2) Mol. Biol. Of the Cell, 4th Ed., pgs. 335-365.The Central Dogma of Molecular Biology: DNAz RNA—— Protein Translation\ noun\ a rendering from one language into another The fundamental problem: How do you get from one language based on 4 nucleotide code to another with a 20 amino acid code? Two Models were proposed: George Gamow: Proposed mechanism mediated by direct templating Francis Crick: Proposed mechanism utilizing adaptorsCrick’s Adaptor Hypothesis Crick’s Predictions Currently Known A: - the RNA of the microsomal particles, regularly arranged, is the mRNA template” — “...whatever went into the template: . : . Codon-Anticodon in a specific way did so by forming hydrogen bond: Interactions — “...the amino acid is carried to the . template by an adaptor...” Aminoacyl-tRNA — “such adaptors... might contain tRNA nucleotides” — “...a separate enzyme would be required to join each adaptor to its Aminoacyl-tRNA Ree own amino acid Synthetase — “...the specificity required to Aa . _ distinguish between ... isoleucine Editing by Aminoacyl and valine would be provided by tRNA synthetases these enzymes” ‘Crick, FHC. 1958. Symp. Soc. Exp. Biol. 12: 138-163.Voet and Voet, Figure 30-10 Z The adaptor hypothesis ; Amino Amino Pewribni tt) Polypeptide acid 1 acid 2 acid 3 Adaptors. Nucleic acid Codon 1 Codon 2 Codon 3‘Voet and Voet, Figure 30-6 Transfer RNA (RNA) vegeVoet and Voet, Figure 30-8 Third reading frame start Potential problem in a triplet genetic code- Second reading s frame start alternate reading frames First reading frame start SUFA*C*U SAC “UPA 10 USA SC DBD BD e210 08 frame | D--G@ tris re21ing frame First reading frameVoet and Voet, Table 30-2] ES, The standard genetic code- 64 codons (including initiation and termination signals) “Nonpolar amino acid residues are ten, basic residues are blue, ‘atidi¢ residues are red, and polar unchanged residues are purple. "AUG forms part of the initiation signal as well as coding for internal Met resigues.oot and Voet, Table 30-4 Rules for anticodon degeneracy 5'-Anticodon 3'-Codon_ Base Base Cc G A U U AorG G Uorc I U,C, or AVestal Vee Fig 39] Tdeniity elements in tRNAS Size of yellow ball is proportional to the fraction of 20 tRNA acceptor types for which the nucleoside observed determinant([Nesrand Vows Figue 30-20tRNA Charging Step 1: Formation of AA-AMP, the Activated Intermediate (AA—AMP) Xen ‘ow - . syle Roo -9 rp? HoN-c-c° OH N oko 4 O-P0— a AminoacylARNA 4 + Syntheuss” 2 sp O-P-O-p-0- HNC o 6 ho ‘OH Pyrophosphate Amino AcidtRNA Charging Step 2: Formation of AA-tRNA Amino acids are attached to the tRNA. By atRNA synthetase via an ester linkage at the 3’ position Voet and Voet, Figure 30-16 | H—C—-R I Nui Aminoacyl+tRNAProkaryatie Eukaryotic Ribosomal Components Assembled Ribacomes 808‘Three dimensional model of the 7 é 30S and 50S ete ribosomal subunits (derived from exyo-EM | ‘And other methods) =a =‘Structure of the 70S ribosome and its functional center| cients Pats etm tae eo BY Lijas 1999) Science 285:2077Large (50S) Subunit Prone pple 238 aRNA-rangs & ite PSSRNA Cop ugundy & white Asie RNA goes Pete RNA- re Nop siden toms thin 18 angst of th peptidy teal: risome ffl ibn Pedy wane emer modes ei bon fami though acon freien 235 RNA? From Cah, Science 209: #78 G000)|3-D Model of the Small (308) Ribosomal Subunit, Key RNA gold re alors pons ) Foot ‘Sesnasn eal, Call 102615 2000)[Voet and Woe, Figure 30-40 | Translation initiates with N. Formylmethionine and tRNA™* Mee EB. coli tRNA/ ¢ Shaded areas illustrate differences between initiator tRNA and other tRNAsVoet and Voet, Figure 30-43 Formation of the 30S initiation complex during the initiation phase of translation pen ca alm ALL Taso tet = eiaipVoet and Voet, Figure 30-43 Formation of the 70S initiation complex during the initiation phase of translation =e ‘_p- A: 3 GDP +P, + 8-8Voet and Voet, Figure 30-44 The elongation cycle of translation ‘vancpoqsianion onsiocot0o7aag + GDP + Fy EG. Pema, aN canoe rr asm 8, 2Voet and Voet, Figure 30-39 Mechanism of peptidyl transferase reaction Paite Asie Peite Aste 1 NH | Amino group of A site Roe amino acid carries out i ed a nucleophilic attack e La on the ester linkage nee ae between the P site (RNA | ——— 1 : o=¢ o=c and the nascent chain, 1 1 resulting in the ne ae transfer of the mi Rage nascent chain to o=c o=c e 1 | ! the A site (RNA So o ou 9 | | ERNA IRNAy. 1) tRNA) tRNA‘aan Peptidyl-tRINA Aminoaeyl-tRNA, Uneharged (RNA. PeptidyltRNAVoet and Voet, Figure 30-48 Mechanism of translation termination Step 1- Recognition of the stop codon by release factors at the Ribosomal A site Nascent polypeptide RF-3 GTPVogt and Voet, Figure 30-48 Mechanism of translation termination Step 2- Hydrolysis of the ester bond between the completed polypeptide chain and the last tRNA. farm 4,0 SH! Polypeptide Mgr Uncharged tRNA Stariisis 4113Voet and Voet, Figure 30-48 Mechanism of translation termination Step 3- Disassembly of The translation complex + GDP +P,Voet and Voet, Figure 30-49 Mechanism of polypeptide chain release During termination, water carries out a nucleophilic attack on the ester linkage between the P site tRNA and the nascent chain, resulting in the release of the completed polypeptide chain.Voet and Voet, Figure 30-46 OH Posttronsiocational Pretransesational GIP. + GDP +P; ‘state ses Or A cQr A sos (a erty ( e 308 el I Binding state: —E PsP PyP AYA The three site tee 8 37” vepticy! ; + teanster “Hybrid States GpP+P; model for niet translation “ @Ay | elongation NN) Pye AP Intermediate Re tit Ei a tia oe moiEnergy requirements for translation Charging: 2 ~P bonds per amino acid Initiation: | ~P bond per polypeptide chain Elongation: 2 ~P bonds per peptide bond (ifn amino acids, n-1 peptide bonds) Termination: 1 ~P bond per polypeptide chain| Voet and Voet, Figure 30-38 Multiple ribosomes can translate an mRNA simultaneously in a polysome complexDifferences in Translational Complexity Between Prokaryotes and EukaryotesVoet and Voet, Figure 30-41 Translation initiation signals Initiation codon. araB GCGAUU- galE AGAGUU- Lael AAACCA- lacZ ACCAUG- QB phage replicase UCUAAG- X174 phage A protein GUUCGU- R17 phage coat protein GCcuuUCcU- Ribosomal $12 GCAACA- Ribosomal L10 GCUUUA- irpE CAAACA- trp leader AAAGCA- 3' end of 165 rRNAMoet ialVbet Era? Base pairing between the Shine Dalgarno sequence and the 3’ end of 16S rRNA facilitates translation initiation fMet -Arg-Ala- R17 phage A protein mRNA -AUUCCUAGGAGGUUUGACCUAUG CGAGCU- CUTTGeT 3! end of 165 rRNA |, UeCuUccA (colicin E3 fragment) 3 yo’ CCACUAG- 5°‘Voet and Voet, Table 30-9 Antil tics That Inhibit Prokaryotic Translation Inhibitor ‘Action Chloramphenicol- Inhibits peptidyl transferase on the prokaryotic large subunit. Erythromycin- Inhibits translocation by the prokaryotic large subunit. Fusidie Acid- Inhibits elongation in prokaryotes by binding to BF-G*GDP in a way that prevents its dissociation from the large subunit. Puromycin- An aminoacyl-tRNA analog that causes premature chain termination in prokaryotes and eukaryotes. Streptomyei Causes mRNA misreading and inhibits chain initiation in prokaryotes, Tetracycline- Inhibits the binding of aminoacyl-RNAs to the prokaryotic small subunit,Voet and Voet, Figure 30-47 Puromycin is a charged tRNA analog aC CH oi al NH, nv ] *y bay —0-P—0-ciiy 6 HH OHStreptomycin Induces Misreading Both str-resistant and str-dependent mutations can be isolated, and both protein $12. Both mutations reduce the level ig and act to compensate for the ‘misreading induced b}Voot and Voet, Figure 30-48 Str inhibits proofreading of codon-anticodon pairing, which leads to the mis-insertion of incorrect aminoacyl-tRNAs into the A site at both sense and nonsense codons. This can lead to mis-incorporation of incorrect amino acids and the suppression of stop codons. Nascent polypeptide it Peptidy-tRN PssiteTranslational control is mediated at the level of translation initiation fMet-Arg-Ala- -AUUCCUAGGAGGUUUGACCUAUG|CGAGCU- T1tittl ee CCACUAG- 5 3 no! The efficiency of translation initiation is determined by: 1) How well the S.D. sequence conforms to the consensus sequence that is complementary to the 3° end of 16S rRNA. 2) The distance between the S.D. sequence and the start codon (a7 base spacer is optimal).Translation Initiation on Polycistronic mRNAs SD- AUG UAA SD - AUG UAA I a Each gene in an operon contains its own independent Shine- Dalgarno sequence, as well as start and stop codons. Thus, under normal conditions, translation initiation of the genes within a polycistronic mRNA occurs independently at each gene.