Nucleic Acids Biochemistry Translation 2 PDF

• • • • • • • • • • • • • • • Recap of last lecture Translation of 4-base RNA code into the code of 20 A.A.s requires adaptors Start & Stop codons define protein-coding ORF in the mRNA mRNAs from bacteria, eukaryotes differ in RBS, ORFs per mRNA, modifications tRNAs have conserved 3’ end that speci...

• • • • • • • • • • • • • • • Recap of last lecture Translation of 4-base RNA code into the code of 20 A.A.s requires adaptors Start & Stop codons define protein-coding ORF in the mRNA mRNAs from bacteria, eukaryotes differ in RBS, ORFs per mRNA, modifications tRNAs have conserved 3’ end that special enzymes append amino acids to tRNAs have functional and structural regions formed by RNA base pairing charging of tRNAs done by sequential addition of ATP, then tRNA to amino acid Different tRNA structures confer a “2nd genetic code” to get the right A.A. added tRNA synthetases use two recognition pockets to prevent mis-charging of tRNAs Pre –ribosome quality control of amino acid-tRNAs needed to prevent errors Ribosomes are complex ribonucleoproteins that convert mRNA info to proteins Eukaryotic ribosomes larger than prokaryotic, have ribonucleoprotein subunits Ribosomes make 1 protein at a time, but can work simultaneously on a mRNA Peptide bond is switched from outgoing to incoming tRNA to lengthen protein Ribosome holoenzyme creates active sites and tunnels for making protein Ribosome provides kink in mRNA which facilitates tRNA entry, enzyme action BIOC 3041 Nucleic Acids Biochemistry Lecture 22 – Translation 2 Initiation Termination Elongation The steps of protein synthesis Initiation Assembly of a functional ribosome in correct place on a mRNA, start protein synthesis Elongation Cycle Correct AA brought to ribosome, joined to polypeptide chain, ribosome moves 1 position along the mRNA. Termination When stop codon reached, there is no AA to be Added, newly-made polypeptide released Disassembly A special factor binds to ribosome, causes release of bound mRNA and tRNA, these are recycled in next round of protein synthesis Ribosome cycle involves initiation, elongation, termination & disassembly The initiation of translation (prokaryotic) An overview of the events of translation initiation Components • Two ribosome subunits • mRNA • Initiator formylmet- tRNAi Met (fMet) • Three Initiation Factors Events: • Recruit ribosome to mRNA • Place a charged tRNAi in the P site • Position the ribosome over the start codon Small ribosomal subunit must align P site on start codon during initiation Prokaryotic mRNAs are initially recruited to the small subunit by base-pairing to rRNA • The 16S rRNA interacts with RBS to position the AUG in the P site. • Only small subunit needed here (has the 16S rRNA as part of it) • Must position mRNA exactly or a frameshift error will occur Small ribosomal subunit must accurately dock at the right site in mRNA A special tRNA charged with a modified methionine binds directly to the prokaryotic small subunit • Initiator tRNA is formyl-Met-tRNAifMet (base-pairs with AUG or GUG or UUG) • Deformylase removes the formyl group during or after the synthesis • NH2-terminal Met, and next 1-2 amino acids commonly removed after too! a peptide bond Only prokaryotes use formyl-Met as the first amino acid of a new protein 3 initiation factors direct assembly of an initiation complex containing mRNAs and initiator tRNA IF1: blocks tRNAs from binding to “A” site (Aminoacyl) IF2: a GTPase; binds to both fMet-tRNAinitiator and IF1, preventing further tRNA binding to small subunits. IF3: binds to small subunit and prevents it from binding to large subunit; this is essential for translation initiation. Initiation Factors promote correct sequential ribosomal assembly Summary of translation initiation in prokaryotes The 2 Initiation Factors (I.F.s) bind at or near tRNA binding sites (A, P, E sites) • IF3 promotes the dissociation of the apo-ribosome… • IF1 binds near the A site, blocking it (want 1st tRNA in Peptidyl site, not Aminoacyl site!) • IF2 (+GTP) assists the fMet-tRNAfMet binding to P site • The 30S pre-initiation complex is formed & ready for mRNA binding IF3 plays key role in keeping small subunit open and ready for other factor, mRNA, tRNA binding Summary of translation initiation in prokaryotes • (The order of mRNA or fMet-tRNAi binding is uncertain…) • mRNA binds and positions the start codon correctly • Conformational changes occur in the small subunit, causing IF3 loss • 50S subunit can now bind and stimulates the GTPase activity of IF2 (IF2-GDP has less affinity for ribosome….) • IF2(GDP) and IF1 are released • The 70S initiation complex is now formed mRNA & tRNA binding sets off causes I.F. loss leading to complete ribosome Eukaryotic protein synthesis Key Differences between eukaryotes & prokaryotes 1) Initiator tRNA always binds small subunit before mRNA does 2) The first Met in eukaryotes is not formylated (for nuclear genes…) 3) A separate set of accessory proteins prepares mRNA before it it is bound by small subunit 4) Ribosome + tRNA scans the mRNA for first start codon Eukaryotic ribosome assembly differs from prokaryotes • eIF1A and eIF3 dissociate the ribosome large subunit • eIF3, eIF5, and eIF1 act as 1 functional unit (1-3-5) • eIF2(GTP)•Met-tRNAi Met binds the small subunit and positions the tRNAi in the P site… • 43S preinitiation complex formed Ternary complex Large subunit needs to be displaced before then bringing in tRNA to make preinitiation complex Eukaryotic ribosomes recruited to mRNA by 5′ Cap secondary structure mRNA cap is recognised by eIF4 subunits 1) eIF4E • eIF4G • eIF4A (in that order, E-G-A) EGAB! 2) eIF4B binds –A, activates RNA helicase activity of A to get rid of unwanted secondary structure 3) Translation can be regulated by proteins interacting with the E subunit 43S preinitiation complex 48S preinitiation complex Capped mRNA proteins bind & unwind mRNA before preinitiation complex made Translation initiation factors hold eukaryotic mRNAs in circles • eIF4G protein that binds to 5′ cap area interacts with the proteins that coat the poly-A tail • This allows small subunit to run on a circular mRNA track to minimize the time needed to find a new mRNA • Maximizes amount of protein made in short time By linking proteins bound to 5′ & 3′ end of mRNA, translation efficiency can be maximized Start codon found by scanning downstream of 5′ mRNA end • identification of start AUG codon by the small ribosomal subunits • 48S preinitiation complex scans from 5′ capped end to 3′ • Uses ATP in helicase activity • Proper base pairing of start codon with tRNAi-Met causes conformational change • eIF2 hydrolyzes GTP to GDP causing eIF loss • eIF5B-GTP binds and recruits large subunit • This causes eIF5B to hydrolyze GTP, cause loss of eIF1A and 5B-GDP Start codon binding sets off cascade leading to eIF loss & large subunit gain Translation elongation • mechanism of elongation is highly conserved in prokaryotes & eukaryotes cyclic process: • an aminoacyl-tRNA is loaded into the A site ribosome • A peptide bond is formed (the peptidyl transferase reaction) • the peptidyl-tRNA•mRNA is translocated in the ribosome • Precise translocation is difficult, helper proteins are needed tRNA-AA slotted into A site to allow peptide bond formation before translocation Aminoacyl-tRNA is delivered to A site by elongation factor EF-Tu (elongation factor thermo unstable) • interaction between EF-Tu and its binding site on large subunit triggers its GTPase activity E P A At the start of each cycle: • the A (aminoacyl) site on the ribosome is empty • the P (peptidyl) site contains a peptidyl-tRNA • the E (exit) site contains an uncharged (empty) tRNA... • EF-Tu (GTP) binds with an aminoacyl-tRNA and brings it to the ribosome EF-Tu is the escort for AA-tRNA and uses GTP to catalyze drop-off reaction E P A EF-Tu helps ensure that the correct aminoacyl-tRNA is in place: E P A 1) Only EF-Tu(GTP) can bind to the aminoacyl end of the aa•tRNA 2) It prevents the aminoacyl end of charged tRNA from entering peptidyl transferase centre early 3) The codon-anticodon pairing is verified 4) GTPase is activated by the ‘factor binding centre’ E P A 5) EF-Tu(GDP) is released EF-Tu is the escort for AA-tRNA and uses GTP to catalyze drop-off reaction E PA The ribosome uses 3 mechanisms to select against incorrect aminoacyl-tRNAs 1. Adenines in 16S rRNA bind to minor groove of codon:anticodon “helix” 2. GTPase is not activated if EF-Tu & tRNA is not positioned correctly by base pairing with mRNA 3. “Accommodation” of tRNA into peptidyl transferase site fails more frequently if tRNA is not paired correctly, tRNA can’t rotate into position Ribosome has structural features to prevent wrong tRNA from binding codon Peptide bond formation and the elongation factor EF-G drive translocation of tRNAs & mRNA Three events: 1) The P-site tRNA must move to the E site since it is now “empty” P E A translocase E P 2) The A-site peptidyl tRNA must move to the P site since it now has a peptidyl-bond 3) The mRNA must move by three nucleotides to expose the next codon E PA EF-G protein needed for this, formerly known as “translocase”, use GTP energy to ‘kick’ tRNAs 1 site over  tRNA base pairing to mRNA drags it along too! After peptidyl bond made, tRNAs and mRNA all need to move to the next site to continue translation E PA A How does EF-G-GDP interact with the A site so well? • EF-G translocase mimics a tRNA to displace the tRNA bound to the A site • EF-G bound to GTP can slip into A site and then use GTP energy to act like a molecular “flicker”  flicks tRNAs one site over EF-Tu-GDP-Phe-tRNA EF-G-GDP • EF-G with GTP bound is activated by entering the A site, stimulates the GTP hydrolysis to GDP EF-G shares tRNA’s molecular shape, allowing it to enter A site and function EF-Tu-GDP and EF-G-GDP must exchange GDP for GTP prior to participating in a new round of elongation • EF-G has a lower affinity for GDP than GTP so it easily loses GDP spontaneously and also spontaneously binds new GTP • For EF-Tu, a GTP-exchange factor EF-Ts is required for the GDP-GTP exchange…. (EF-Ts=EF-Transfer……according to Waller) • A cycle of peptide bond formation consumes two molecules of GTP and one molecule of ATP • 1 ATP for adenylylation, 2 GTP for tRNAs EF-G spontaneously exchanges GDP, EF-Tu needs EF-Ts GTP exchange factor Termination of translation • release factors (RF) terminate translation in response to stop codons • Release factors activates the hydrolysis of polypeptide from peptidyl-tRNA Class I RF: recognizes stop codon, break protein off tRNA Class II RF: stimulate dissociation of class I RF from ribosome Class I RF: prokaryotes: RF1 (UAG, UAA); RF2 (UGA, UAA) eukaryotes: eRF1 (UAG; UGA; UAA) Class II RF: regulated by GTP binding and hydrolysis prokaryotes: RF3 eukaryotes: eRF3 Eukaryotic and prokaryotic release factors almost identical in number, function Short regions of class I release factors recognize stop codons and trigger release of the peptidyl chain CCA • RF1 structure very similar to tRNA structure, RF1s seem to have a peptide anticodon GGQ • RF1s have strictly conserved GGQ (Gly Gly Gln) • GGQ: involved in polypeptide hydrolysis; close to peptidyl-transferase center E A P • Model of a RF1 bound to the A site • SPF: peptide anticodon; interacts with stop codon Release Factor1 has an anticodon and a peptide bond hydrolysis end GDP-GTP exchange and GTP hydrolysis control the function of the class II RFs RF-3 has a higher affinity to GDP than to GTP Class I release factor binds to A site • SPF peptide anticodon pairs with stop codon • GGQ is positioned in peptidyl transferase site • Hydrolysis occurs • Polypeptide dissociates RF3•GDP binds to a ribosome containing a Class I RF • RF3 exchanges GDP for GTP (kind of like EF-Ts does for EF-Tu..) • Class I factor is displaced caused by RF3-GTP’s new shape • GTP is hydrolysed • RF3•GDP dissociates (has low affinity for ribosome on its own) Class 2 RF evicts Class 1 RF from ribosome by using Conformation change caused by GDP GTP binding The ribosome recycling factor (RRF) mimics a tRNA • Polypeptide, RF1 (RF2), RF3 are released • RRF mimics a tRNA to stimulate the release of tRNA and mRNA from a terminated ribosome • RRF and EF-G use GTP hydrolysis to push out the tRNA and mRNA (P site tRNA leaves directly from P site, not E site) • RRF and EF-G then lost, allowing IF3 to bind to small subunit and displace the large subunit • This process effectively recycles ribosome RRF and EF-G use GTP to clear out ribosome and prepare it for recycling by IF3 Recap of lecture www.youtube.com/watch?v=8Hsz_Vmcy-Y • Ribosome cycle involves initiation, elongation, termination & disassembly • Small ribosomal subunit must align P site on start codon, dock to mRNA during initiation • Only prokaryotes use formyl-Met as the first amino acid of a new protein • • Initiation Factors promote correct sequential ribosomal assembly IF3 plays key role in keeping small subunit open and ready for other factor, mRNA, tRNA binding • mRNA & tRNA binding sets off causes I.F. loss leading to complete ribosome • Large subunit needs to be displaced before then bringing in tRNA to make preinitiation complex • Capped mRNA proteins bind & unwind mRNA before preinitiation complex made • Start codon binding sets off cascade leading to eIF loss & large subunit gain • tRNA-AA slotted into A site to allow peptide bond formation before translocation • EF-Tu is the escort for AA-tRNA and uses GTP to catalyze drop-off reaction • Ribosome has structural features to prevent wrong tRNA from binding codon • After peptidyl bond made, tRNAs and mRNA all need to move to the next site to continue translation • EF-G shares tRNA’s molecular shape, allowing it to enter A site and function • EF-G spontaneously exchanges GDP, EF-Tu needs EF-Ts GTP exchange factor • Eukaryotic and prokaryotic release factors almost identical in number, function • Release Factor1 has an anticodon and a peptide bond hydrolysis end • Class 2 RF evicts Class 1 RF from ribosome by using conformation change caused by GDP  GTP binding • RRF and EF-G use GTP to clear out ribosome and prepare it for recycling by IF3 Reading – MBPGF p 449-474, 498- ,MBOG7 pg 528 -549, MBOG6 pg 479-503