically requires the local recruitment of chromatin- modifying enzymes. Both types of enzymes can allow greater access to the DNA present in chromatin, and by doing so, they facilitate the role of these enzymes in transcription initiation. Transcription Elongation Produces Superhelical Tension in DNA Once it has initiated transcription, RNA polymerase does not proceed smoothly along a DNA molecule; rather, it moves jerkily, pausing at some sequences and rapidly transcribing through others. A moving polymerase generates positive superhelical tension in the DNA in front of it an negative helical tension behind it. Transcription Elongation in Eukaryotes is Tightly Coupled to RNA Processing Transcription is only the first of several steps needed to produce an mRNA. RNA Splicing: the modification of an RNA after transcription, in which introns are removed and exons are joined. This is needed for the mRNA before it can be used to produce a correct protein through translation. www.notesolution.com How Cells Read the Genome: From DNA to Protein 21:54 RNA Capping is the First Modification of Eukaryotic Pre-mRNAs As soon as RNA polymerase II has produced about 25 nucleotides of RNA, the 5’ end of the new RNA molecule is modified by addition of a cap that consists of a modified guanine nucleotide. Three enzymes, acting in succession, perform the capping reaction: 1) A phosphotase removes a phosphate from the 5’ end of the nascent RNA. 2) A guanyl transferase adds a GMP in a reverse linkage (5’ to 5’ instead of 5’ to 3’) 3) A methyl transferase adds a methyl group to the guanosine. RNA Splicing Removes Intron Sequences from Newly Transcribed Pre-mRNAs Both intron and exon sequences are transcribed into RNA. Only after 5’ and 3’ end processing and splicing have taken place is such RNA termed mRNA. Nucleotide Sequences Signal Where Splicing Occurs The splicing machinery must recognize three portions of the precursor RNA molecule: the 5’ splice site, the 3’ splice site, and the branch point in the intron sequence that forms the base of the excised lariat. Key steps in RNA splicing are performed by RNA molecules rather than proteins. These RNA molecules are relatively short and there are five of them (U1, U2, U4, U5, and U6; known as snRNAs) involved in the major form of pre-mRNA splicing. Each is complexed with at least seven protein subunits to form a snRNP (small nuclear ribonucleoprotein) These snRNPs form the core of the spliceosome, the large assembly of RNA and protein molecules that performs pre-mRNA splicing in the cell. The Spliceosome Uses ATP Hydrolysis to Produce a Complex Series of RNA-RNA Rearrangements Although ATP hydrolysis is not required for the chemistry of RNA splicing, it is required for the assembly and rearrangements of the spliceosome. Trans-splicing: a type of RNA splicing present in a few eukaryotic organisms in which exons from two separate RNA transcripts are joined together to form an mRNA. Group I Self-Splicing Sequences: aided by proteins in the cell that speed up the reaction. This intron sequence binds a free G nucleotide to a specific site on the RNA to initiate splicing. www.notesolution.com How Cells Read the Genome: From DNA to Protein 21:54 Group II Self-Splicing Sequences: uses an especially reactive A nucleotide in the intron sequence itself for the same purpose. Both types of self-splicing reactions require the intron to be folded into a highly specific 3D structure that provides the catalytic activity for the reaction. Translation: From RNA to Protein Most genes in a cell produce mRNA molecules that serve as intermediaries on the pathway to proteins. In this section, we examine how the cell converts the info carried in an mRNA molecule into a protein molecule. An mRNA Sequence is Decoded in Sets of Three Nucleotide Once an mRNA has been produced by transcription and processing, the information present in its nucleotide sequence is used to synthesize a protein. Reading Frame: phase in which nucleotides are read in sets of three to encode a protein. A mRNA molecule can be read in any one of three reading frames, only one of which will give the required protein. tRNA Molecules Match Amino Acids to Codons in mRNA tRNA: set of small RNA molecules used in protein synthesis as an interface (adaptor) between mRNA and amino acids. Each type of tRNA molecule is covalently inked to a particular amino acid. Four short segments of the tRNA molecules are folded double-helically, producing a molecule that looks like a four-leaf clover. Anticodon: set of three consecutive nucleotides that pairs with the complementary codon in an mRNA molecule. tRNAs are Covalently Modified Before They Exit from the Nucleus Eukaryotic tRNAs are synthesized by RNA polymerase III. tRNA splicing uses a cut-and-paste mechanism that is catalyzed by proteins. All tRNAs are modified chemically – nearly 1 in 10 nucleotides in each mature tRNA molecule is an altered version of a standard G, U, C, or A ribonucleotide. Specific Enzymes Couple Each Amino Acid to its Appropriate tRNA Molecule www.notesolution.com How Cells Read the Genome: From DNA to Protein 21:54 Recognition and attachment of the correct amino acid depends on enzymes called aminoacyl- tRNA synthetases, which covalently couple each amino acid to its appropriate set of tRNA molecules. Most cells have a different synthetase enzyme for each amino acid; one attaches glycine to all tRNAs that recognize codons for glycine, another attaches alanine to all tRNAs that recognize codons for alanine, and so on. Editing by tRNA Synthetases Ensures Accuracy Most synthetases select the correct amino acids by a two-step mechanism: 1) The correct amino acid has the highest affinity for the active-site pocket of it synthetase and is therefore favored over the other 19 (analogy: fits like a glove). 2) For amino acids where it is difficult to distinguish by size, a second step occurs after the amino acid has been covalently linked to AMP. When tRNA binds the synthetase, it tries to force the amino acid into a second pocket in the synthetase, the precise dimensions of which exclude the correct amino acid but allow access by closely related amino acids. The RNA Message is Decoded in Ribosomes A ribosome contains four binding sites for RNA molecules: one is for the mRNA and three (called the A-site, the P-site, and the E-site) are for tRNAs. A tRNA molecule is held tightly at the A- and P- sites only if its anticodon forms base pairs with a complementary codon on the mRNA molecule that is threaded through the ribosome. A-site: aminoacyl-tRNA P-site: peptidyl-tRNA E-site: exit-tRNA Elongation Factors Drive Translation Forward and Improve Its Accuracy Elongation Factors: a set of proteins that facilitate the events of translational elongation. Types of Elongation Factors in Prokaryotes and Eukaryotes: EF-Tu/EF-1: mediates the entry of aminoacyl tRNA into a free site in the ribosome. EF-Tu functions by binding an aminoacylated, or charged, tRNA molecule in the cytoplasm. Helps to move translation forward; also increases accuracy of translation in several ways: www.notesolution.com How Cells Read the Genome: From DNA to Protein 21:54 As it escorts an incoming aminoacyl-tRNA to the ribosome, EF-Tu/EF-1 checks whether the tRNA-amino acid match is correct. EF-Tu/EF-1 monitors the initial interaction between the anticodon of an incoming aminoacyl- tRNA and the codon of the mRNA in the A-site. EF-G/EF-2: catalyzes the translocation of the tRNA and mRNA down the ribosome at the end of each round of polypeptide elongation. Causes shifting from the A site to the P site. The Ribosome is a Ribozyme Ribozyme: RNA with catalytic activity. Nucleotide Sequences in mRNA Signal Where to Start Protein Synthesis Initiator tRNA: initiates protein synthesis by binding to the amino acid methionine and delivering it to the small ribosomal subunit. Eukaryotic Initiation Factors: proteins involved in the initiation phase of eukaryotic translation. Stop Codons Mark the End of Translation Release Factors: a protein (not a tRNA molecule) that allows for the termination of translation by recognizing the stop codon. Proteins are Made on Polyribosomes Polysomes (polyribosomes): clusters of ribosomes spaced as close as 80 nucleotides apart along a single mRNA molecule. These multiple initiations allow the cell to make many more protein molecules in a given time than would be possible if each had to be completed before the next could start. There are Minor Variations in the Standard Genetic Code Translational recoding: Other nucleotide sequence info present in an mRNA can change the meaning of the genetic code at a particular site in the mRNA molecule. Bacteria, archaea, and eukaryotes have available to them a 21 amino acid that can be incorporated directly into a growing polypeptide chain through translation recoding. Translational Frameshifting: allows more than one protein to be synthesized from a single mRNA. Inhibitors of Prokaryotic Protein Synthesis are Useful as Antibiotics www.notesolution.com How Cells Read the Genome: From DNA to Protein 21:54 Many of the most effective antibiotics used in modern medicine are compounds made by fungi that inhibit bacterial protein synthesis. Quality Control Mechanisms Act to Prevent Translation of Damaged mRNAs Non-sense Mediated mRNA Decay: eliminates defective mRNAs before they can be effectively translated into protein. It does this when the cell determines that an mRNA molecule has a stop codon in the wrong place – a situation likely to arise in an mRNA molecule that has been improperly spliced. The surveillance mechanism begins as an mRNA molecule is being transported from the nucleus to the cytosol. As the 5’ end emerges, a ribosome attaches itself and if it senses more exon junction complexes when it reaches the stop codon, the mRNA will be degraded. Some Proteins Begin to Fold While Still Being Synthesized When a protein folds into a compact structure, it buries most of its hydrophobic residues in an interior core. Molecular Chaperones Help Guide the Folding of Most Proteins Most proteins probably do not begin to fold during their synthesis. Instead, they are met at the ribosome by a special class of proteins called molecular chaperones. Many molecular chaperones are called heat-shock proteins, because they are synthesized in dramatically increased amounts after a brief exposure of cells to an elevated temperature. Several major families of eukaryotic molecular chaperones: Hsp60: form a large barrel-shaped structure that acts after a protein has been fully synthesized. This type of chaperone forms an isolation chamber into which mis-folded proteins are fed, preventing their aggregation and providing them with a favourable environment in which to attempt to refold. Hsp70: the Hsp70 machinery acts early in the life of many proteins, binding to a string of about seven hydrophobic amino acids before the protein leaves the ribosome. www.notesolution.com 21:54 www.notesolution.com Manipulating Proteins, DNA, and RNA 21:54 Section: Analyzing and Manipulating Data; page 532 – 534, 540 – 541 Genetic Engineering: the ability to manipulate DNA with precision in a test tube or an organism. Recombinant DNA Technology: comprises a mixture of techniques, some newly developed and some borrowed from other fields such as microbial genetics: Cleavage of DNA at specific sites by restriction nucleases, which greatly facilitates the isolation and manipulation of individual genes. DNA ligation, which makes it possible to design and construct DNA molecules that are not found in nature. DNA cloning through the use of either cloning vectors or the polymerase chain reaction, in which a portion of DNA is repeatedly copied to generate many billions of identical molecules. Nucleic acid hybridization, which makes it possible to find a specific sequence of DNA or RNA with great accuracy and sensitivity on the basis of its ability to selectively bind a complementary nucleic acid sequence. Rapid determination of the sequence of nucleotides of any DNA, making it possible to identify genes and to deuce the amino acid sequence of the proteins they encode. Simultaneous monitoring of the level of mRNA produced by every gene in a cell, using nucleic acid microarrays, in which tens of thousands of hybridization reactions take place simultaneously. Restriction Nucleases: enzymes that can be purified from bacteria cut the DNA double helix at specific sites defined by the local nucleotide sequence, thereby cleaving a long double-stranded DNA molecule into fragments of strictly defined sizes. Cohesive Ends: some restriction nucleases produce staggered cuts, which leave short single- stranded tails at the two ends of each fragment. At these cohesive ends, each tail can form complementary base pairs with the tail at any other end produced by the same enzyme. DNA molecules produced by splicing together two or more DNA fragments with cohesive ends are known as recombinant DNA molecules. Genes Can be Cloned Using DNA Libraries DNA Cloning: literally refers to the act of making many identical copies of a DNA molecules, but it also describes the isolation of a particular stretch of DNA (often a particular gene) from the rest of a cell’s DNA, because this isolation is greatly facilitated by making many identical copies of the DNA of interest. DNA cloning can be accomplished in several ways: Inserting a particular fragment of DNA into the purified DNA genome of a self-replicating genetic element – generally a virus or a plasmid. A virus or plasmid used to house cloned genetic www.notesolution.com Manipulating Proteins, DNA, and RNA 21:54 material is called a cloning vector, and the DNA propagated by insertion into it is said to have been cloned. To isolate a specific gene, one often begins by constructing a DNA library – a comprehensive collection of cloned DNA fragments from a cell, tissue, or organism. This library often includes at least one fragment that contains the gene of interest. The plasmid vectors most widely used for gene cloning are small circular molecules of double- stranded DNA derived from larger plasmids that occur naturally in bacterial cells. For use as cloning vectors, the purified plasmid DNA circles are first cut with a restriction nuclease to create linear DNA molecules. Gel Electrophoresis Separates DNA Molecules of Different Sizes The pores in polyacrylamide gels are too small to permit very large DNA molecules to pass, it is for this reason that the much more porous gels formed by dilute solutions and agarose are used. www.notesolution.com Membrane Transport of Small Molecules and the Electrical Properties of Membranes: Part 2 21:54 Protein-free Lipid Bilayers are Highly Impermeable to Ions In general, the smaller the molecule and the more soluble it is in oil (the more hydrophobic, or nonpolar, it is), the more rapidly it will diffuse across a lipid bilayer. By contrast, lipid bilayers are highly impermeable to charged molecules, no matter how small: the charge and high degree of hydration of such molecules prevents them from entering the hydrocarbon phase of the bilayer. Transporters and Active Membrane Transport Symporter: the simultaneous coupled transfer of a second solute in the same direction. Antiporter: the coupled transfer of a second solute in the opposite direction. Lactose Permease: a well-studied H+ driven symporter. It transports lactose across the plasma membrane of E.coli. The Permease consists of 12 loosely packed transmembrane α-helices. During the transport cycle, some of the helices undergo sliding motions that cause them to tilt. These motions alternately open and close a crevice between the helices, exposing the binding sites for lactose and H+, first on one side of the membrane then on the other. Transporters in the Plasma Membrane Regulate Cytosolic pH Most cells have one or more types of Na+ driven antiporters in their plasma membrane that help to maintain the Cytosolic pH at about 7.2. These transporters use the energy stored in the Na+ gradient to pump out excess H+, which either leaks in or is produced in the cell by acid-forming reactions. Two mechanisms are used: either H+ is directly transported out of the cell or HCO3- is brought into the cell to neutralize H+ in the cytosol. When H+ is transported directly into the cell, the Na+ - H+ exchanger couples an influx of Na+ to an efflux of H+. Another, that uses a combination of both mechanisms, is a Na+ driven Cl- - HCO3- exchanger that couples an influx of Na+ and HCO3- to an efflux of Cl- and H+. (So that NaHCO3 comes in and HCl goes out). This mechanism is twice as effective as the Na+ - H+ exchanger. An Na+ independent Cl- -HCO3- exchanger adjusts the Cytosolic pH in the reverse direction. There are Three Classes of ATP Driven Pumps 1. P-type Pumps: are structurally and functionally related multipass transmembrane proteins. They are called P-type because they phosphorylate themselves during the pumping cycle. This class includes many of the ion pumps. www.notesolution.com Membrane Transport of Small Molecules and the Electrical Properties of Membranes: Part 2 21:54 2. F-type Pumps: are turbine-like proteins, constructed from multiple different subunits. They are found in the plasma membrane of bacteria, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts. They are often called ATP synthases because they normally work in reverse: instead of using ATP hydrolysis to drive H+ transport, they use the H+ gradient across the membrane to drive the synthesis of ATP from ADP and phosphate. V-type Pumps: normally pump H
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