Lecture 18: Translation II

Translation II Side notes: Elongation: sequential addition of amino acids (1) delivery of next aminoacyl-tRNA to the A site based on codon indicated by mRNA Elongation factors (EFTu in bacteria, eEF1A in eukaryotes) help load the tRNA (2) catalysis of peptide bond formation between the amino acid in the A site and the polypeptide chain in the P site (3) translocation: GTP-dependent elongation factor (EFG bacteria, eEF2 in eukaryotes) mediates the movement of mRNA-tRNA through the ribosome, moving it from the A site to the P site, freeing up the A site The tRNA in the E site then leaves the ribosome Elongation: decoding, peptide bond formation, and translocation Upon initiation, the initiator tRNA is at the P site, leaving an empty A site and priming elongation Elongation in bacteria and eukaryotes is very similar in terms of protein factors and their functions The first step in elongation is decoding: an aminoacyl-tRNA with an anticodon that is complementary to the mRNA codon is chosen by the ribosome The aminoacyl-tRNA is bound by an elongation factor (EFTu-GTP in bacteria or eEF1A-GTP in eukaryotes), which ensures high fidelity (on the order of 10^-3) The codon/anti-codon interaction is cognate (fully accurate). nearcognate (single mismatch) or non-cognate (more than one mismatch) Decoding step: ensuring high-fidelity Cognate aminoacyl-tRNAs associate more tightly with ribosome complex than non-cognate ones These binding differences are recognized by a two-step selection process: initial selection and proofreading These cognate-discriminated binding steps are thermodynamic contributors to fidelity Ribosomes have a decoding center in the small subunit that surrounds the codon-anticodon complex Recognition of the cognate helix induces a conformational change that triggers GTPase activation by EFTu These structural changes are stabilized for cognate interactions These cognate-stimulated forward steps are kinetic contributors to fidelity Peptide bond formation Once the aminoacyl-tRNA has been positioned in the A site, aminoacyl-tRNA and peptidyl-tRNAs are ready for peptide bond formation, where the growing polypeptide chain is transferred to the aminoacyl-tRNA The peptidyl transferase active site is packed with highly conserved rRNA nucleotides that surround the two tRNAs and help position them Binding of an aminoacyl-tRNA in the A site allows the peptidyl-tRNA to be positioned in the active site Chemistry involves nucleophilic attack of amino group of aminoacyltRNA on carbonyl of peptidyl tRNA. The 2’ OH group of peptidyl tRNA facilitates the transfer Final step of elongation: translocation Translation II Side notes: In bacterial elongation, which protein factor is required to translocate the mRNA-tRNA complex? • • • • EFG eEF2 EFTu eEF1A The complex of mRNA bound to tRNAs in A and P sites moves through the ribosome, freeing jo the A site and thus undergoing translocation Translocation involves large-scale movements of tRNAs, conformational changes to ribosome, and promoted by GTPase (EFG in bacteria, eEF2 in eukaryotes) Translocation thus associated with a hybrid ribosome state, where the acceptor end of tRNA moves with the large subunit first, followed by the anticodon moving with the small subunit The GTPase EFG acts as a “pawl” in a motor, where its domain mimics tRNA as if binds in the small subunit decoding center, promoting forward motion of mRNA:tRNA complex, driven by GTP hydrolysis Termination and ribosome recycling: release of mRNA from ribosome Termination occurs when the ribosome reaches a stop codon (UAG, UAA, or UGA). Stop codons are recognized by release factors, not tRNAS! Class I release factors (RF) recognize stop codons In bacteria, RF1 recognizes UAA and UAG and RF2 recognizes UAA and UGA In eukaryotes, eRF1 recognizes all three stop codons Interactions between RF and ribosome promotes release of the polypeptide Class II release factors (GTPase) are also involved RF3 in bacteria and eRF3 in eukaryotes The final step is ribosome recycling where the large and small subunits dissociate, releasing the remaining tRNA and mRNA RRF and EFG in bacteria and Rli1 or ABCE1 in eukaryotes Termination of translation requires class 1 release factors Class 1 release factors are bifunctional They associate with the small subunit decoding center They associate with the catalytic event of the large subunit RFs and tRNA are similar in structure and RFs position in the A site in the ribosome RF evolved independently in bacteria and eukaryotes Bacteria have two: RF1 (UAA, UAG) and RF2 (UAA, UGA), while eukaryotes have one: eRF1 (UAA, UAG, UGA) Class 1 RFs in bacteria and eukaryotes are unrelated with different secondary structural motifs, but have the same GGQ motif that is needed for catalysis In eukaryotic termination, binding of which factor releases the polypeptide chain from the ribosome? • • • • eRF1 eRF3 ABCE1 RF1 Termination is also mediated by class 2 release factors Class 2 RFs are GTPases that are also needed for termination, but have different roles in bacteria and eukaryotes In bacteria, GTPase RF3 promotes dissociation of RF1/2 after peptide release by coupling GTP hydrolysis In eukaryotes, GTPase eRF3 behaves as an escort for eRF1 and uses GTP hydrolysis for its own departure Eukaryotes have another factor ABCE1 (ATP-binding cassette ATPase) that replaces eRF3 to promote peptide release eRF1 remains bound to the ribosome after the peptide release reaction Polysomes form when multiple ribosomes are translating on a single mRNA With elongation, the initiation codon is released, allowing a new ribosome to assemble Translation II Side notes: Ribosomes start to pile up on the mRNA, forming structures called polysomes Abortive termination in response to decoding errors Decoding errors are more likely during stressful conditions, when there may be a shortage of some of the aminoacyl-tRNAs or from frameshifting events In bacteria, when the codon/anticodon helix at the P site has a mismatch, it triggers a conformational change that decreases the fidelity in the A site this leads to lots of errors and promiscuous recruitment of class 1 RF, causing premature termination The faulty peptide is detected, and then degraded, likely by the cellular peptidases and proteases in bacteria. This acts as a quality control mechanism. No evidence of post-peptidyl transfer quality control in eukaryotes Ribosome recycling is the final step in protein synthesis After translation is finished, the ribosome must be released so it can be reused The recycling substrate is a complex of the ribosome, mRNA and deacylated tRNA In bacteria, the ribosome recycling factor (RRF) inserts itself into ribosome and acts with EFG and promotes complex disassembly Initiation factor IF3 binds to small subunit to stabilize the disassociated state In eukaryotes, eRF1 remains bound after the peptide is released ATPase ABCE1 promotes ribosome recycling with ATP hydrolysis eIF2D helps eject mRNA and deacylated tRNA Core initiation factors eIF1, eIF1A, and eIF3 bind to and stabilize the dissociated ribosomal subunits There is no eukaryotic homolog of bacterial RRF Consequences of a stalled ribosome Ribosomes can sometimes stall or arrest, and these arrested complexes must be rescued (1) the associated mRNA is targeted for mRNA decay (2) the protein being synthesized is targeted for proteolysis (3) the ribosome is recycled tmRNA:SmpB as a quality control mechanism for stalled ribosomes in bacteria In general, ribosomes stall when they reach the end of an mRNA. Truncated mRNAs arise from incomplete transcription or mRNA decay.This is less of a problem in eukaryotes, since transcription and translation are separated and mRNA has a 5’ cap and 3’ poly(A) tail Instead, bacteria use tmRNA and its partner protein SmpB First, tmRNA first acts as a tRNA and adds alanine to peptide Second, tmRNA acts as an mRNA with its short ORF and termination codon, which translate to an 11 amino acid rage that signals for peptide degradation SmpB is positioned where the anticodon loop is placed in a tRNA tmRNA-SmpB mimics intact tRNA and binds in the empty ribosomal A site the tail of SmpB inserts into the mRNA channel This occurs rapidly when there is a truncated mRNA, but tRNA-SmpB binding is blocked when mRNA is present in the A site and mRNA channel Translation II Side notes: Non-stop or no-go decay as a quality control mechanism for stalled ribosomes in eukaryotes Eukaryotes are more likely to have full length mRNA being translated based on its 5’ cap, 3’ poly(A) tail, and the associated protein factors Instead, eukaryotic ribosomes stall on mRNAs that are endonucleolytically cleaved, exonucleolytically processed, have substantial secondary structure, etc. One mRNA decay pathway is non-stop/no-go decay (NSD/NGD) to deal with 1) premature polyadenylation (a long string of As in an ORF) and 2) no stop codon In NSG/NGD, ribosomes start to stack up upon a long string of adenosines with no stop codon, which triggers successive cleavage of mRNA in the A site of stalled ribosome Each successive cleavage results i. accumulation of ribosomes at end of truncated mRNA This leads to complete mRNA degradation Stalled ribosomes can be dissociated by Pelota and Hbs1L termination factor homologs, which recruit ABCE1 to promote ribosomal recycling The ribosome quality control (RQC) pathway in eukaryotes The ribosome quality control (RQC) system targets incomplete proteins for degradation NEMF and Ltn1 bind to the 60S subunit with the incomplete protein This stimulates addition of a string of alanines and threonines on the Cterminus (CAT tails) The CAT tails facilitate ubiquitination, which leads to degradation of the incomplete protein There are functional similarities between eukaryotic NEMF and bacterial tmRNA, underscoring importance of degrading incomplete protein products Recoding: read-out of genetic code is reprogrammed through mRNA Normally, a codon defines a specific amino acid Recoding is where the readout is reprogrammed in an mRNA specific fashion: a codon is interpreted differently in a specific mRNA, and in competition with the normal reading of the codon Recoding ca. produce several proteins from a single gene Nonsense suppression is where stop codons are misread and termination fails to occur Frameshifting is where the mRNA shifts so that peptide synthesis proceeds in a different reading frame Nonsense suppression Nonsense suppression is rare, but is more common when associated with specific mRNA elements A hexanucleotide motif (CARYYA) downstream of the stop codon in some viral mRNAs triggers increased read through GalPol precursor in Murine Leukemia Virus is made by nonsense suppression In GalPol synthesis, a 3’ proximal pseudoknot forms. This encourages misreading of the stop codon UAG by Gln-tRNA instead of stop Stop codons can also allow incorporation of non-standard amino acids Selenocysteine is similar to cysteine, but has a selenium instead of a sulfur tRNA has an anticodon that matches UGA translation ii Side notes: In E. coli, the selenocysteine insertion sequence (SECIS) stem loop is found 3’ of the UGA that will be decoded To determine the UGAs that will be decoded, SelB delivers the charged tRNA to the codon, and also acts as a guide by interacting with the SECIS stem loop Programmed frameshifting is triggered by local mRNA elements Ribosomes typically move along mRNA, reading codons (3 nucleotides) sequentially If the ribosome moves by a different number of nucleotides (a number that is not a multiple of three), the reading frame is changed, producing peptides with different composition This change of reading frame is called frameshifting and occurs most often +1 or -1 nucleotides from the original Specific mRNA sequence features that cause frameshifting are called programmed frameshifts Examples of programmed frameshifting The frameshifting occurs at the sequence CUU UGA C When there are lots of RF2 in the cell, it recognizes the stop codon (UGA) When there is insufficient RF2, binding to UGA is slower and a +1 frameshifting occurs because Leu-tRNA recognizes near-cognate UUU (+1) in the +1 frame as well as CUU (original) Most of the ribosomes terminate at the end of the gag gene but some undergo a -1 shift, resulting in a Gag-Pol fusion protein The shift is thought to occur due to slipping of the mRNA in the ribosome, facilitated by a downstream psuedoknot The mRNA sequence is said to be “slippery” because the tRNA in the P site can bind reasonably in the -1 frame as well as binding in the initial frame