Origins and Molecular Evolution of the NusG Paralog RfaH

Abstract

The only universally conserved family of transcription factors comprises housekeeping regulators and their specialized paralogs, represented by well-studied NusG and RfaH. Despite their ubiquity, little information is available on the evolutionary origins, functions, and gene targets of the NusG family members. We built a hidden Markov model profile of RfaH and identified its homologs in sequenced genomes. While NusG is widespread among bacterial phyla and coresides with genes encoding RNA polymerase and ribosome in all except extremely reduced genomes, RfaH is mostly limited to Proteobacteria and lacks common gene neighbors. RfaH activates only a few xenogeneic operons that are otherwise silenced by NusG and Rho. Phylogenetic reconstructions reveal extensive duplications and horizontal transfer of rfaH genes, including those borne by plasmids, and the molecular evolution pathway of RfaH, from “early” exclusion of the Rho terminator and tightened RNA polymerase binding to “late” interactions with the ops DNA element and autoinhibition, which together define the RfaH regulon. Remarkably, NusG is not only ubiquitous in Bacteria but also common in plants, where it likely modulates the transcription of plastid genes. IMPORTANCE In all domains of life, NusG-like proteins make contacts similar to those of RNA polymerase and promote pause-free transcription yet may play different roles, defined by their divergent interactions with nucleic acids and accessory proteins, in the same cell. This duality is illustrated by Escherichia coli NusG and RfaH, which silence and activate xenogenes, respectively. We combined sequence analysis and recent functional and structural insights to envision the evolutionary transformation of NusG, a core regulator that we show is present in all cells using bacterial RNA polymerase, into a virulence factor, RfaH. Our results suggest a stepwise conversion of a NusG duplicate copy into a sequence-specific regulator which excludes NusG from its targets but does not compromise the regulation of housekeeping genes. We find that gene duplication and lateral transfer give rise to a surprising diversity within the only ubiquitous family of transcription factors.

NusG, an Ancient Yet Rapidly Evolving Transcription Factor

“…Consequently, NusG/Spt5 proteins compete with the cognate initiation factors for binding to RNAP, reducing pausing during transcription elongation and potentially facilitating promoter escape ( Sevostyanova et al, 2008 ; Grohmann et al, 2011 ). Along with the housekeeping NusG present in every free-living cell, many species also contain NusG paralogs ( Wang B. et al, 2020 ) that regulate expression of selected genes in a sequence- or condition-specific fashion.…”

“…The universal conservation of the NusG structure and its binding site on the RNAP, as well as perceived common principles of gene expression control in bacteria, justified using the E. coli NusG as a paradigm. However, early and recent data suggest that, beyond occupying the same site on RNAP, even housekeeping NusGs, which are encoded within the conserved genomic locus, secE - nusG - rplK - rplA in evolutionary distant bacterial phyla ( Wang B. et al, 2020 ), have relatively few common features. Comparison of NusG proteins from E. coli and B. subtilis , the best studied Gram-negative and Gram-positive model bacteria that grow very similarly in the lab, illustrates these differences.…”

“…In addition to housekeeping NusG/Spt5 proteins present in all free-living cells, many genomes encode one or more NusG paralogs ( Wang B. et al, 2020 ). While the primary sequences of these proteins are very diverse, the high conservation of residues that comprise the high-affinity RNAP binding site suggests that all of them bind to the TEC similarly.…”

“…located in long horizontally acquired operons ( Figure 10 ), which are silenced by Rho. RfaH abolishes Rho-dependent termination ( Sevostyanova et al, 2011 ) and the ability to bind Rho appears to be lost early in RfaH evolution ( Wang B. et al, 2020 ). RfaH elicits dramatic, 50 + fold activation of gene expression in vivo , an effect that was initially assumed to be mediated by its direct antitermination effects on RNAP ( Artsimovitch and Landick, 2002 ).…”

“…While functional data are available for just a few NusG SP , recent bioinformatics analysis suggests that these proteins fall into eight different clusters, which differ in their primary sequence signatures as well as regulatory contexts. Some NusG SP , such as RfaH, form one group and are encoded by single cistrons, whereas others (e.g., loaP , taA , and upxY ) are adjacent to their target operons ( Wang B. et al, 2020 ). ActX, which is closely related to RfaH ( Figure 11 ), is encoded within pilus biosynthesis operons on antibiotic-resistant plasmids in E. coli and Klebsiella pneumoniae ( Núñez et al, 1997 ), but its regulatory function remains unknown.…”