Role of conserved features of initiator tRNA and ribosome heterogeneity in translation initiation in Escherichia coli

Abstract

Translation is one of the fundamental and core cellular processes catalysed by a ribonucleoprotein complex called ribosome. The process involves four major steps: initiation, elongation, termination and recycling. Initiation is the rate limiting step in translation, which determines the correct reading frame in an mRNA. Initiation occurs by formation of an initiation complex comprising 30S ribosomal subunit, mRNA, initiator tRNA, and initiation factors. The recruitment of 30S ribosomal subunit to the mRNA is aided by interaction between conserved RNA sequence called anti-Shine Dalgarno (aSD) in 16S rRNA and the Shine Dalgarno (SD) sequence in an mRNA present upstream of the start codon. Initiator tRNA (i-tRNA) is recruited directly to the ribosomal P-site with the help of initiation factor 2 (IF2). On the other hand all elongator tRNAs are brought to the ribosomal A-site by elongation factor Tu (EF-Tu). The P-site binding of i-tRNA has been attributed to two of its unique features. First, the CxA mismatch (in Escherichia coli) at 1x72 position, which is a major determinant for formylation of amino acid attached to i-tRNA. Formylation increases the affinity of i-tRNA to IF2 and prevents its binding to EF-Tu. Second, the presence of 3 consecutive GC base pairs (3GC pairs) in the anticodon stem of i-tRNA which is conserved in all the three domains of life. The i-tRNA lacking this feature is incompetent in initiation. However, the exact mechanism of how these two conserved features play a role in the fidelity of translation initiation is still not fully understood. The work described in the thesis attempts to uncover the finer details of the fidelity at the step of initiation of protein synthesis using molecular genetics and biochemical tools