BIOINFORMATIC ANALYSIS OF ACCELERATED SEQUENCE EVOLUTION OF SPECIFIC EUBACTERIA

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The two specific aims of this effort are to identify and quantitate the level of sequence variation among a set of four rapidly evolving RNA polymerase (RNAP) genes that constitute targets for antibiotic therapy, and to identify highly conserved structural motifs in the four RNAP core complex subunit proteins that could be suitable candidates for new antibiotic development. RNA polymerase subunits normally evolve at comparatively slow rates, but in one family of flowering plants, the Geraniaceae, the cyanobacterial-type RNAP in chloroplasts are evolving at a much higher rate. Nucleotide and deduced-amino acid sequence analysis of full length and partial (ca. 1 kb) genomic sequences of 8 a (rpoA), 19 beta (rpoB), 16 beta¿ (rpoC1) and one beta¿ (rpoC2) subunit genes from several species of the genus Pelargonium revealed a gradient of sequence evolution, with rpoA (encoding an assembly or scaffolding protein) evolving most rapidly, followed by rpoC1 , rpoC2, and rpoB (encoding subunits involved in catalysis and contact between the RNAP and the DNA template). These genes appear to be the most rapidly evolving RNAP genes yet sequenced. Remarkably, there is as much sequence divergence between rpo genes in this genus of plants (representing about 20 million years of evolution) as between rpo genes from distantly-related groups of eubacteria representing at least 2 billion years of evolution. As such, the rpo genes from these organisms might represent a natural experiment in saturation mutagenesis, and may help define immutable portions of the RNAP structure. Several core motifs in all genes were found to be highly conserved, and may represent suitable targets for ¿smart drug¿ design against the eubacterial RNAP homologues.