Study of common Vibrio 5S ribosomal RNA sequence variants and development of a new methodology to identify the phenotypes of Vibrio proteolyticus 5S rRNA variants

Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. In principle, a comprehensive understanding of the evolutionary process at the molecular level would make it possible to enumerate which specific RNA sequences could be successfully chosen and which could not. Bacterial 5S rRNA is used as a model system to attempt to develop the insight needed to make such predictions. One such principle is the intuitive notion that specific sequence changes that are seen frequently in aligned datasets of naturally occurring 5S rRNAs might be widely accepted in many other 5S rRNA sequences.; To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 single changes and 4 base-pair changes) were identified in this way which, by the hypothesis, might be expected to be successfully incorporated in many of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence and evaluated for their ability to, function as a valid 5S rRNA in an Escherichia coli cellular context. The results demonstrate that changes which are known to have been successfully incorporated several times in a local region of the structure space are very likely to be compatible with essentially all the sequence in the local region.; In addition, the work also demonstrates that the system, composed of an E. coli strain whose five copies of its eight genomic 5S rRNA genes were deleted and an active plasmid-borne 5S rRNA gene that is able to compensate the slow growth rate of this knockout strain, can be exploited in conjunction with random mutagenesis to rapidly collect 5S rRNA mutation data in the service of rRNA phylogeny study.