, 1995). IF3 is known to promote subunit dissociation and to discriminate other tRNAs from the initiator tRNA; mutations at positions 787, 791, 792, and 795

result in a decrease in subunit association (Tapprich et al., 1989; Santer et al., 1990; Lee et al., 1997). The decreased binding affinity of IF3 to 30S when residues at 791 and 792 are altered (Tapprich et al., 1989; Santer et al., 1990) indicates that this is not due to a lack of antiassociation activity, but rather a loss of ability of the initiator tRNA to select or bind to the P-site, resulting in a decrease in subunit association. The footprinting and structural data suggest that Crizotinib order these residues are heavily involved in tRNA selection in the P-site and it is likely that these sites may form a structural motif that interacts with IF3 and recruits the initiator 5FU tRNA to the P-site. The P-site-specific antibiotics edeine, pactamycin, and kasugamycin, which stabilize

the P-site-bound tRNA, show a footprint in the 790 loop; this also supports the involvement of the 790 loop in the recruitment of initiator tRNA to the P-site. Here, we describe how we functionally analyzed the role of G791 in protein synthesis. This residue has been shown to be an invariant and essential residue for ribosome function (Lee et al., 1997; Song et al., 2007). To investigate the functional role played by G791 during the process of protein synthesis, we adopted a novel genetic approach (Lee et al., 1997, 2001; Kim et al., 2009) by introducing a base substitution at position 791 and then selecting multicopy suppressors that partially restored the protein synthesis ability of the mutant ribosomes. We identified IF1 as a multicopy suppressor of the mutant ribosome bearing a G to U substitution at position 791. Based on functional analyses of the effects of IF1 on the mutant ribosome, we suggest the involvement Tau-protein kinase of IF1 in the restoration of the P-site that

was perturbed by a nucleotide substitution at position 791. All plasmids were maintained and expressed in E. coli DH5α (Hanahan, 1983). Cultures were grown in Luria–Bertani (LB) medium (Luria & Burrous, 1957) or LB medium containing 100 μg mL−1 ampicillin (LB–Amp100). To induce the synthesis of plasmid-derived rRNA from the lacUV5 promoter, IPTG was added to a final concentration of 1 mM and 0.1% of l-arabinose was used to induce the synthesis of initiation factors from the BAD promoter. Plasmids pRNA122 and pRNA16 ST were described previously (Lee et al., 1997, 2001). The construction of pKAN3, pKAN4, and pKAN6 was described previously (Tamura et al., 2006). To construct pRNA122-U791U1192 and pRNA122-G770U1192, the XbaI and SexAI fragment from pRNA122-U1192 (Lee et al., 1996) was subcloned into the same sites in pRNA122-U791 (Song et al., 2007) and pRNA122-G770 (Kim et al., 2007).