Francisella RNA polymerase contains a heterodimer of non-identical α subunits

Abstract

BackgroundAll sequenced genomes of representatives of the Francisella genus contain two rpoA genes, which encode non-identical RNA polymerase (RNAP) subunits, α1 and α2. In all other bacteria studied to date, a dimer of identical α subunits initiates the assembly of the catalytically proficient RNAP core (subunit composition α2ββ'). Based on an observation that both α1 and α2 are incorporated into Francisella RNAP, Charity et al. (2007) previously suggested that up to four different species of RNAP core enzyme might form in the same Francisella cell.ResultsBy in vitro assembly from fully denatured state, we determined that both Francisella α subunits are required for efficient dimerization; no homodimer formation was detected. Bacterial two-hybrid system analysis likewise indicated strong interactions between the α1 and α2 N-terminal domains (NTDs, responsible for dimerization). NTDs of α2 did not interact detectably, while weak interaction between α1 NTDs was observed. This weak homotypic interaction may explain low-level transcription activity observed in in vitro RNAP reconstitution reactions containing Francisella large subunits (β', β) and α1. No activity was observed with RNAP reconstitution reactions containing α2, while robust transcription activity was detected in reactions containing α1 and α2. Phylogenetic analysis based on RpoA resulted in a tree compatible with standard bacterial taxonomy with both Francisella RpoA branches positioned within γ-proteobacteria. The observed phylogeny and analysis of constrained trees are compatible with Francisella lineage-specific rpoA duplication followed by acceleration of evolutionary rate and subfunctionalization.ConclusionsThe results strongly suggest that most Francisella RNAP contains α heterodimer with a minor subfraction possibly containing α1 homodimer. Comparative sequence analysis suggests that this heterodimer is oriented, in a sense that only one monomer, α1, interacts with the β subunit during the α2β RNAP subassembly formation. Most likely the two rpoA copies in Francisella have emerged through a lineage-specific duplication followed by subfunctionalization of interacting paralogs.

Faster evolving Drosophila paralogs lose expression rate and ubiquity and accumulate more non-synonymous SNPs

“…However, because neofunctionalization may also result in sequence evolution asymmetry while subfunctionalization may also result in loss of overall expression or expression uniformity in one of the copies, it is difficult to detect these processes by the genome-wide analysis of sequence and expression divergence as any signal from these processes occurring in a particular gene is going to be indiscernible from similar patterns caused by unfinished pseudogenization alone. A clear-cut demonstration of the signature of neo- or subfunctionalization would therefore require a detailed gene-specific structural and expression analysis [ 17 - 19 , 57 ].…”

Riboswitches for Intracellular Study of Genes Involved in Francisella Pathogenesis

“…In most bacteria, the RNAP holoenzyme contains a dimer of identical α subunits (24). However, in the Francisella genus, the α subunits are encoded by two distinct genes, yielding two unique variants of the RNAP α subunit (25). Given the fundamental role of the α subunit in transcriptional regulation (26), the inefficient transcription of foreign promoters in Francisella may be attributed to the differences in RNAP.…”

Basic mechanism of transcription by RNA polymerase II

“…1A). Variant forms of the core are described for some Eubacteria, such as Francisella (featuring α 1 α 2 heterodimer 6 ), and Bacillus (adding δ and alternate ω subunits 7 ), whereas in Helicobacter, Wolbachia and Wolinella RNAPs β and β′ subunits are fused together 1 . RNAP subunit initially described as γ in Nostoc , Anabaena , and other Cyanobacteria turned out to be a product of a split in the ancestral gene, coding for β′ ortholog 8,9 .…”