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Translation Initiation, Elongation, Termination Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Protein synthesis very complex • Requires: – Various tRNAs with their attached amino acids – Ribosomes – mRNA – Numerous proteins with different functions – Cations – GTP Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Protein synthesis very complex • Bacterial/eukaryotic translation similar – eukaryotes need more nonribosomal proteins – Both involve initiation, elongation & termination • Ribosome starts translation at initiation codon – Establishes reading frame – GUG can be initiator (f-met inserted here; valine if codon is internal) Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Prok. Step 1: small ribosomal subunit binds to mRNA • Bacterial mRNAs have Shine-Dalgarno (S-D) sequence – 5 - 10 nucleotides before initiation codon; – S-D complementary to 16S small subunit sequence near 3' end Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Prok. Step 1: small ribosomal subunit binds to mRNA • AUG recognition by complementarity • Initiation factors (IFs in prok; eIFs in euk) – – prokaryotic cells require 3 initiation factors – IF1, IF2, & IF3 bind 30S subunit & help it attach to mRNA – IF2 is GTP-binding, required for adding first aminoacyl-tRNA – IF3 may prevent the large (50S) subunit from joining early – IF1 may stop aa-tRNA from entering wrong site on the ribosome Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Prok. Step 2: first aa-tRNA binds to ribosome at AUG • Methionine is always first amino acid incorporated in protein chain – In prokaryotes, formyl group (Nformylmethionine) – Usually removed enzymatically; ~50% of time it remains – 2 distinct methionyl-tRNAs: tRNAiMet ; tRNAMet – Mitochondria & chloroplasts initiate with Nformylmethionine Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.47 Prok. Step 2: first aa-tRNA binds to ribosome at AUG • Aminoacylated initiator tRNA enters the preinitiation complex – binds to both the AUG codon of mRNA & the IF2 initiation factor – IF1 & IF3 are released Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Prok. Step 3: complete initiation complex • Large subunit joins • GTP bound to IF2 is hydrolyzed – GTP hydrolysis drives ribosome conformational shift – Causes release of IF2-GDP Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.47 Eukaryotic Initiation • 10 initiation factors: 25 polypeptides in total – Many (eIF1, eIF1A, eIF3) bind to 40S subunit – prepares the subunit for binding to mRNA • Initiator tRNAfMet also binds the 40S subunit prior to its interaction with mRNA – Initiator tRNA enters subunit in association with eIF2-GTP, which is homologous to the bacterial IF2-GTP – Next, 43S preinitiation complex binds to 5' end of mRNA – methylguanosine cap aids in recognition Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.48 Eukaryotic Initiation • mRNA already bound with initiation factors – eIF4E binds to 5' cap of eukaryotic mRNA – eIF4A moves along 5’end • removes any double-stranded regions – eIF4G links 5' capped end & 3' polyadenylated end • circularizes message (reason not clear) Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.48 Eukaryotic Initiation • 43S complex scans to AUG – Kozak consensus: 5'-CCACCAUGC-3' – Then, eIF2-GTP is hydrolyzed – eIF-GDP & other eIFs are released – large 60S subunit joins the complex to complete initiation Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E The Role of the Ribosome • EM: highly irregular shape with bulges, lobes, channels & bridges • X-ray crystallographic studies (1990’s) – 3 sites for association with tRNAs – the sites receive each tRNA in successive steps – A (aminoacyl) site – tRNA enters here (except tRNAiMet) – P (peptidyl) site - tRNAs donate aa of growing chain – E (exit) site - tRNA leaves from here after losing aa Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.49 The Role of the Ribosome • tRNAs span the gap between the 2 ribosomal subunits – anticodon end of tRNAs contacts the small subunit – plays a key role in decoding the information contained in the mRNA – aa end of tRNAs contact the large subunit, – plays a key role in catalyzing peptide bond formation Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E The Role of the Ribosome • Interface between small & large subunits – spacious cavity lined almost exclusively by RNA – small subunit facing cavity: single ds RNA helix – bring together mRNA & incoming tRNAs – primordial ribosomes made of RNA? (ribozymes) Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E The Role of the Ribosome • Peptidase active site also largely RNA – deep cleft – protects peptide bond from hydrolysis • A tunnel through the large subunit – Begins at the active site – Path of elongating polypeptide Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.49 Elongation • Step 1: Aminoacyl-tRNA selection – after initiation, initiator tRNA is in P site with A site empty – Second aminoacyl-tRNA enters A site • tRNA already bound with EF-Tu (prok) or eEF1a (euk) • EF-Tu associated with GTP • rRNA small subunit verifies proper codon-anticodon • Then, GTP is hydrolyzed • Then, the Tu-GDP complex is released • Regeneration of Tu-GTP from Tu-GDP requires EF-Ts Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Elongation • Step 2: Peptide bond formation – Amino group in A site reacts with carboxyl group in P site – P site tRNA no longer charged (deacylated) – fMet transferred to dipeptide on tRNA in A site – No energy required – Catalyzed by peptidyl transferase of large subunit (ribozyme) Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Elongation • Step 3: Translocation – requires GTP-bound elongation factor & GTP hydrolysis • G (prokaryotes) • eEF2 (eukaryotes) – ribosome moves along mRNA in 5'—>3' direction • tRNA-dipeptide moves to P site • deacylated tRNA moves from P to E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.50 Elongation • Step 4: Releasing the deacylated tRNA – Leaves E site • Elongation cycle takes ~0.05 sec – aa-tRNAs from cytosol probably rate limiting – at least 2 molecules of GTP are hydrolyzed per cycle– • one during aminoacyl-tRNA selection • one during translocation – new aminoacyl-tRNA binds • new peptide bond, etc. – Elongation cycle continues until termination Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Elongation • Reading frame and elongation – Most destructive mutations are frameshift mutations – Some mRNAs have recoding signal • Causes the ribosome to change its reading frame • shift to –1 frame or to +1 frame • common in viral mRNA’s Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Termination • Stop codons (UAA, UGA, UAG) – Selenocysteine in ~12 mammalian proteins • Selenocysteine rare, contains selenium (the 21st amino acid) • Has its own tRNA - tRNASec, but lacks its own AAS • seryl-tRNA synthetase attaches serine to 3' end of tRNASec • After attachment, serine is altered enzymatically • encoded by the stop codon UGA • UGA followed by folded region of the mRNA that binds special EF • EF recruits a tRNASec into the A site rather than termination factor Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Termination • Termination requires release factors – Bacteria have 3 • RF1 recognizes UAA & UAG • RF2 recognizes UGA & UAA • RF3 merely increases activity of other factors – Eukaryotes have 2 (eRF1 & eRF3) • work together & recognize all of the stop codons • Release factors superficially resemble tRNA • Enter A site and interact directly with stop codon • A tripeptide acts as anticodon Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Termination • Termination requires release factors – RF3 (or eRF3) carries a bound GTP that is hydrolyzed later – Then polypeptide is severed from its attachment to tRNA – both deacylated tRNA & the release factor are then released – then ribosome separates from mRNA & dissociates Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Non-sense mutations • Cause a variety of inherited diseases • Partial polypeptides can result • Many such mRNAs destroyed by nonsense-mediated decay (NMD) Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Polyribosomes • Also known as polysomes – Greatly increases protein synthesis rate – Polysomes free in cytosol synthesize soluble proteins – also seen on cytosolic surface of ER • make membrane, secretory and/or organelle proteins • Coupled transcription/translation – Possible only in Prokaryotes – In eukaryotes, mRNA must leave nucleus Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Copyright, ©, 2002, John Wiley & Sons, Inc., Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.51a