Initiator tRNAs are special in their direct binding to the ribosomal P-site due to the hallmark occurrence of the three consecutive G-C base pairs (3GC pairs) in their anticodon stems. How the 3GC pairs function in this role, has remained unsolved. We show that mutations in either the mRNA or 16S rRNA leading to extended interaction between the Shine-Dalgarno (SD) and anti-SD sequences compensate for the vital need of the 3GC pairs in tRNA fMet for its function in Escherichia coli. In vivo, the 3GC mutant tRNA fMet occurred less abundantly in 70S ribosomes but normally on 30S subunits. However, the extended SD:anti-SD interaction increased its occurrence in 70S ribosomes. We propose that the 3GC pairs play a critical role in tRNA fMet retention in ribosome during the conformational changes that mark the transition of 30S preinitiation complex into elongation competent 70S complex. Furthermore, treating cells with kasugamycin, decreasing ribosome recycling factor (RRF) activity or increasing initiation factor 2 (IF2) levels enhanced initiation with the 3GC mutant tRNA fMet , suggesting that the 70S mode of initiation is less dependent on the 3GC pairs in tRNA fMet . 3GC base pairsnitiation of protein synthesis, assisted by initiation factors, is a highly regulated process in all life forms. In eubacteria, binding of both the initiator tRNA (tRNA fMet ) and mRNA to the small ribosomal subunit (30S) leads to the formation of a 30S preinitiation complex primarily with the help of the three initiation factors (IF1, IF2, and IF3). This stage is then followed by docking of the large ribosomal subunit (50S) to ultimately produce an elongation competent 70S complex upon the departure of all of the three initiation factors (1). The localization of mRNA onto the 30S subunit is facilitated by a purine rich sequence (Shine-Dalgarno, SD sequence), located upstream of the start codon, by its pairing with a complementary sequence (anti-SD sequence) at the 3′-terminus of the 16S rRNA (1, 2). The tRNA fMet binding to ribosome is aided by the unique features it possesses. A virtually universal feature of all of the initiator tRNAs, the presence of three consecutive G-C base pairs (G 29 G 30 G 31 :C 39 C 40 C 41 , referred to as 3GC pairs) in the anticodon stem is known to be important for their preferential binding in the ribosomal P-site (3, 4). Mutations in the 3GC pairs result in poor binding of tRNA fMet to the ribosomal P-site (3, 4).However, the mechanism of how the 3GC pairs help in binding of tRNA fMet into the ribosome has remained unclear. In the crystal structure of the initiator tRNA bound 70S ribosome, it was seen that the universally conserved A1339 and G1338 residues of 16S rRNA establish A-minor interactions with the first GC (G 29 -C 41 ) and middle GC (G 30 -C 40 ) pairs, respectively (5). Although these interactions were seen as suboptimal, the IF3 induced conformational changes may optimize these interactions (6). Another study showed that a major role of IF3 is to uniformly increase the rate of dissociation of...