Escherichia coli NusG protein stimulates transcription elongation rates in vivo and in vitro

Abstract

The rate of transcription elongation in Escherichia coli was reduced when cells were depleted of NusG. In a purified in vitro system, NusG accelerated the transcription elongation rate. The stimulation of the rate of transcription elongation by NusG appears to result from the suppression of specific transcription pause sites.The Escherichia coli nusG gene encodes an essential transcription factor that has been implicated in a variety of cellular and viral termination and antitermination processes (2,9,10,21,22). NusG is required, along with other Nus factors, for effective antitermination by N protein of phage in vitro (2,8,10,11,15). The nusG4 mutation restores N activity in a nusA1 host, suggesting that NusG is also involved in antitermination in vivo (22). NusG has been shown to stimulate Rho-dependent termination in vivo (21).In vitro experiments indicate that NusG binds directly and selectively to Rho (9) and more weakly to core RNA polymerase (8). NusG both stimulates and changes the pattern of Rho-dependent termination at the tR1 and trp tЈ terminators (9,14). A model has been proposed in which NusG serves as a bridge between RNA polymerase and Rho, thus helping to recruit Rho into the termination complex (9). Indeed, recent evidence indicates that NusG stably associates with stalled elongation complexes only if Rho is bound to the nascent RNA and that the presence of NusG in the complex leads to a slower off-rate of Rho from the transcript (13).The participation of NusG in factor-dependent termination and antitermination processes, both of which involve modulation of transcription elongation, suggests the possibility that NusG may directly affect the rate at which RNA polymerase synthesizes RNA molecules. In this paper, we demonstrate that NusG increases the rate of transcription elongation both in vivo and in vitro. The ability of NusG to suppress transcriptional pausing probably accounts for its acceleration of the overall rate of transcription elongation.NusG depletion slows the rate of transcription elongation. We first asked if depletion of NusG affected the rate of transcription elongation in E. coli cells. Strains SS287 and SS294 are derivatives of N99 that carry a nusG::Kn chromosomal insertion and a nusG ϩ plasmid. SS287 carries the rep ts plasmid pSS119; SS294 carries the rep ϩ plasmid pSS120. Depletion of NusG in SS287 occurs when the cells are shifted from 32 to 42ЊC, inducing the loss of the pSS119 plasmid (1, 21).The rate of RNA chain growth can be estimated from the time of appearance of ␤-galactosidase after IPTG (isopropyl ␤-D-thiogalactosidase) induction of lacZ (5). This technique has been used recently to demonstrate altered rates of elongation by certain RNA polymerase mutants (3). To measure the effect of NusG depletion on the initial kinetics of ␤-galactosidase induction, SS287 and SS294 were grown in LuriaBertani medium at 32ЊC until early log phase and shifted to 42ЊC for 2.5 h. Cultures were maintained in exponential growth by dilution into fresh media. Optical density at 650 nm...

Enhancing transcription through the Escherichia coli hemolysin operon, hlyCABD: RfaH and upstream JUMPStart DNA sequences function together via a postinitiation mechanism

“…RfaH has significant amino acid sequence similarity with NusG (1, 18), a protein shown in E. coli to be involved in both transcriptional termination and antitermination (6,7,21,22,30,31). The role of NusG in transcription by E. coli RNAP is controversial.…”

Modulation of Rho-Dependent Transcription Termination in Escherichia coli by the H-NS Family of Proteins

“…We measured the in vivo rates of transcription elongation at the wild-type lacZ locus of different strains after IPTG addition, as described previously (10,29). The results are depicted in Fig.…”

“…Transcription elongation rate measurements at lacZ locus in vivo. ␤-Galactosidase specific activities were determined at various times after addition of IPTG to each culture grown in 0.4% glycerol-minimal A medium supplemented with Casamino Acids at 0.5% (and additionally 0.2% Ara in the case of strains in panel B), and the inflection point was identified on a curve plotting square root of enzyme specific activity against time, as described previously (10,29). The strains used were follows: derivatives of MG1655 (wild type, squares), GJ3171 (rho, triangles), and GJ10729 (nusG, circles) transformed with either the vector pLG339 (open symbols) or its derivative expressing H-NS⌬64 (solid symbols) (A) and MG1655 (wild-type) with either pBAD18 (vector, Ⅺ) or pHYD2525 (YdgT, f), and GJ3171 (rho) with pHYD2525 (YdgT, OE) (B).…”

Highly Divergent RfaH Orthologs from Pathogenic Proteobacteria Can Substitute for Escherichia coli RfaH both In Vivo and In Vitro

“…In addition, RfaH is also homologous to NusG, a general elongation factor, and the percent identity approaches that of distant RfaH orthologs. Although, like RfaH, NusG also increases the RNA chain elongation rates in vitro (8) and in vivo (43), its other effects on transcription are opposite to those of RfaH: NusG increases Rho-dependent termination (36), reduces pausing at the ops site but does not affect pausing at the hairpin-dependent sites (2), participates in formation of macromolecular antitermination complexes (22,34), and lacks the FIG. 1.…”