Replication and gene expression in pro kary otes VFU Brno 10.10.2018 Structure of prokaryotic genome NUCLEOID • Not bounded by a nuclear membrane • DNA, HLP (histone-like proteins), non-histone proteins • Prokaryotic chromosome - DNA is usually one circular molecule of dsDNA • E. coli - 4,7 Mbp - >4000 genes • Attached to cellular membrane in several places • Origin of replication oriC and termination region ter PLASMIDS Small circular molecules of dsDNA (1-200 kbp) - up to several thousands per cell (usually 100s) They carry genes that are essential for survival (resistence to antibiotics e.g.) Every plasmid is a replicon (has its own oriC) and replicates independently of the genomic DNA Bacteria can acquire plasmids by conjugation or from the environment Great importance in biotechnology Prokaryotic chromosome Usually circular molecule Organized into looped domains Each loop is independently supercoiled RNA cleaved (e) Partially unfolded chromosome ^/^tEach loop is independently supercoiled. Nicked (a) Circular, unfolded (b) Folded chromosome, (c) Supercoiled, Dtik. chromosome actually 40 to 50 loops folded chromosome (d) Partially uncoiled chromosome Partial RNase digestion Partial DNase digestion Nicked DNA Figure 9-15 Principles of Genetics, 4/e O 2006 John Wiley & Sons a Exponential phaie of growth k RNA polymeraEe dl RNA fjrunoler. Oh-ns | Transcription ' fdUurtei OFis doi:10.1038/nrmicro2261 Nature Reviews | Microbiology a | The folded chromosome is organized into looped domains that are negatively supercoiled during the exponential phase of growth. In tins phase, the abundant nucleoid-associated proteins histone-like nucleoid structuring protein (H-NS) and factor for inversion stimulation (Fis) bind throughout the nucleoid and are associated with the seven ribosomal RNA operons. As shown here in tvvo cases, these are organized into superstructures called transcription factories, b | In stationary phase the rRNA operons are quiescent and Fis is almost undetectable. The chromosome has fewer looped domains, and those that are irsible consist of relaxed DNA. y DNA replication Protein Replication goes in both directions • Bacterial chromosome has one origin of replication (oriC) • Replication goes in both directions from oriC • Replication ends in ter region i_i 1 jim Figure 5-6 Molecular Biology of the Cell 5/e (©Garland Science 2008) Replication fork origin of replication leading strand lagging strand sliding clamp DNA polymerase I leading strand template single-stranded binding protein helicase leading strand overall direction ^ or replication Us' continuous synthesis parental DNA topoisomerase/ gyrase Okazaki fragment #3 Okazaki fragment #2 Okazaki fragment #1 lagging strand template sliding clamp lagging strand/ DNA discontinuous polymerase I synthesis DNAIigase https://courses.lumenlearning.com/microbiology/chapter/dna-replication/ DNA replication rules • Template - DNA strand used for synthesis • Primer - free 3'0H end • Replication proteins - polymerase,... • dNTP - deoxyribonucleosidtriphosphates (dATP, dGTP, dCTP, dTTP) DNA replication is 5'-3' directed Strand growth can occur in both directions - 5'-3' or 3'-5' from template 3'-5'- different polymerase (artificial) Problem is in the proofreading mechanism - repair of wrongly incorporated base: in the case of 3'-5' polymerization - termination of replication primer strand p p HYPOTHETICAL 3'-TO-5' TRAND GROWTH p p Ss-»- p p ACTUAL 5'-TO-3' STRAND GROWTH i mwTU PROOFREADING 5' end produced if one nucleotide p is removed by proofreading p p pj 5' : 3' 3' end produced when one nucleotide is removed by proofreading incoming correct deoxyribonucleotide triphosphate 5' P P incoming correct deoxyribonucleotide triphosphate 3' REACTION DOES NOT PROCEED, AS NO HIGH-ENERGY BOND WOULD BE CLEAVED HIGH-ENERGY BOND IS CLEAVED, PROVIDING THE ENERGY FOR POLYMERIZATION Figure 5-10 Molecular Biology of the Cell 5/e (© Garland Science 2008) DNA replication is SEMI-DISCONTINUOUS • Replication proceeds on both strands simultaneously - template strands have opposite polarity (5'-3' and 3'-5') - they are antiparallel: • leading strand - continuous synthesis from one primer lagging strand - discontinuous, Okazaki fragments from multiple different primers leading- newly strand synthesized template strand DNA polymerase on leading strand DNA primase slldin<,c,amp IZZL and clamp loadera^I/ una neux single-strand DNA- binding protein g- ^ —DNAhelicase lagging-strand template DNA polymerase \ primer / on lagging strand newly new Okazaki (just finishing an synthesized fragment Okazaki fragment) strand Figure 5-19a Molecular Biology of the Cell S.'e ID Garland Science 2008) DNA replication is SEMI-CONSERVATIVE • New molecule of dsDNA contains one strand from template DNA and one strand of newly synthesised DNA • Meselson-Stahl experiment - 15N labeled DNA in bacteria further cultivated in 14N environment • www.youtube.com/watch?v=JcUQ TZCGOw a) Semiconservative model b) Conservative model c) Dispersive model DNA synthesis during replication 3' end of strand OH 5'end of strand Figure 5-3 Molecular Biology of the Cell 5/e(© Garland Science 2008) Replication proteins - polymerases family Pol I A 5'--3r polymerase Pol II 0 5-3' polymerase .'! :■ i i-ioiuiclrii^f- PolA PolB,. Number Mil -SOS + SOS In Irtrj w\l 400 50-75 350 - 1000 DNA replication, ONA replication Oksz-iiki frog men r ^backup DNA maturation, polymerase). d.n A repair DNA repair. TLS Pol III 5'-3" rKrtynwws* y-S' ejionuelea-se Pol IV Pol V Y Y S-3' polymerase S'-3'po4ymcrnse a .V' 10-20 150-250 * 15 10-20 1200- 2500 200 □NAreplicaltan DNA repair TLS TLS {Fijalkowska J. et al., FEMS Microbiology Reviews, 2012} Replication proteins - polymerases DNA polymerase I • Globular protein; Mw = 109 000 • Multidomain structure - own polymerase activity 3'-5' exonuclease (proofreading) and 5'-3' exonuclease activity • Replication in spaces between Okazaki fragments • 5'-3' exonuclease activity ensures degradation of RNA primers during ligation of Okazaki fragments • Low processivity (20-25 nt), low synthesis speed (10-20 nt/s), high fidelity (104-105) DNA polymerase II • Monomer; Mw = 90 000 • Polymerase and 3'-5' exonuclease (proofreading) activity • DNA repair DNA polymerase III Replication proteins - polymerases Processivity • Ability to synthesize the reaction without releasing the template - in case of DNA synthesis it is incorporation of nucleotides - measured in nucleotides, without dissiciation of polymerase - high processivity (leading strand synthesis), low (synthesis of spacers between Okazaki fragments) Fidelity • Ability of DNA polymerase to copy template strand, how it incorporates complementary bases - relates to polymerase errors (e.g. 105 correctly incorporated nucleotides to 1 error) - higher because of proofreading activity of polymerase = ability to cut out incorrectly incorporated nucleotide (3'-5' exonuclease activity) Speed • Average amount of nucleotides incorporated per second - connected to processivity Polymerase III • Complex of several proteins ; Mw = 900 000 • Basic complex of subunits a, sad, through t subunits form a dimer, further associates with subunits y and (3 clamps which raise the processivity of enzyme • Replication of leading and lagging strand • Processivity and speed of synthesis depends on the structure of complex (monomer x dimer; presence of (3 clamp) - whole complex syntesizes DNA 1000 nt/s with high processivity • Polymerase and 3'-5' exonuclease (proofreading) activity LEADING STRAND {Fijalkowska J. et al., FEMS Microbiology Reviews, 2012} Exo- and endo-nuclease activity Replication proteins - other DNA ligase • Creates phosphodiester bond between 5' end and 3' end of 2 polynucleotide strands • Joins Okazaki fragments DNA primase • DNA-dependent RNA-polymerase • Syntesizes RNA primers (one for leading strand and one for each Okazaki fragment) origin of replication leading strand lagging strand DNA helicase DNA gyrase (topoisomerase II) • Unwinds superhelical twists created by replication fork progression • Converts positive superhelicity to negative Replication proteins form complex - replisome Unwinds DNA strands in duplex Replication progress • initiation: requires primer in repilcation origin: short RNA synthesized by primase • elongation: DNA polymerase creates new strand in 5'—>3' direction • termination: in ter region Replication initiation 1. Recognition of replication origin (oriC) by DnaA proteins, relaxation of hydrogen bonds at oriC 2. Binding of helicase to unwound DNA strands - unwinding of duplex DNA in 5'-3' direction - creates replication fork 3. SSB proteins bind to ssDNA (single-strand binding), they keep DNA in unwound state AT-rich region DnaA boxes DnaA proteins bind to DnaA boxes and to each other. Additional proteins that cause the DNA to bend also bind (not shown). This causes the region to wrap around the DnaA proteins and separates the AT-rich region. T DNA helicase (DnaB protein) binds to the origin. DnaC protein (not shown) assists this process. DNA helicase separates the DNA in both directions, creating 2 replication forts. Replication progress Unwinding of dsDNA by helicase Relaxation of superhelicity by topoisomerase Binding of SSB proteins to ssDNA Synthesis of RNA primers on the lagging strands in Okazaki fragments by RNA primase Synthesis of DNA on leading and lagging strands by DNA polymerase III Cleaving of RNA and synthesis of spacers in Okazaki fragments by DNA polymerase I Ligation of DNA segments on lagging strand by DNA ligase origin of replication leading strand lagging strand dna sliding clamp polymerase III sing le-stranded binding protein 3- t parental DNA topoisomerase/ gyrase RNA primer lagging strand template lagging strand/ DNA sliding clamp djscontjnuous polymerase I synthesis DNA ligase Replication termination • Termination region = ter • Tus protein binds to ter • Tus - inhibits the activity of helicases proteins) oriC (DnaB oriC http://reasonandscience.heavenforum.org/tl849p25-dna-replication-of-prokaryotes Replication of plasmid DNA • Plasmids are circular molecules of double-stranded DNA • Plasmid = circular replicon (origin of replication) • Replication by rolling circle mechanism - can result in product containing multiple copies of DNA - concatemer • Smaller plasmids are replicated by host cell replication machinery • Different types/lengths of plasmids linear UNA open circular DNA ',■ supercoiled DNA 1 • '. .' / * .......-_________Ki https://www.youtube.com/watch?v=zOPMLofObxk Rolling circle replication Rolling circle replication Conjugative plasmid Donor cell Recipient cell oom asm Pww keywordsking.com learning.uonbi.ac.ke Transcription RNA synthesis from rNTP on DNA templates by DNA-dependent RNA-polymerase Rules of base complementarity Essential for life - gene expression Nucleic acids Trar Replication "anscription (retrovi Replication Transl Protein Transcription Messenger RNA (mRNA) • Transcription of genetic information in structural genes (then translated to amino acids in proteins) Precursor ribosomal RNA (pre-rRNA) • Primary transcript for rRNA, subsequently posttranscriptionally edited to rRNA Precursor transfer RNA (pre-tRNA) • Primary transcript for tRNA, subsequently posttranscriptionally edited to tRNA Primary transcripts of regulator RNAs Different types of transcripts are synthesized in transcriptional units Prokaryotic genes do not contain introns Prokaryotic genes are polycystronic Cistron ~ gene Transcriptional unit Transcription occurs in forms of transcriptional units - polycystronic RNA * Non-operon transcriptional units • Operons Non-operon transcriptional unit ;otide of inator Operon DNA I^H^ RNA ^^^B polymerase ^^^k ^REPRESSOR ^ r_ promoter operator Initiator) codon < TRANSCRIPTION Last nucleotide of terminator RNA - primary transcript aug aug terminator • PROMOTER CAN OVERLAP WITH OPERATOR Prokaryotic promoter consensus sequences upstream downstream V-- -► (transcription start site = TSS) i -35 -10 +1 TTGACA promoter gene -35 element Pribnow box • Consensus sequences are „average" of all promoters • Promoter similarity ensures affinity to one RNA polymerase • Differences in consensus sequences modify affinity to RNA polymerase = promoter strength = how often will transcription be initiated from the promoter-accurate consensus sequences are rare • Strong bacterial promoter = more similar with consensus • Weak bacterial promoter = differs more from consensus • More detailed regulation - substitutions in -35 and -10 elements, distance of -35 and -10 elements, additional elements in some promoters Prokaryotic RNA polymerase • Recognizes promoters of all transcriptional units • Consists of 5 types of subunits 2xcc • 40 kDa - maintains the stability of complex lx(3 • 155 kDa - key subunit for rNTP binding to enzyme lx P' • 160 kDa - key subunit for promoter binding lx 00 • 160 kDa - regulation and stability lx a • 85 kDa - o-factor (sigma factor) - key subunit for promoter binding -few ^exchangeable" variants, for different types of promoters • Polymerization speed cca 15-20 nt/s - depends on o-factor Transcription progress Initiation of transcription Binding of RNA polymerase to promoter - template DNA strand • Binding of first and second NMPs - rules of complementarity Elongation • Begins by creating of first phosphodiester bond • Connection of nucleoside monophosphates to growing RNA strands Termination of transcription • RNA polymerase stalling • Release of RNA • Release of RNA polymerase from DNA RNA polymerase Promoter-^" ~~~^Sigma factor Attachment of RNA polymerase Unzipping of DNA, movement of RNA polymerase [a) Initiation of transcription 3' 5' Template DNA strand (c) Termination of transcription Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings. Initiation of transcription 1. Binding of RNA polymerase on -35 element and Pribnow box (closed transcriptional binary complex) 2. Abolishment of hydrogen bonds in Pribnow box (open transcriptional binary complex) - transcriptional bubble 3. Transcription of first two nucleotides (open transcriptional ternary complex) The B subunit removed to reveal the transcription bubble and the flipped bases in their pockets. Template DNA is in green and nontemplate DNA is in magenta, with the flipped-out bases in yellow. Bases -11A and -7T interact solely with the a subunit. Base -6G is at the a-B subunit interface. Base +2G interacts solely with the B subunit (insert). (Karpen a deHaseth, Biomolecules, 2015) Elongation 1. Formation of the first phosphodiester bond 2. Connecting of nucleoside monophosphates to the 3 'end of the newly synthesized RNA molecule - energy from rNTP cleavage to rNMP + PP 3. The nucleotides are joined according to the rules of complementarity of the bases according to the sequence of the template strand (similar to replication) 4. After the first 10 nucleotides are added, the a-factor will be released - the stability of the polymerase and matrix DNA complex will increase 5' I I I I I I I O ATGCCGCA v T-l I I C A C T C A H—P Nontemplate strand i II I I I I 3' TACCACG TA 3" q T%>>-Ofi of synthesis- TAC C TTA AC . U cTT 1 ' 1 1 'j- ICTT ^ , AT T CAT i..........1111 \JIA ^C CACUCA" | | | j | | | | C T A ^^£>s ST | 1 I I I I ' ^^-^ \ DHjA RNA polymerase Template strand Termination of transcription 1. RNA polymerase stalling 2. Release of synthesized RNA 3. Release of RNA polymerase from DNA Termination of transcription occurs in terminator of transcription: 1. Terminator independent of p-factor (rho) - terminator type 1 2. Terminator dependent on p-factor (rho) - terminator type 2 Terminator independent of p-factor • The DNA matrix and therefore the terminator transcript contains palindromic GC-rich followed by poly-U sequences • After transcription, synthesized RNA results in the formation of a hairpin with a loop that slows the RNA polymerase • The RNA polymerase synthesizes the poly-U region and drops out of the DNA • Free RNA polymerase is re-associated with o-factor hairpin loop Rho-dependent transcription termination • Structure similar to a p-factor independent termination • The DNA matrix and therefore the transcription terminator contains palindromic GC-rich region • After transcription, synthesized RNA results in the formation of a hairpin with a loop that slows the RNA polymerase • The poly-U sequence is replaced by another - the polymerase has no release signal • The newly generated RNA is bound by p-factor at the rut site , which slides towards the end of the RNA and RNA polymerase • After contact of the p-factor with the RNA polymerase, the polymerase is released • Free RNA polymerase is re-associated with o-factor Rho-Dependent Termination Transcription of structural genes • The structural genes contain the so-called leader sequence (structural genes only) - before the first structural gene within the transcription unit - behind the promoter / operator • The leader sequence (Shine-Delgarn sequence) AGGA (GGU) (RNA) sequence - facilitates binding of RNA to ribosome to the 3'-end of 16S rRNA Transcription of mRNA - summary • The primary transcript of the transcription unit containing the structural genes: • Contains a leader sequence at the 5 'end • It contains transcripts of structural genes that are translated into the polypeptide chain • At the 3'-end UUUUUUUU = end sequence • Contains transcriptions of several genes = polycistronic (multigenic) mRNA • Posttranscriptionally not modified - does not contain introns = no splicing • Life span for several minutes, ribonuclease decomposition in 5'-3' • At any given time, it accounts for 3% of total RNA in the cell Coupling of transcription with translation • Coupled synthesis • Ribosomes are already attached to mRNA during transcription • On the same mRNA molecule both processes (transcription + translation) are simultaneous + degradation of mRNA from the beginning of transcription • For some transcription units up to 15 transcription initiations per minute = 15 new mRNA molecules • For each mRNA up to 30 ribosomes = 30 new polypeptide chains • Binding and sliding of ribosomes along mRNA increases the synthesis rate of RNA polymerase • Absence of ribozomes, on the other hand, causes slowing of RNA polymerase, waiting for ribosomes Figure 17.22 Coupied transcription and translation in bacteria 0.25 u.m Direction of CcipyfJgm £ Pearson ErJucallcm. Inc., publishing as. Benjamin Cumriungs Transcription of functional RNAs • Transcription proceeds from several rRNAs (7 in E. coli) to form precursor rRNA (pre-rRNA) • Each transcript contains 16S, 23S and 5S rRNA along with several tRNAs • Pre-rRNA is posttranscriptionally methylated and cleaved by RNase (RNase III) to the respective rRNA and tRNA Pre-rRNA transcript (30S) Intermediates Mature RNAs 1 16S tRNA 23S 5S (4S) (l) I met hy I a ti on 1 1 2 It 13 3 41 1 --in—!—ii—n—1—^---n—m—n—i—n—i—n—r —■ N methyl groups (2)^t lea vage CD 17S i—m—n—i—9i—i—ir tRNA 25 S "T!-1-f □ 1t5S rRNA tRNA i-t—:—r,—i—r—rf 235 rRNA 5S □ SSrRNA Translation Synthesis of the polypeptide chain (protein, amino acid sequence) according to the nucleotide sequence in mRNA Nucleic Replication DNA^ Transcription Reverse transcription (retroviruses, Genetic code Second nt First nt U C A G Third nt Phe Ser Tyr Cys U U Phe Ser Tyr Cys C Leu Ser STOP STOP/Sel A Leu Ser STOP Trp G Leu Pro His Arg U C Leu Pro His Arg C Leu Pro Gin Arg A Leu Pro Gin Arg G lie Thr Asn Ser U A lie Thr Asn Ser C lie Thr Lys Arg A Met/START Thr Lys Arg G Val Ala Asp Gly U G Val Ala Asp Gly C Val Ala Glu Gly A Val Ala Glu Gly G Genetic code • the genetic code is a triplet code - one amino acid in the protein is encoded by a sequence of three nucleotides Triplet - Codon x anticodon = complementary sequence on tRNA carrying aminoacid mRNA CGUGGUACGAUUGGAUGU Protein Arg Gly Thr Me Gly Cys • the genetic code is universal - the meaning of individual triplets is almost always universal and therefore independent of the species of organism CGU = Arginine CGU = Arginine CGU = Arginine • the genetic code is degenerate - one amino acid can be encoded by several different triplets (but not vice versa) Translation progress Activation of aminoacids • Binding of amino acids to tRNA by aminoacyl-tRNA synthetases Initiation of translation • A sequence of actions to create the initiation complex - 70S ribosome, mRNA, initiation tRNA - initiation factors Elongation • Polypeptide chain extension - elongation factors Termination of translation • Termination of synthesis on a senseless codon, release of the polypeptide from the ribosome - termination factors Translation conditions in prokaryotes • mRNA template • 22 Standard aminoacids • tRNA • Aminoacyl-tRNA-synthetase • Ribosomes • Translation factors • ATP and GTP tRNA 74 to 95 nucleotides MW = 80 000 3'- end = 5'- CCA-3' part of the sequence are unusual bases arising from enzyme modification after transcription (dihydrouridine, ...) names: tRNA: tRNAAla, tRNALeu tRNA + AA: Ala ~ tRNAAla, Leu ~ tRNALeu attached amino acid (Phe) Dihydrouridine loop anticodon a clover leaf HN'' ^NH I I HO—H2C 0, 'ribose> OH OH pseudouridine If riboseN two methyl groups added to G (W.W-dimethyl G) HO — H2C O OH O I CH, 2' O methylated nucleotide two hydrogens added to U (dihydro U) tribose^l wibosey sulfur replaces oxygen in U deamination of A (4-thiouridine) (inosine) FlgultS-iS Molecular B-iologycf the GelISM* Garland 5ti«tc* ifiOE: (A) (B) (C) 5' GCGGAUUUAGCUCAGDDGGGAGAGCGCCAGACUGAAYA'1'CUGGAGGUCCUGUGT'J'CGAUCCACAGAAUUCGCACCA 3' lDl anticodon Figure 6-52 Molecular Biology of the Cell 5/e (© Garland Science 2008) CCA end of tRNA this triplet of nucleotides is present at the 3-end of each tRNA on the last A the amino acid is bound during activation CCA is added by nucleotidyltransferases using ATP and CTP in post-transcriptional modifications of the primary transcript (DNA or RNA) nucleotidyltransferases are in two classes: • class I - Archae • class II - prokaryotes a eukaryotes Nucleotidyltransferases recognize immature tRNA (does not have CCA) from mature (CCA-terminated) and catalyze only immature tRNA Aminoacids H O i ii H,N—C—C—OH i I I Glycine (Gly) aa ~ tRNA + AMP amino acid (tryptophan) | o h,n —c — cf tRNA (tRNA H,N —C-C Trp. CH AMP + 2P, linkage of amino acid to tRNA tRNA synthetase (tryptophanyl tRNA synthetase) Figure 6-58 Molecular Biology of the Cell 5/e I© Garland Science 2008} high-energy |^bond tRNA binds to its codon in RNA 3'H ■ ■ 5' Tl base-pairing LP G G 5' 3' mRNA NET RESULT: AMINO ACID SELECTED BY ITS CODON DIS Prokaryotic ribosome Prokaryotic ribosome 34 proteins Figure 6-64 Molecular Biology of the Cell 5/e [Q Garland Science 20GB} Prokaryotic ribosome • The ribosomal subunits are composed separately after the synthesis of the individual components • The subunits associate together with the synthesis of the protein by binding to mRNA near the 5'-end of the mRNA • The small subunit provides pairing of anticodon (tRNA) - codon (mRNA) • The large subunit provides the synthesis of peptide bonds • The key catalytic activities of the ribosome are assured by rRNA - ribozyme • Bacterial ribosomes synthesize a binding of approximately 20 amino acids per second • 4 key binding sites: • lx for mRNA • 3x for tRNA - A (acceptor / aminoacyl), P (peptidyl) a E (exit) site • Additional binding sites for translational factors (initiation and elongation) Nobel Prize in Chemistry The Nobel Prize in Chemistry 2009 was awarded jointly to Venkatraman Ramakrishnan, Thomas A. Steitz and Ada E. Yonath "for studies of the structure and function of the ribosome." Venkatraman Ramakrishnan Affiliation at the time of the award: MRC Laboratory of Molecular Biology, Cambridge, United Kingdom Thomas A. Steitz Affiliation at the time of the award: Yale University, New Haven, CT, USA, Howard Hughes Medical Institute Ada E. Yonath Affiliation at the time of the award: Weizmann Institute of Science, Rehovot, Israel Prokaryotic translation Initiation of translation • Creation of a ternary complex : fMet (A/-Formylmethionine) ~ tRNAfMet + GTP + IF2 • Identification of the Shine-Delgarno mRNA sequence associated with the IF3, the 30S ribosomal subunit - the exact localization of the AUG codon mRNA into the ribosome P site by the complementarity of the Shine-Delgarno sequence and the 16S rRNA region • Binding of the ternary complex via tRNA-fMet to the ribosome P-site - IFl-containing formation of the pre-initiation complex • Cleavage of GTP to GDP + Pi, releasing IF1-3, and initiation complex formation with the 50S subunit where fMet ~ tRNA-fMet is at the ribosome P site Elongation • Repeated binding of tRNAaa to the ribosome A site with EF-Tu and GTP, creation of a peptide bond between aa at the P and A site, ribosome translocation (content of A to P, P to E) with EF-G and GTP Termination of translation • Recognizing a senseless codon at the A site by RF factor • Cleavage of the synthesized peptide • Release of tRNA, mRNA and dissociation of ribosomal subunits https://www.voutube.com/watch?v=KZBIiAM6Bls Prokaryotic translation - initiation Identification of the Shine-Delgarno mRNA sequence associated with the IF3, the 30S ribosomal subunit - the exact localization of the AUG codon mRNA into the ribosome P site by the complementarity of the Shine-Delgarno sequence and the 16S rRNA region Shine-Delgarno (BJaUG sequence Creation of a ternary complex: fMet ~ tRNAfMet + GTP + IF2 Ternary complex E Binary complex fMet~tRNAfMet i fMet Binding of the ternary complex via tRNAfMet to the ribosome P-site - IFl-containing formation of the pre-initiation complex V J Pt tn""---- Clearing GTP to GDP + Pi, releasing IF1-3, and initiating complex formation with the 50S subunit where fMet ~ tRNAfMet is at the ribosome P site Shina-Dalgarno sequence mRNA f~ I S—^KTjA Q UAft GCAGGU Start codon Preinitiation complex 16S rRNA Initiation complex AUG codon and formylmethionin AUG codon • It encodes the first amino acid in the peptide • It also occurs within the mRNA chain • AUG encodes for methionine, but at the beginning of the polypeptide is formylmethionine • There are 2 different methionine tRNAs - tRNAMet a tRNAfMet H H2 N- C-COOH I CH5 I CH, I S I H H I I 0=C— N — C— COOH I H transformylaza deformylaza CH. CH. CH, • At the beginning of mRNA Met ~ tRNAfMet binds AUG codon which is formylated to fMet ~ tRNAfMet by IF2 • Inside mRNA Met ~ tRNAMet binds AUG codon via EF-Tu Elongation of polypeptide chain 1. aminoacyl-tRNA binds to the A site and the tRNA used is released from the E site 2. A new peptide bond is created between the amino acids at the E and at the A site 3. The large subunit of the ribosome moves and the so-called hybrid sites P / A and E / P are created 4. The small subunit moves one codon - releases from the A site, tRNAs are shifted from P to E and from A to P growing polypeptide chain Figure 6-66 Molecular Biology of the Cell 5/e 1® Garland Science 2008) Elongation of polypeptide chai Binding of aminoacyl-tRNA to site A is mediated by the elongation factor ET-Tu, which is "driven" by hydrolysis of GTP to GDP (hydrolysis catalyses conformational protein change) • EF-Tu increases the speed and accuracy of translation - proofreading mechanisms EF-G elongation factor catalyses large ribosome subunits movement - energy from GTP hydrolysis Figure 6-67 Molecular Biology of the Cell S/e (© Garland Science 2008) Wobble base pairing 22 AA - 64 codons - 40 tRNA Some tRNAs bind to multiple codons In 1966 F. Crick postulated the wobble hypothesis The 1st and 2nd nucleotide (5'-XXo-3 ') are strictly bound by Watson-Crick pairing to tRNA 3rd nucleotide (5'-ooX-3 ') binds alternatively to non-Watson-Crick pairing tRNAs carrying the same AA but recognizing the different codons are called isoacceptor Codon Anti-Codon A U or I G C or U or I C Gor I U A or G or I Wobble Base-Pairing between anticodon & codon H Gj*>** Adonma W-c base pairing Pebble pairing H Gb»r*ie 9«. 7 Mwl pwnonly m«h A I no ITU SutWI GTPase and GTP • The process involves GTPase, also a part of the EF-Tu factor • The GTP-binding domain is evolutionarily highly conserved and occurs from bacteria to higher eukaryotes • GTP hydrolysis is associated with a conformational change involving A2662 from 23S-rRNA R. M. Voorhees et al., Science 330, 835-838 (2010) • His84 acts as a base that removes the proton from H20 • OH- attacks y-phosphate in GTP • GDP is released A2662 ■ W (^ A £ Switch 1 f ,^^fT His64 J '.] Gly33 ^Thr61 EF-Tu V oh oh q- jVp" ' Switch 1 / / y 1 "^p^ A2B62 ? SRL Termination of translation • the presence of an nonsense codon where AA does not bind, but the termination factors do • presence of termination factors RF1 (for UAG and UAA), RF2 (for UGA and UAA) and RF3 (stimulates the effect of RFland RF2) • From the carboxyl terminus of the polypeptide chain, the tRNA is released to stop its elongation • This frees both the polypeptide chain and the ribosome, which then breaks down into its subunits BINDING OF RELEASE FACTOR TO THE A-SITE TERMINATION AUCAACUGGUflGCGAU« Regulation of translation speed A-standard ribosome movement B - slowing of ribosome due to codons for less frequent AA C - slowing of the ribosome due to secondary mRNA structure D - slowing of the ribosome due to positively charged AA - electrostatic interactions reduce the speed of movement in the tunnel Mechanism of peptide bond formation • P-loop and the A-loop are part of 23S-rRNA • There is no protein in the vicinity of 18A from the catalytic site • N3 nitrogen of adenine A2451 is decisive P-loop (2246-2259) A-loop (2547-2561) G2061 02P, 24515" ,i,-jpuromycin Aft. 34S1 pK^8 aa-tRNA 7-6 X tRNA Good nudeophile ^ aa-tRNA 2151 a" tRNA aa-tRNA-.. V- \ O —* ?v —* 2l tRNA pK^-2 (A1 Ml tRNA] — -> aa-tRNA—*( 3j Peptidyl transfer product S* with new amide bond formed JR^A_ NH, i <" II O—i N 0 OH 0=c r1 -CH nh NH O OH 0 -O-C-N-CH-C-0 nh 'J N n h o oh 0=c r2-ch nh: 0 OH 1 <' o—i n Nl I ^__6 OH o -0-C-N-CH-C-O— 7^ "— N r1-ch r2 nh ■ 0 = c >-N 0 OH 1 o=c r2-ch nh 0 = 0 RI-CH NH ■ 0 = 0 nh,, Genes are expressed differently gene A TRANSCRIPTION RNA i TRANSLATION ^A/ V£z \A/ KQ/ v& v5P \£/ <&/ Figure 6-3 Molecular Biology of the Cell 5/e {© Garland Science 2008] gene B i a DNA TRANSCRIPTION RNA TRANSLATION