Role of the Ribosomal P-Site Elements of m2G966, m5C967, and the S9 C-Terminal Tail in Maintenance of the Reading Frame during Translational Elongation in Escherichia coli

The ribosomal P-site hosts the peptidyl-tRNAs during translation elongation.Which P-site elements support these tRNA species to maintain codon-anticodon interactions has remained unclear. We investigated the effects of P-site features of methylations of G966, C967, and the conserved C-terminal tail sequence of Ser, Lys, and Arg (SKR) of the S9 ribosomal protein in maintenance of the translational reading frame of an mRNA.We generated Escherichia coli strains deleted for the SKR sequence in S9 ribosomal protein, RsmB (which methylates C967), and RsmD (which methylates G966) and used them to translate LacZ from its1 and 1 out-of-frame constructs. We show that the S9 SKR tail prevents both the1 and1 frameshifts and plays a general role in holding the P-site tRNA/peptidyl-tRNA in place. In contrast, the G966 and C967methylations did not make a direct contribu-tion to the maintenance of the translational frame of anmRNA. However, deletion of rsmB in the S93 background caused sig-nificantly increased1 frameshifting at 37°C. Interestingly, the effects of the deficiency of C967 methylation were annulled when the E. coli strain was grown at 30°C, supporting its context-dependent role. Ribosomes use elegant mechanisms to ensure that only the cor-rect aminoacyl-tRNAs with anticodons cognate to the codon triplets displayed in the A-site are selected and accommodated to extend the polypeptide chain (1). However, for the accuracy of protein synthesis, maintenance of the codon-anticodon interac-tions during translocation and subsequently during the residenc