Translation Initiation Complex Formation with 30 S Ribosomal Particles Mutated at Conserved Positions in the 3′-Minor Domain of 16 S RNA

Abstract

Escherichia coli 30 S ribosomal subunits containing in vitro (phage T7 RNA polymerase-generated) 16 S rRNA, both wild-type and mutant, were examined by toeprinting. These synthetic particles were used to compare the effects of the absence of base modification and of specific nucleotide substitutions in conserved sequence regions of the RNA on the assembly of mRNA, tRNAs and 30 S particles into a translational initiation complex. Initiation factor-3-dependent selection of tRNA Met f from a mixture of tRNA Met f and tRNA Phe occurred with all particles, although 20 times less initiation factor-3 was needed for the synthetic particles, including the mutants. Whereas isolated 30 S particles or those reconstituted with isolated RNA did not distinguish between tRNA Met f and tRNA Phe for ternary complex formation in the absence of initiation factor-3 (intrinsic selection ability), the synthetic particles preferred tRNA Met f. The difference between the natural and synthetic particles appears to be due to the absence of certain base modifications, but not m 2 6A, in the synthetic RNA. Synthetic particles containing the mutation U1512C, which converts the universal U·G pair to C·G enhanced both tRNA Met f binding and selectivity, although other mutations at that site, namely U1512G, G1523A and U1512C/C1524U, had no such effect. Mutants U1498G and G1401C/C1501G, both located in a highly conserved single-stranded region of the 3′-minor domain, also enhanced tRNA Met f selectivity, in this case by reducing complex formation with elongator tRNA. Complex formation between elongator tRNA and the G1401C/C1501G mutant was reduced to almost undetectable levels. The results also indicated that the association rate for initiation complex formation for G1401C/C1501G was considerably lower than for the wild-type sequence. This result had not been detected by standard tRNA-30 S binding assays. Overall, the data suggest that (some of) the 16 S rRNA base modifications as well as the tertiary structure around the decoding site act to desensitize the intrinsic selection ability of the ribosome for tRNA Met f.