Dominant lethal mutations in a conserved loop in 16S rRNA.

Abstract

The 530 stem-loop region in 16S rRNA is among the most phylogenetically conserved structural elements in all rRNAs, yet its role in protein synthesis remains mysterious. G-530 is protected from kethoxal attack when tRNA, or its 15-nucleotide anticodon stem-loop fragment, is bound to the ribosomal A site. Based on presently available evidence, however, this region is believed to be too remote from the decoding site for this protection to be the result of direct contact. In this study, we use a conditional rRNA expression system to demonstrate that plasmid-encoded 16S rRNA genes carrying A, C, and T point mutations at position G-530 confer a dominant lethal phenotype when expressed in Escherichia coli. Analysis of the distribution of plasmid-encoded 16S rRNA in ribosomal particles, following induction of the A-530 mutation, shows that mutant rRNA is present both in 30S subunits and in 70S ribosomes. Little mutant rRNA is found in polyribosomes, however, indicating that the mutant ribosomes are severely impaired at the stage of polysome formation and/or stability. Detailed chemical probing of mutant ribosomal particles reveals no evidence of structural perturbation within the 16S rRNA. Taken together, these results argue for the direct participation of G-530 in ribosomal function and, furthermore, suggest that the dominant lethal phenotype caused by these mutations is due primarily to the mutant ribosomes blocking a crucial step in protein synthesis after translational initiation.