MCB L8
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| Puromycin | Puromycin enters the vacant A site without the involvement of EF-Tu. It is a substrate for peptidyl transferase through its amino group forming peptidyl puromycin. Peptidyl puromycin is not anchored to the A site, and dissociates from the ribosome, resulting in premature chain termination. About 50% of ribosomes react with puromycin – those with vacant A sites. |
| Puromycin | Puromycin enters the vacant A site without the involvement of EF-Tu. It is a substrate for peptidyl transferase through its amino group forming peptidyl puromycin. Peptidyl puromycin is not anchored to the A site, and dissociates from the ribosome, resulting in premature chain termination. About 50% of ribosomes react with puromycin – those with vacant A sites. |
| Translation – A recap | Converts the genetic code (DNA) into protein sequence. Translation template = mRNA The ribosome catalyses polypeptide chain formation. The joining of aminoacyl residues by the ribosome to form a polypeptide. The primary sequence of the polypeptide is specified by the triplet codons in mRNA. |
| Puromycin | Puromycin enters the vacant A site without the involvement of EF-Tu. It is a substrate for peptidyl transferase through its amino group forming peptidyl puromycin. Peptidyl puromycin is not anchored to the A site, and dissociates from the ribosome, resulting in premature chain termination. About 50% of ribosomes react with puromycin – those with vacant A sites. |
| Secondary structure map of bacterial 16S rRNA | RRNAs from several hundred species have now been sequenced. All show same defined structures. Conserved regions of rRNA, mutations in these are often lethal. Variable regions, can tolerate mutations. Base –paired stems (A-form helix) common, compensating base changes between species. |
| TRNA binding in the Ribosome | Ribosomes have three binding sites for tRNA that span the 30S and 50S subunits. A = Acceptor site of codon-directed binding of incoming aa tRNA P = Peptidyl site; holds codon directed peptidyl tRNA E = Exit site; not associated with mRNA |
| 23S rRNA in the peptidyl transferase reaction | N3 of A2486 accepts a proton from the amino group of the aminoacyl tRNA in the A site. This enhances the negative charge of the amino group, allowing it to attack the bond between the peptide and tRNA in the P site. The N3 of A2486 H-bonds to the oxyanion in the tetrahedral intermediate stabilising it and accelerating the reaction. The 3’-OH of the tRNA in the P site accepts the proton from A2486, completing the reaction. – Ribozyme activity |
| TRNA structure (cloverleaf shape) | TRNAs are named according to the amino acid with which they become charged. Isoaccepting tRNAs: several different tRNAs become charged with the same amino acid. The aminoacyl-tRNA synthetases show specificity for the tRNAs they charge, and the correct interaction is with cognate tRNAs. tRNA contains a number of modified nucleosides. |
| Cloverleaf model for tRNA | The D loop contains 8-12 unpaired bases and 2-3 dihydrouricil residues. The Anticodon loop of 7 unpaired bases, Contains the three anticodon bases. The anticodon is flanked on its 5’ side by U, and on 3’ side by an alkylated purine. The TψC loop -7 unpaired bases 5’ TψCG 3’ present. Involved in binding to the ribosome ‘A’ site |
| Yeast tRNAPhe | CCA-3’ is located ~70 Å away from the anticodon. The DHU and TψC loops form the corner of the “L”. Most bases are stacked, a major factor in stabilisation. The 3 anticodon bases and the –CCA-3’ bases are unstacked, allowing interaction with the codon base, or the aminoacyl-tRNA synthetase. |
| Shared reactions of tRNAs | 1. Interaction with elongation factor (except initiator tRNA) 2. Binding to the ribosome ‘A’ site 3. CCA terminal addition 4. ‘Invariant’ modifications to bases |
| Unique reactions of tRNAs | 1. Amino acylation by synthetases 2. Codon-Anticodon interaction 3. Recognition of initiator (fmet tRNA) by initiation factor 4. Recognition of initiator by transformylase 5. Unique base modifications. |
| Aminoacyl-tRNA synthetases | Each synthetase recognises a single amino acid and all of the tRNAs that should be charged by it – called COGNATE tRNAs. Cognate tRNAs may differ in their anticodons and in other parts of the molecule. Class 1: contacts tRNA at minor groove. Class 2: Contacts tRNA at major groove. |
| TRNA charging by aminoacyl-tRNA synthetases | This is of equal importance to the accuracy of protein synthesis as the selection of the amino acid. The features of individual tRNAs which are recognised by their cognate synthetase are called IDENTITY ELEMENTS. |
| Proofreading by aminoacyl-tRNA synthetases | Proofreading can occur at two stages: 1. By hydrolysis of the ester bond of an “incorrect” aminoacyl-AMP intermediate triggered by the binding of the cognate tRNA, 2. Hydrolysis of the ester bond of a “miss-matched” aminoacyl-tRNA. Most aminoacyl-tRNA synthetases possess editing (hydrolytic) sites in addition to the acylation site. Usually, the acylation site rejects an amino acid larger than the cognate aa, due to insufficient room. The editing site hydrolyses aminoacyltRNAs which are smaller than the cognate aa. |
| Editing of Aminoacyl-tRNA | The flexible CCA arm of an aminoacyl-tRNA can move the amino acid between the activation site and the editing site. If the amino acid fits well into the editing site, the amino acid is removed by hydrolysis. |
| Antibiotics | Several antibiotics that inhibit the peptidyl transferase activity of ribosomes bind directly to the peptidyl transferase centre of the 23S rRNA. Single base mutations in the antibiotic binding sites results in antibiotic resistance. Naked 23S rRNA retains peptidyl transferase activity. Colicin E3 is a protein that inhibits growth of bacterial cells that lack the Col plasmid. It acts by cleaving 16S rRNA 50 nucleotides from its 3’ end. The cleaved fragment contains the a stretch of ~6 nucleotides complementary to the ribosome binding site of the mRNA. Hence ribosomes cannot initiate protein synthesis. The ribotoxins ricin and α-sarcin both act on a conserved stem-loop structure in 28S rRNA involved in binding aminoacyl tRNA and inhibit protein synthesis. Ricin removes a single adenine residue, α-sarcin causes a single cleavage in the sugar-phosphate backbone. |
| Diphtheria toxin | Diphtheria toxin is produced by pathogenic strains of Corynebacterium diphtheriae. It acts catalytically on elongation factor 2 (EF-2), the eukaryotic homologue of EF-G. All EF-2s contain a postranslationally modified histidine residue called diphthamide. The toxin transfers ADP ribose from NAD+ to the imidazole ring. This completely inhibits translocation. |
| Streptomycin | Streptomycin is a highly basic trisaccharide. Streptomycin binds to the 16S rRNA of the 30S subunit of the bacterial ribosome, which interferes with the binding of formylmethionyl-tRNA to ribosomes and thereby prevents the correct initiation of protein synthesis. |
| Puromycin | Puromycin enters the vacant A site without the involvement of EF-Tu. It is a substrate for peptidyl transferase through its amino group forming peptidyl puromycin. Peptidyl puromycin is not anchored to the A site, and dissociates from the ribosome, resulting in premature chain termination. About 50% of ribosomes react with puromycin – those with vacant A sites. |