No abstract

The transcriptional enhancer protein RfaH positively regulates production of virulence factors in Escherichia coli and Salmonella enterica serovar Typhimurium via a cis element, ops. Genes coding for RfaH orthologs were identified in conceptually translated genomes of bacterial pathogens, including Vibrio and Yersinia spp. We cloned the rfaH genes from Vibrio cholerae, Yersinia enterocolitica, S. enterica serovar Typhimurium, and Klebsiella pneumoniae into E. coli expression vectors. Purified RfaH orthologs, including the most divergent one from V. cholerae, were readily recruited to the E. coli transcription elongation complex. Postrecruitment stimulation of transcript elongation appeared to vary with the degree of similarity to E. coli RfaH. V. cholerae RfaH was particularly defective in reducing downstream pausing and termination; this defect was substantially alleviated by an increase in its concentration. When overexpressed episomally, all of the rfaH genes complemented the disruption of the chromosomal copy of the E. coli gene. Thus, despite the apparently accelerated divergent evolution of the RfaH proteins, the mechanism of their action is conserved well enough to make them transcriptionally active in the E. coli system.Bacterial RNA polymerase (RNAP) carries out transcription of the entire genome and is one of the most regulated cellular enzymes. During synthesis of a single RNA molecule, RNAP transitions through a series of mechanistically and conformationally distinct stages: recruitment, initiation, elongation, and termination (37). At each of these stages RNAP activity is regulated by a host of cis-and trans-acting signals, although historically it was the regulation of entry into the catalytic cycle (recruitment and initiation) that received the most attention. In the cell, RNAP cannot splice individual RNA molecules nor can it initiate synthesis of each RNA chain de novo in the absence of a promoter, and thus RNAP must remain associated with its product for thousands of nucleotide addition cycles. However, RNAP movement along the template is not monotonous as RNAP can pause or terminate transcription at distinct sites (37); some of these sites play regulatory roles, whereas others exist as an emergent property of the energetics of transcription (13). In combination these sites determine not only the rate of transcription elongation but also, at least in certain cases, the folding pathways of the RNA molecules (19,28).Elongating RNAP may release the nascent RNA chain either stochastically (with low frequency) or at specific sites, terminators (with comparatively high frequency ranging from 5 to Ͼ99%). Numerous bacterial protein factors, acting alone or as a part of large macromolecular complexes, regulate transcription termination, either negatively or positively (26,38). While some of these factors act upon a single terminator, others are able to exert a processive antitermination effect that persists over thousands of nucleotides and allows bypass of several tandem terminators (38); these mechani...