Distinctive contributions of the ribosomal P-site elements m ²G966, m ⁵C967 and the C-terminal tail of the S9 protein in the fidelity of initiation of translation in Escherichia coli

Abstract

The accuracy of pairing of the anticodon of the initiator tRNA (tRNA ^fMet) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m ²G966 (methylated by RsmD), m ⁵C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNA ^fMet. We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA (tRNA ^fMet with CAU anticodon); CAC and CAU with tRNA ; UAG with tRNA ; UAC with tRNA ; and AUC with tRNA using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold). Also, the S9 tail suppressed initiation with tRNA lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.