A-site , ribosome

During the normal process of termination of translation, stop codons are recognized by protein release factors (RF). Although the details of the process are not fully understood, it is believed that when a termination codon reaches the ribosomal A-site, the RF associates with the ribosomal-mRNA complex, inducing the peptidyl-transferase center to hydrolyze the ester bond of the pepti-... 

Figure 14.8 Four self-complementary RNA fragments containing a tandem array of two E. coli 16S ribosomal A site modules.

Erythromycin and the other macrolides prevent release of tRNAs from the ribosomal A site after peptide bond formation. 

Puromycin, made by the mold Streptomyces al-boniger, is one of the best-understood inhibitory antibiotics. Its structure is very similar to the 3 end of an aminoacyl-tRNA, enabling it to bind to the ribosomal A site and participate in peptide bond formation, producing peptidyl-puromycin (Fig. 27-31). However, because puromycin resembles only the 3 end of the tRNA, it does not engage in translocation and dissociates from the ribosome shortly after it is linked to the carboxyl terminus of the peptide. This prematurely terminates polypeptide synthesis. 

Figure 29-16 Schematic diagram of the tmRNA structure and its function in the rescue of ribosomes stalled at the end of a messenger RNA that has been broken and has lost its inframe termination codon. After it binds into the ribosomal A site the tmRNA, which has been charged with alanine, undergoes the peptidyltransferase reaction and translocation to the P site. Then it lays down its mRNA-like coding sequence, which is used by the ribosome to add ten more amino acids to form the 11-residue C-terminal degradation signal A ANDENYALAA. This induces rapid degradation of the imperfect protein that has been formed.4363...

Fig. 4. Role of the stop codon and lOSa-RNA in E. coli translation. (A) When a stop codon is encountered, a complex of two release factors, RF-1 and RF-3 or RF-2 and RF-3, binds instead of the tRNA. The release factor RF-1 recognizes the stop codons UAA and UAG, while RF-2 recognizes UAA and UGA. The binding of the release factor complex results in hydrolysis of the peptidyl-tRNA and release of the peptide. (B) The role of lOSa-RNA. If truncated mRNA without a stop codon is translated in E. coli, the ribosome stops at the end of the mRNA. lOSa-RNA can then bind to the ribosomal A site and lOSa-RNA can act as tRNA by transferring an alanine to the truncated protein. Subsequently, lOSa-RNA acts as mRNA and a peptide tag with the indicated sequence is added to the truncated protein. lOSa-RNA encodes a stop codon and therefore the protein is released and then degraded by proteases specifically recognizing this C-terminal tag.

Fig. 5. Protein-RNA fusion. Covalent RNA-protein complexes can be generated by ligation of a DNA-puromycin linker to the in vitro transcribed mRNA. During in vitro translation, the ribosome stalls at the RNA-DNA junction. Puromycin can then bind to the ribosomal A-site. The nascent polypeptide is thereby transferred to puromycin. The resulting covalendy linked complex of mRNA, puromycin, and peptide can be used for selection experiments. After affinity selection, the bound complexes are eluted and subsequently the mRNA is amplified by RT-PCR.

Synthesis of fMet occurs on its tRNA. The tRNA is charged by MetRS with methionine, which then is formylated by methionyl-tRNA formyltransferase (MTF) in the presence of the formyl donor NlO-formyltetrahydrofolate (27). Initiation factor IF2 sequesters the fMet-tRNA and excludes it from the ribosomal A site. Instead, IF2-bound fMet-tRNA is transported to the ribosomal P site. EF-Tu GTP can bind methionyl-tRNA , although at a lower affinity than... 

Figure 7 General chemical scheme for peptidyl transfer by the ribosome. The scheme shows nucleophilic attack by the amine group of the amino acid (with side chain R2) esterified to the tRNA in the ribosomal A site (right) on the ester linkage of the aminoacyl tRNA (with amino acid chain Rl). The 2 OH of the peptidyl tRNA participates in the reaction and seems to transfer a proton to the 3 0 leaving group, either directly or potentially via a solvent bridge. Adapted from Reference 76.

Foloppe, N., Chen, I.J., Davis, B., Hold, A., Morley, D., Howes, R. A structure-based strategy to identify new molecular scaffolds targeting the bacterial ribosomal A-site. Bioorg. Med. Chem. 

Figure 4 Puromycin (A) is an antibiotic analog of tyrosyl tRNAthat differs by the groups in red. Once joined to a DNA linker at the 3 -end and a psoralen (B) at the 5 -end it is ready to covalently link the mRNA to nascent peptide. Photoactivation of the psoralen (green), cross-links the 5 -end of the linker and the mRNA. Once the ribosome stalls at the end of the mRNA, the puramycin enters the ribosome A-site and is transferred to the end of the newly formed protein. However, the ribosome is unable to hydrolyze the ribamine (red) amide bond thus forming a permanent link between mRNA and the encoded protein (C).

Mansouri, S., NouroUahzadeh, E. and Hudak, K.A. (2006) Pokeweed antiviral protein depurinates the sarcin/ricin loop of the rRNA prior to binding of aminoacyl-tRNA to the ribosomal A-site. RNA, 12, 1683-1692. 

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