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Dudi Engelberg Room 1-517 Tel: 658 4718 e-mail: [email protected] 1 The central dogma of molecular biology 2 DNA Transcription RNA Translation Protein 3 Could proteins multiply ? 4 What do we have RNA for? 5 Same DNA content in all cells of the mulicellular organism? What is the function of DNA? Can cells function without DNA? 6 7 8 9 10 Are these all nucleotides that appear in DNA and RNA? 11 12 13 What are the cellular functions of nucleotides? 14 Some cellular functions of nucleotides 1. Building blocks of nucleic acids. 2. Energy carrier (ATP, GTP). 3. Building parts of enzymes co-factors (e.g., NAD, FAD, CoenzymeA, S-adenosylmethionine). 4. Regulators in signal transduction processes. 5. Second messengers in signal transduction (cAMP, cGMP). 6. Phosphate donors in phosphorylation reactions. Involved in many more pottranslational modifications. 7. Serve as structural molecules (rRNA). 15 8. Activators of carbohydrates for synthesis (glycogen for example). Some cellular functions of deoxynucleotides 1. Building blocks of nucleic acids (DNA). 16 Some cellular functions of deoxynucleotides 1. Building blocks of nucleic acids (DNA). 2. Energy carrier (ATP, GTP). 3. Building parts of enzymes co-factors (e.g., NAD, FAD, CoenzymeA, S-adenosylmethionine). 4. Regulators in signal transduction processes (GTP). 5. Second messengers in signal transduction (cAMP, cGMP). 6. Phosphate donors in phosphorylation reactions. 7. Serve as structural molecules (rRNA). 8. Activators of carbohydrates for synthesis (glycogen for example). 17 18 19 Some deviations from the averaged Watson & Crick model The pitch angle between base pairs could be 28o - 42o. Bases could propel (deviate from planarity). Damages: kinks and covalent bonding inside the helix (usually Between bases). Presence of unusual bases (in tRNA for example) allows unusual base pairing and novel structural motifs. Presence of specific sequences (stretch of purines, palindromes, sequence repeats). 22 23 24 25 26 27 The driving force towards synthesis is the breakdown of PPi. Phosphodiester bond 28 29 30 31 32 Mechanism of the basic synthesis reaction of nucleic acids Addition of nucleotide involves an attack by the 3’-hydroxyl group at the end of the growing RNA molecule on the a phosphate of the oncoming NTP. Two Mg2+ ions coordinated to the phosphate groups of the NTP and to three Asp residues of the subunit of E. coli RNA polymerase (conserved in most RNA polymerasess in nature). One Mg2+ ion facilitate the attack by the 3’-hydroxyl group on the a phosphate and the other ion facilitates the displacement of pyrophosphate. 33 The Mg2+ ions stabilize in fact the transition (intermediate) state. Polymerization of nucleotides - DNA and RNA biosynthesis 1. The reaction is directional; proceeds from 5’end to 3’end. As a result the product is asymetric (5’end different than 3’end. 2. The nucleotides (of the same strand) are always linked in a phospho-di-ester bond (a covalent bond). 3. Energy is wasted in addition of each monomer. The driving force towards synthesis is degradation of pyrophosphate. 4. The precursors are always nucleotides tri-phosphates (NTPs or dNTPs). 6. The reaction is directed by a pre exist plan (a template). (No polymerase is capable of adding nucleotides randomly). May be there are some - quite important 34 35 Basic characteristics of DNA Pol 1. Is not capable of de novo synthesis. Requires: A. A template (as any other polymerase). B. A primer (RNA oligo, nicked DNA, protein?) 2. Possesses two catalytic activities: A. A 5’ to 3’ polymerase activity. B. A 3’ to 5’ exonuclease actiivty. 3. Substrates are only dNTPs. 36 How DNA Pol is regulated? Does it possess regulatory sites? 37 DNA replication is semi-conservative DNA replication is bi-directional 38 Schematic structure of E. coli replication origin (OriC) 245 bp. 3 repeats of 13 bp sequences + 4 repeats of 9 bp sequence. These elements are highly conserved in replicationorigins of bacteria. 39 Initiation step: “opening” DNA “preparing the template before any DNA synthesis occurs. 40 First key step in replication: binding of DnaA protein molecules to the four 9 bp repeats. DnaA binding requires ATP and HU Second step: binding of DnaB (hexamerix helicase). Two hexamers bind to unwind DNA at two points creating two potential replicating forks. Third step: binding of SSBs (essential for stabilizing single strand throughout the replication process) and DNA gyrase (DNA topoII) - this step allows DnaB helicase to unwind thousands of base-pairs. 41 DnaA binds cooperatively to form a core around which OriC DNA is wrapped. At the presence of ATP DnaA melts the DNA of the A-T rich 13 bp tandem repeats. DnaA molecules recruit two DnaB-DnaC complexes, one for each replication forks. (6 DnaC monomers bind the DnaB hexamer.) Gyrase must be present to relieve topological Stress - otherwise helicase cannot further catalyze unwinding. Altogether a pre-priming complex is formed: 480 kD, 6 nm radius. 42 Initiation step has prepared the template. Moving to elongation step: Priming is required. A mechanism for bi-directionality is required. Leading strand synthesis begins with The synthesis of a short primer (10-60 n) catalyzed by primase (DnaG - special RNA Pol). 43 Both strands are sybthesized by DNA Pol3. Lagging strand: A new primer is synthesized near the replication fork. Synthesis continues until the Fragment extends as far as the primer of the previous fragment. 44 45 Specific structural capabilities of DNA Pol 3. 46 DnaB (helicase) + DnaG (primase) form a functional unit within the replication fork, called primosome. DNA pol3 - a dimer - one set of subunits synthesize the leading strand and other set the lagging strand. Once DNA is unwound by DnaB, DnaG associates occasionally with DnaB and synthesizes a short RNA primer. A new sliding clamp is then positioned at the primer by the clamp-loading complex of Pol 3. When a synthesis of a fragment is completed, replication halts and the core subunits of Pol 3 dissociate from their sliding clamp and from the new fragment. 47 subunits on DNA 48 49 50 51 Exonuclease activity is located ahead of pol activity 52 53 54 55 56 57 58 59 Sequence of the RNA is identical to that of the coding strand (with the replacements of Us for Ts). 60 Products of the transcription reaction (primary transcript): In prokaryotes: an unstable RNA- rapidly degraded (mRNA or cleaved to give mature products (rRNA, tRNA). In eukaryotes: modified at the ends (mRNA) and/or cleaved 61 to give mature products (all RNAs). With the exception of the RNA genomes of certain viruses, all RNA molecules in nature (mRNA, tRNA, rRNA, miRNA, snRNA) are derived from information stored in DNA and obtained via transcription. Namely, just like DNA during replication, RNA is synthesized on DNA template (DNA-dependent RNA synthesis). 62 Transcription=DNA-dependent RNA synthesis 63 Polymerization of nucleotides - DNA and RNA biosynthesis 1. The reaction is directional; proceeds from 5’end to 3’end. As a result the product is asymetric (5’end different than 3’end. 2. The nucleotides (of the same strand) are always linked in a phospho-di-ester bond (a saturated covalent bond). 3. Energy is consumed during addition of each monomer. The driving force towards synthesis is degradation of pyrophosphate. 4. The precursors are always nucleotides tri-phosphates (NTPs or dNTPs). 6. The reaction is directed by a pre exist plan (a template). No plymerase is capable of adding nucleotides randomly. 64 At its basic enzymatic level, transcription is a reaction highly similar to replication 65 Comparison of replication to transcription (some aspects) Replication Quantity: The whole genome Transcription Parts of the genome Timing: One time per life cycle (time is determined by the checkpoint system) some parts - all life time some parts - some time some parts - never Location: From origin to end Many starts and many stops (starts and stops must be most accurate) DNA substrate: The two strands One strand (could be a different for each particular case Nucleotide substrates: dNTPs NTPs 66 Comparison of replication to transcription (some aspects) Replication Proofreading: Post-reaction repair: Fate of product: Always Always Remains attached to template Processivity: High or low Ligating fragments: Yes Transcription Never Never Released from template High (from start to termination) No - products are independent molecules 67 Sequence of the RNA is identical to that of the coding strand (with the replacements of Us for Ts). 68 Products of the transcription reaction (primary transcript): In prokaryotes: an unstable RNA- rapidly degraded (mRNA or cleaved to give mature products (rRNA, tRNA). In eukaryotes: modified at the ends (mRNA) and/or cleaved 69 to give mature products (all RNAs). RNA Polymerase - general properties 1. Properties similar to DNA Polymerases: - Basic chemical mechanisms: addition of ribonucleotides to the 3’-OH of the chain. Consequently determination of a 5’ to 3’ directionality. - Requires a template. - Adding nucleotides on the basis of optimal hydrogen bonds with the template strand (A-U, C-G). 2. Properties specific to RNA Pol - Using only one strand as a template (must make a choice). - Does not require a primer (pppN 5’ end). - Very complex regulation for “choosing” the starting points (which may be different in every cell, in every developmental stage and in ageing. - Does not have a 3’ 5’ exonuclease activity. - The rate of mistake in high (1/104-105). 70 During a successful round: RNA Pol associates with the starting point and dissociates at the termination point, defining a transcription unit. A transcription unit may include more than one gene Nomenclature: Upstream. Downstream; numbers; left to right; no base is defined as base zero. 71 Rates (in E. coli): Transcription: 40 nuc../sec. Similar to rate of translation. Replication: 1,000nuc./sec/strand RNA pol creates the ‘transcription bubble’ when It binds to a DNA. The bubble moves with it. Displacing of the product (RNA), reforming the dsDNA 72 About 17 bp are unwound at any given time. Length of RNA:DNA hybrid within the bubble: up to 12 bp. Length of RNA within the bubble: ~25 b. 73 Within the transcriptional bubble (in bacteria), RNA Pol : Unwinds and rewinds DNA Maintains the conditions of the template and coding strands. Synthesizes RNA. 74 The transcription reaction can be divided into the Following stages: Template recognition - binding of RNA pol to DNA at a sequence known as promoter forming a “closed complex”, unwind the DNA to form an “open complex”, creating the ‘bubble’. Initiation - synthesis of the first nucleotide bond. RNA pol Does not move while it synthesizes the first ~9 bases. Abortive events may occur, forcing initiation to start again. Initiation phase ends when the enzyme succeeds in extending the chain and clears the promoter. 75 Elongation - enzymes moves along the DNA, extending the RNA, unwinding the DNA exposing new segments of the template and displace the RNA-DNA hybrid to re-form the original double stranded DNA. RNA emerges as a free single strand. Termination - recognition of the point at which no further bases should be added to the chain. The enzyme and the RNA should be released and the DNA re-forms the original duplex state. 76 Initiation of transcription: a crucial (some time the only) regulatory step in gene expression. Some key questions: How starting point is recognized? How initiation rate is determined? The transcription bubble: transiently and shortly separation of the DNA to single strands. The process of transcription: the usual complementary base pairing process. 77 Stages in which the bubble is created Template recognition. Closed complex. Local unwinding: open complex (template strand is available) Initiation (up to 9 bases that could be released; no move) Promoter clearance Elongation - Movement of the bubble. (inchworm move or fluent?) Termination:1. Cease addition of nucleotides. 2. Set complex apart. Just like initiation, termination is 78 sequence-dependent. Defines the terminator. Promoter: The sequence of DNA needed for RNA polymerase to bind to the template and accomplish the initiation reaction (synthesis of the first nucleotide bonds). Terminator: The sequence of DNA required for disrupting the bubble and reforming the DNA duplex (after the last base is added). 79 an ’ subunits have a channel for the DNA 80 Yeast RNA Pol II is composed of 12 subunits (holoenzyme). Two subunits form a different sub-complex. Two subunits are not essential for 81 viability. Following DNA binding and melting, the “clamp” swings back to force a turn. [note, colors of subunits82are the same as in the crystal structure] “wall” protein is enforcing a turn. The length of RNA hybrid is limited by the activity of the “rudder” protein. The RNA is forced to leave the DNA When it hits the protein rudder. 83 The bridge protein is found in different conformations In different crystal structures. Probably, breaking and re-making of contacts is mediated by conformational changes of the “bridge” protein: A nucleotide addition cycle: 1. The bridge is in a straight conformation adjacent to the nucleotide entry site. 2. After adding a nucleotide to the RNA the bridge protein is in contacts with the newly added nucleotide, undergoes a conformational change and moves one base pair along the template, obscuring the nucleotide entry site. 3. The bridge returns to its straight conformation, allowing Entry of next nucleotide of the template - namely, the bridge acts as a ratchet. 84 Properties of the core enzyme The core enzyme of E. coli has a general affinity for DNA (driven by electrostatic attraction between the basic protein and the acidic DNA). Yet, it does not distinguish between promoters and other sequences. Any random sequence bound by core enzyme is described as a “loose binding site”. No change occurs in the DNA which remains duplex. Such a core enzyme-DNA complex is stable (half life for dissociation is 60 min.). 85 Properties of the holoenzyme The holoenzyme has a drastically reduced ability to recognize “loose binding sites” (half life of <1sec. Kd reduced by a factor of 104). The holoenzyme binds promoters with Kds 1,000 time higher than core enzyme with half lives of hours. However, it manifests a specific Kd to any specific promoter. Sigma confers the ability to recognize specific sites. It is also involved in “melting”, creating an “open” complex. 86 Depending of specific promoter the Kd for DNA:RNA pol association is 106 - 1012 (first level of regulation of rate of transcription). Formation of an open complex by melting (that is driven by sigma) allows tight binding that is not reversible. Initiation rate (frequency of initiation) also differs (dependent on other factors in addition to RNA pol:DNA associatio. Frequencies can range between 1/sec (rRNA genes to 1/30 min. (lacI promoter). 87 The holoenzyme binds promoters with Kds 1,000 time higher than core enzyme with half lives of hours. This property assists with promoter recognition, but significantly interferes with elongation. Therefore, sigma dissociates from the enzyme when elongation starts. Sigma factor is recycled. It becomes unnecessary when abortive initiation is concluded. 88 (sigma) (promoter region) Sigma contacts mainly bases of the coding strand and continues to hold these contacts - an important step in melting (forming an “open 89 complex and recognition of template strand. What is responsible for the ability of holoenzyme to bind specifically to promoters? Sigma has domains that recognize promoter DNA, but as an independent protein Sigma does not bind to DNA. There is major change in conformation of sigma when it binds core enzyme. The N-terminal region of free sigma suppresses the activity of the DNA-binding region - it is an autoinhibitory domain. 90 How holoenzyme finds a specific promoter (60bp in a 4x106 stretch)? The forward rate constant for RNA Pol binding to promoters is faster than random diffusion (that limits the constant to 108/M-1Sec-1). The measured rate constant for association with a 60 bp target is 1014/M-1Sec-1. If the target is the whole genome the rate constant is around 1014/M-1Sec-1. But how does the polymerase move from random binding sites to promoters? Perhaps RNA Pol binds DNA and remains contact (no simple diffusion that relies on random binding). Rather, a direct Displacement with other sequence occurs (no sliding). 91 The “diffusion model: random association with loose sites on DNA, dissociation and re-bind, until occasionally (statistically) interacting with a promoter, and remains associated. 92 93 A direct displacement model - diffusion is not required 94 Promoter’s function is provided directly by its DNA sequence/structure (it does not need to be transcribed or translated). It is a cis-acting site. [in genetic terminology, sites that are located on the same DNA are said to be in cis. Sites that are located on two different molecules of DNA are being in trans.] 95 Conserved - a base most often present at a position. Perhaps the most striking feature of E. coli promoters is the lack of extensive conservation of sequence over the 60 bp associated with RNA Pol. Promoter elements (in E. coli): Start point (a purine in 90% of the RNAs). -10 sequence -35 sequence The distance separating the -35 and the -10 sites. 96 97 The -10 sequence: T80A95T45A60A50T96 Sequence that resides in poistions of -18 to -9 in all known E. coli promoters. Subscripts denote the percent occurrence of the most frequent found base 98 The -35 sequence: T82T84G78A65C54A45 The distance separating the -35 and -10 sites is between 16-18 bp in 90% of promoters. In the exceptions it can go down to 15 or up to 20. Sequence itself is not important. Some promoters have an A-T-rich sequence located farther upstream. It is called UP element and interacts with a subunit of RNA pol. It Is typically found in promoters that are highly expressed, such as the promoters of the rRNA genes. 99 Up elements are associated with a subunit of RNA pol. Found in promoters that are highly expressed. 100 In spite of conservation of promoters there is ~1000 fold variation in the rate at which RNA polymerase binds to different promoters in vitro. Binding rates correlate well with the frequencies of transcription in vivo. 101 Sequences at prokaryotic terminators show no similarities. Many terminators require a hairpin to form. Termination involves recognition of signals on the transcript. 102 Hairpin structure + U rich sequence (1100 sequences in E. Coli fit these criteria. Intrinsic terminator - other factors are not required. Works in vitro too. Hairpins may cause polymerase to slow or even to stop. Antitermination process may allow RNA Pol to continue (readthrough). Downstream U-rich destabilizes RNA-DNA hybrid. Hairpin + U-rich are Necessary, but not sufficient. 103 The weakest base-pair is the rU-dA 104 Rho: A 275 Kd homo-hexamer. RNA binding domain + ATPase domain. Belong to a family of ATP-dependent helicases. Functions as an ancillary factor for RNA Pol. Most efficient at 10% concentration. Accounts for about 50% of terminations in E. coli. Rho-dependent termination sequences are rich in Cs and poor in Gs. Reside 50-90 bases from termination sites. Acts processively on a single RNA substrate. Moves faster than RNA Pol. Pausing is important for Rho-dependent termination too. 105 106 107 Translation Components involved in translation account for 35% of the dry weight of E. coli cells. 108 109 A condensation reaction: formation of the peptide bond by removal of water (dehydration) from the -carboxyl group of one amino acid and the -amino group of another ----------------------------------------------------------------------------------- 110 111 To make the reaction thermodynamically more favorable, the carboxyl group must be chemically modified or activated so that the hydroxyl group can be more readily eliminated 112 113 (Dihydrouridine) 114 115 First stage in translation: aminoacyl-tRNA synthetases esterify the 20 amino acids to their corresponding tRNA. Each enzyme is specific for one amino acid and one or more tRNAs. 116 Step 1: An enzyme-bound intermediate Aminoacyl-AMP forms when the carboxyl group of the amino acid reacts with the -phosphoryl group of ATP, creating an anhydride linkage, with displacement of pyrphosphate. Step 2: The aminoacyl group is transferred to its corresponding tRNA. The resulting ester linkage has a highly negative standard free energy of hydrolysis. 117 118 Valine and isoleucine differ in only a single methylene group 119 Proofreading by aminoacyl-tRNA synthetases Two active sites in the Ile-tRNAIle synthetase: - binding of the amino acid to the enzyme (affinity to Ile is only a little higher than affinity to Val (error in 1/200 entries. - binding of aminoacyl-AMP product. This site has higher affinity to AMP-Val. A hydrolytic site. 120 What is accomplished by aminoacylation of tRNA? 1. Activation of the amino acid for peptide bond formation. 2. Attachment of the amino acid to an adaptor tRNA that ensures appropriate placement of the amino acid in a growing polypeptide. 121 122 123 N-formyl group is added to the amino group of methionine by transformylase. Transformylase is specific to Met attached to tRNAfMet 124 125 Translation initiation in prokaryotes IF-3 prevents combining of the 30S and 50S subunits The initiating 5’AUG is guided to its correct position by the Shine-Delgarno sequence in the 5’UTR of the mRNA (AUG is the beginning of an ‘open reading frame’). The initiating 5’AUG is positioned at a site called the P site, the only site in the ribosome to which fMet-tRNAfMet can bind. The fMet-tRNAfMet is the only aminoacyl-tRNA that binds first to the P site. 126 Step 2 in translation initiation: GTP-bound IF-2 and the fMet-tRNAfMet join the ribosome, guided by the anticodon that pairs with the mRNA initiation codon. Step 3 in translation initiation: The complex (30S + IF1,IF2-GTP,IF3 + fMet-tRNAfMet) combines with the 50S ribosomal subunit; simultaneously, the GTP bound to IF-2 is hydrolyzed to GDP and Pi which are released from the complex. All 3 initiation factors are also released from the complex. IF-2-GDP is re-loaded with GTP via a GDP/GTP exchange reaction. 127 128 129 130 131 132 Translational elongation Step 1: Appropriate incoming aminoacyl-tRNA binds to a complex of GTP bound EF-Tu. The GTP-EF-Tu-aminacyl-tRNA complex binds the A site of the 70S complex. The GTP is hydrolyzed and the EF-Tu-GDP is released. EF-Tu-GTP complex is regenerated via a GDP/GTP exchange reaction catalyzed by EF-Ts. AA2 133 Translation elongation, Step 2: Formation of the peptide bond: The -amino group of the amino acid in the A site acts as a nucleophile, displacing the tRNA in the P site to form a peptide bond. The tRNAfMet at the P site is now uncharged. The peptidyl transferase reaction is probably catalyzed by the 23S rRNA 134 Translation elongation, Step 3: Translocation Move of the ribosome. The ribosome moves one codon towards the 3’ end of the mRNA. Translocation is catalyzed by EF-G-GTP (translocase). The ribosome is now ready for the next elongation cycle. 135 Termination Catalyzed by RF1 or RF2. (depending on the particular stop codon). RF1 and RF2 are proposed to mimic the structure of tRNA. RF-1 recognizes UAG and UAA. RF-2 Recognizes UGA and UAA. In eukaryotes, a single RF, eRF, recognizes all 3 termination codons. Releasing factors: 1. Hydrolyze the ester linkage of the peptydil-tRNA bond. 2. Release the polypeptide and the last tRNA (now uncharged). 3. Dissociate the ribosome to 30S and 50S subunits. 136 EF-Tu EF-G The carboxy terminal domain of EF-G mimics the structure of tRNA. Altogether EF-G mimics the structure of EF-Tu-tRNA complex 137and probably binds to the A site and displacing the peptidyl-tRNA. Translation is energy consuming: On average, hydrolysis of more than 4 NTPs to NDPs is required for the formation of each peptide bond of a polypeptide. 138 Bacterial ribosome’s M.W.: ~2.7 million Components in the ribosome structure: Proteins: blue (in large subunit); Yellow (in small subunit). Bases of rRNA in large subunit: white. Backbone of rRNA in large subunit: green. rRNA in small subunit: white. tRNAs: purpule, 139 mauve, gray. mRNA contacting tRNAs: red. In the 50S subunit, the 5S and 23S rRNAs form the structural core. The proteins are secondary elements in the complex, decorating the surface. No protein is detected within 18A of the active site for peptide bond formation. 140 141 50S subunit of a bacterial ribosome. Red - a puromycine molecule at the active peptidyl transferase site. Note no proteins in the vicinity. 142 143 Steady state level of a protein (expression level) is determined by a combination of regulation of: Transcription initiation mRNA degradation (mRNA stability) mRNA processing Transport to cytoplasm Translational control Folding and protein processing Protein degradation (protein stability) 144 145