Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 – 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 (N- formylmethionine) –Usually removed enzymatically; ~50% of time it remains –2 distinct methionyl-tRNAs: tRNA i Met ; tRNA Met –Mitochondria & chloroplasts initiate with N- formylmethionine

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E Figure 11.47

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 Figure 11.47

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 tRNA f Met 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 Figure 11.48

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 Figure 11.48

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 tRNA i Met ) –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 Figure 11.49

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 Figure 11.49

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 eEF1  (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 Figure 11.50

Copyright, ©, 2002, John Wiley & Sons, Inc.,Karp/CELL & MOLECULAR BIOLOGY 3E 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 21 st amino acid) Has its own tRNA - tRNA Sec, but lacks its own AAS seryl-tRNA synthetase attaches serine to 3' end of tRNA Sec 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 tRNA Sec 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 Figure 11.51a