Bacterial NusG is a highly conserved transcription factor that is required for most Rho activity in vivo. We show by nuclear magnetic resonance spectroscopy that Escherichia coli NusG carboxyl-terminal domain forms a complex alternatively with Rho or with transcription factor NusE, a protein identical to 30S ribosomal protein S10. Because NusG amino-terminal domain contacts RNA polymerase and the NusG carboxy-terminal domain interaction site of NusE is accessible in the ribosomal 30S subunit, NusG may act as a link between transcription and translation. Uncoupling of transcription and translation at the ends of bacterial operons enables transcription termination by Rho factor, and competition between ribosomal NusE and Rho for NusG helps to explain why Rho cannot terminate translated transcripts.

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...

Escherichia coli transcription termination protein Rho aids in the release of newly synthesized RNA from paused transcription complexes (reviewed in Ref. 1). The homohexameric protein binds nascent RNA and, with the RNA-dependent hydrolysis of ATP, disrupts the ternary transcription complex, releasing product RNA and allowing RNA polymerase to recycle. The discovery of a 5Ј 3 3Ј RNA-DNA helicase activity of Rho (2) suggested that Rho might disrupt the RNA-DNA duplex of the transcription bubble. Recent studies of ternary transcription complexes (Refs. 3-5 and reviewed in Ref. 6) suggest that such disruption could be important in transcription termination, as could be the release of the nascent RNA just 5Ј of the RNA-DNA duplex from its interactions with RNA polymerase. As described by Nudler et al. (7), the interaction of RNA with RNA polymerase immediately 5Ј from the RNA-DNA hybrid may control the opening and closing of an RNA polymerase clamp around the DNA template near the leading edge of the enzyme, and contribute to the stability of the ternary transcription complex. An appealing model for Rho is one in which the enzyme binds to exposed mRNA behind RNA polymerase and travels 5Ј 3 3Ј along the RNA as it hydrolyzes ATP, binding and releasing RNA from different parts of the hexamer to accomplish movement (8). Such activity could release nascent RNA from RNA polymerase-binding sites and could constitute the basis for its RNA-DNA helicase activity, both of which might be involved in transcript release from paused ternary transcription complexes. The finding that the same number of ATP molecules per RNA length is hydrolyzed by Rho traveling along RNA and Rho unwinding RNA-DNA hybrids (8) supports this hypothesis.Rho binds single-stranded RNA, showing preferred entry regions on RNA upstream of eventual transcription termination sites. However, the characteristics of these regions, beyond low secondary structure and some preference for a C-rich, G-poor base composition (9), are too poorly understood to permit their identification by sequence inspection. When bound to RNA, Rho protects 80 bases from ribonuclease degradation (10, 11). The binding of Rho to 10-base RNA oligomers was reported as best fit by three tight and three weaker sites per hexamer (12, 13).The RNA-dependent hydrolysis of ATP is essential for the transcription termination function of Rho. Two components of ternary transcription complexes, the DNA template and RNA polymerase, are not required to elicit this ATPase activity, thus considerably simplifying study of the reaction (14). The reaction is particularly well stimulated by the RNA homopolymer poly(C), and Rho is frequently assayed in vitro by measuring its poly(C)-dependent ATPase activity. Previous work has shown that the Rho hexamer binds three molecules of MgATP in a single class of catalytically competent sites (15,16). An additional class of three ATP-binding sites of lower affinity has also been suggested (16), although the catalytic activity of these sites was not assessed. The stoic...

Porphyromonas gingivalis peptidylarginine deiminase (PAD) catalyzes the deimination of peptidylarginine residues of various peptides to produce peptidylcitrulline and ammonia. P. gingivalis is associated with adult-onset periodontitis and cardiovascular disease, and its proliferation depends on secretion of PAD. We have expressed two recombinant forms of the P. gingivalis PAD in Escherichia coli, a truncated form with a 43-amino acid N-terminal deletion and the full-length form of PAD as predicted from the DNA sequence. Both forms contain a poly-His tag and Xpress epitope at the N-terminus to aid in detection and purification. The activities and stabilities of these two forms have been evaluated. PAD is cold sensitive; it aggregates within 30 min at 4 °C, and optimal storage conditions are at 25 °C in the presence of a reducing agent. PAD is not a metalloenzyme and does not need a co-factor for catalysis or stability. Multiple L-arginine analogs, various arginine-containing peptides, and free L-arginine were used to evaluate substrate specificity and determine kinetic parameters.

Escherichia coli transcription termination factor Rho uses the energy of ATP hydrolysis to travel 5' --> 3' along RNA. We previously showed that the hexameric Rho protein binds three molecules of ATP in active sites and that hydrolysis of the three bound ATP molecules upon RNA binding is sequential. Other models of Rho ATP hydrolysis activity have arisen from reports of additional ATP binding sites on Rho. Here we present further evidence from binding, isotope partitioning, and rapid mix/chemical quench experiments, in support of the presence of only three equivalent ATP binding sites on Rho that are catalytic sites and that fire sequentially. These results are incorporated into a proposed mechanism for directional Rho tracking along RNA.