Lecture 9 Translation.

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1 1 Translation Summary of important events in translation. 2 Translation Reactions involved in peptide bond formation. Lecture 9 3 Genetic code Three types of RNA molecules perform different but complementary roles in protein synthesis (mrna carries three base words termed codons; trna deciphers the code and delivers the specified amino acid; rrna associates with a set of proteins to form ribosomes, structures that function as protein-synthesizing machines). 4 Genetic code Two different frames (genetic code nonoverlapping, commaless). 5 Genetic code Data of filter paper assay (binding of lysyl-trna to ribosome in response to various codons). 6 Genetic code Two-step decoding (1 st step: aminoacyl-trna synthetase couples aa to its corresponding trna; 2 nd step: anticodon in the trna base-pairs with correct codon). 7 trna General structure of trna (cloverleaf pattern, 73 to 93 nt, ~25kD, 7 to 15 unusual bases, 1/2 of nt base paired, CCA terminus/acceptor stem, TΨC loop, extra arm, DHU loop, and anticodon loop are not paired; 5'-G phosphorylated). 8 trna yeast trna structure suggested by Holley in trna Yeast alanine-trna (76 nt, Holley R. did sequencing in 1965). 10 trna A skeletal model of yeast phe-trna (L-shaped). 11 trna Computer-generated 3D model (generalized backbone of all trnas). Helix stacking in trna (4 helices form L-shaped structure). 12 trna Aminoacyl-tRNA (aa coupled to trnas through ester linkages to either 2'- or 3'-OH of the 3'-A). 13 Aminoacylation of trna Two steps reaction (activation of aa to aminoacyl adenylate, transfer it to the A of CCA, Class I to 2'-OH, Class II to 3'-OH). 14 Aminoacylation of trna Two classes of synthetases in E. coli (class I monomeric, class II dimeric, bind ATP in different conformations, class I to 2'-OH, class II to 3'-OH).

2 15 Aminoacylation of trna Two classes of synthetases in E. coli (class I monomeric, class II dimeric, bind ATP in different conformations, class I to 2'-OH, class II to 3'-OH). 16 Aminoacylation of trna Synthetase binding to trna (I and II synthetase recognize different faces of trna). 17 Aminoacylation of trna Microhelix with acceptor stem and a loop (24 nt out of 76 nt) can be recognized by alanyltrna synthetase. 18 Synthetase to trna binding Threonyl-tRNA synthetase and trna Thr (recognizes both the acceptor stem and the anticodon loop, CCA arm extends into the zinc-containing activation site, CGU anticodon H- bonding to enzyme). 19 Synthetase to trna binding Glutaminyl-tRNA synthetase and trna Gln (in addition to acceptor stem and anticodon loop, contact at G10:C25 bp). 20 Synthetase to trna binding Proofreading (fidelity is <1/10 4, Ser-tRNA Thr + Thr-tRNA synthetase immediate hydrolysis of Ser-tRNA Thr, editing site identified 20 A from active center by X-ray and mutagenesis). 21. Synthetase to trna binding Flexible CCA arm (if aa fits into editing site, it is removed). 22 Synthetase to trna binding Active site (large fragment of one subunit of Thr-tRNA synthetase, Zn coordinates with incoming Thr). 23 Ribosome Low resolution EM. 24 Ribosome X-ray structure (23S RNA=yellow, 5S=orange, 16S=green, L proteins=red, S proteins=blue). (30S = S1 S S RNA, 50S = L1 L S and 5S RNA, 70S = 30S + 50S, S20=L26, 2 copies L7, L12). 25 Ribosome Composition of prokaryotic and mammalian ribosome. 26 Ribosome EM pictures of prokaryotic ribosomes. 27 Ribosome CryoEM at 25 A (4300 projections analysed). 28 Ribosomes Stereoview of A, P, E sites (anticodon loops and mrna codons on 30S). 29 Ribosomes

3 16S rrna (2 nd structure showing regions involved in three sites). Tertiary structure of 16S (1542 nt, 5'-part=red, center=green, 3'-part=blue). 30 Ribosomes X-ray structure of 50S at 2.4 A (Proteins that appear on the surface of the large ribosomal subunit shown in gold, rrna in gray; a, front or crown view, b, back view, the 180 o rotated crown view orientation; c, A view from the bottom of the subunit down the polypeptide tunnel exit which lies in the center; The proteins visible in each image are identified in the small images at the lower left of the figure. Figures were generated using RIBBONS proteins identified at lower left). 31 Ribosomes L19 (extended structure to fit into cavities within 23S rrna). 32 Ribosome, more detailed view 30S and 50S (two orientations). 33 Ribosome, more detailed view 70S (EM and two oritentations). 34 Ribosome, more detailed view 30S+50S (the cavern in between). 35 Ribosome, more detailed view Schematic view of ribosome (a, 70S where the large cavern between subunits can accommodate 3 trnas; b, 30 S, c. 50S; trna anticodon end touches 30S and acceptor end touches 50S). 36 Ribosome, more detailed view 2D-analysis (S and L proteins). 37 Ribosome, in action A ribosome's true colors (50S seen from the viewpoint of 30S, proteins in purple, 23S rrna in orange and white, 5S rrna in burgundy and white, A-site trna (green) and P-site trna (red). The peptidyl transfer mechanism catalyzed by RNA. The general base (adenine 2451 in Escherichia coli 23S rrna) is rendered unusually basic by its environment within the folded structure; it could abstract the proton at any of several steps, one of which is shown here). 38 Ribosome, in action The polypeptide exit tunnel (The tunnel surface is shown with backbone atoms of the RNA color coded by domain. Domains I (yellow), II (light blue), III (orange), IV (green), V (light red), 5S ( pink), and proteins are blue. 39 Ribosome, in action A space-filling representation of the large subunit surface at the tunnel exit showing the arrangement of proteins, some of which might play roles in protein secretion. The RNA is in white (bases) and orange (backbone) and the numbered proteins are blue. A modeled polypeptide is exiting the tunnel in red. 40 Ribosome, in action 50S + trnas (A space-filling representations of the 50S with the three trna molecules, in the same relative orientation that they are found in the 70S, docked by model building onto

4 the CCA's bound in the A- and P-site. The proteins are in pink and the rrna in blue. A backbone ribbon representation of the A-, P-, and E-sites are shown in yellow, red, and white, respectively. (A) The whole subunit in a rotated crown view. (B) A closer view shows the numbered proteins close to the trnas. 41 Snapshot of protein synthesis Another view of three sites (anticodons are in 30S, acceptors in 50S). 42 Snapshot of protein synthesis Sie of peptide bond formation. 43 Snapshot of protein synthesis Peptide tunnel (A, P site trna acceptor arms converge at the active site, a tunnel for the growing polypeptide chain to exit). 44 Snapshot of protein synthesis Mechanism (Peptide bond formation starts when peptidyl-trna in the P site and aminoacyltrna in the A site. Translocation occurs via the action of EF-G and deacylated trna is pushed to E site to be freed). 45 Snapshot of protein synthesis Peptide formation (a tetrahedral intermediate is formed). 46 Codon-anticodon recognition Wobble base pairs (1 st and 2 nd base of codon form Watson-Crick pairing, 3 rd base form wobble pairing; i.e. U:G, A:I, C:I, U:I). Alanyl-tRNA has anticodon IGC and it recognizes three codons: GCU, GCC, and GCA. Thus, the degeneracy of the genetic code arises from the wobble in the pairing of the third base of the codon with the first base of the anticodon. 47 Codon-anticodon recognition Condon-anticodon determine the binding (Ala-tRNACys recognizes UGC, a Cys codon, but incorporates Ala). 48 Codon-anticodon recognition Wobble base pairing table. 49 Initiation Two types of Met-tRNA (trnai Met used exclusively for starting protein chains, trna Met delivers Met to internal sites. In bacteria, a formyl group is added to Met-tRNAi Met ). 50 Initiation Formation of N-formylmethionyl-tRNA (the same synthetase attaches met to trnaf and trnam, but transformylase only formylates met-trnaf). 51 Initiation Initiation in prokaryotes (Initiation factors IF1 and IF3 form complex with 30S, IF2-GTP binds fmet-trnaf and mrna and displace IF3 to bring fmet-trnaf and mrna to the 30S and forms the 30S initiation complex, fmet-trnaf at the P site). 52 Initiation Initiation in eukaryotes (eif2 and eif3 have similar counterparts in prokaryotes, eif4 is the cap binding protein, eif1 and 1A scan for initiation codon, eif5 stimulates association between 60S and 48S initiation complex, eif6 binds to 60S to prevent premature associatio, 60S+40S=80S (4200 kd)).

5 53 Initiation Adapter eif4g (it binds to many proteins and help recruiting 40S to the mrna). (vi) Initiation sites (AUG or GUG preceded by purine-rich bases for 16S pairing, in bacteria called Shine-Dalgarno sequence, for eukaryotes the Kozak sequence -ACCAUGG- defines the initiation site). AUG is the only initiation codon. 40S binds to the cap and searches for AUG, a scanning process powered by helicase and ATP. Many initiation factors are involved. For example, eif4e binds to the 7-mG cap. eif4a is a helicase. 54 Initiation sites 55 Initiation The sequence for translation initiation. 56 Elongation Three steps (EF-Tu with GTP brings an aa-trna to A site, peptidyl transferase forms a peptide bond, EF-G with GTP translocates the growing peptide and its mrna codon to the P site). EF-G (molecular mimicry, the N-terminal region is similar to that of EF-Tu). Elongation factor Tu (a G-protein family member, binds to aminoacyl-trna when it is in GTP form and then deliver the aminoacyl-trna to the A site. EF-Tu cannot bind the fmettrnaf, but bind Met-tRNAm. Once the complex is at the A site GTP will be hydrolyzed and EF-Ts will join the complex to release GDP. Peptide bond formation is spontaneous. Once formed, mrna must move by a distance of 3 nt and the new peptidyl-trna must move to the P site. This translocation is mediated by EF-G (aka translocase). 57 Elongation Translocation mechanism (EF-G/GTP binds to 50S EF-Tu site, GTP hydrolysis induces conformational change and drives the stem of EF-G to A site and pushes trnas and mrna by one codon). 58 Elongation EF-Tu (EF-Tu forms complex with trna). 59 Elongation EF-G structure is remarkably similar to that of EF-Tu-tRNA complex. GTP hydrolysis force EF-G to push peptidyl-trna and its associated mrna to move through ribosome by one codon. 60 Initiation and Elongation Eukaryotic protein synthesis and sites of action for initiation and elongation factors. Factors are abbreviated as: 2, eif2; 2B, eif2b; A, eif4a; E, eif4e; 4F, eif4f; G, eif4g, E1, eef1; E2, eef2; S6, ribosomal protein S6; PHAS, phosphorylated, heat- and acidstable protein. Those factors shown in color are targets of the signaling pathways. 61 Termination Stop codons (UAA, UGA, or UAG is recognized by RFs, RF1 for UAA or UAG; RF2 for UAA or UGA; RF3 G protein homologous to EF-Tu, mediates interaction of RF1 or 2 with ribosome). 62 Termination RF brings a H 2 O molecule to hydrolyze the ester bond in peptidyl-trna 63 Termination Ribosome release factor from E. coli. 64 Termination

6 RF1 and RF2 recognize the stop codon (how the tripeptide binds to the stop codon). Release factor: RF1 binds UAA, UAG; RF2 binds UAA, UGA; RF3 mediates the interaction between RF1 or RF2 with the ribosome. 65 Termination Assay of releasing factors (Nirenberg's assay, using AUG polymer and [3H]fMet-tRNAf and ribosome). 66 Nobel Prize Winners Robert Holley, Gobind Khorana, Marshall Nirenberg