Two Structurally Independent Domains of E. coli NusG Create Regulatory Plasticity via Distinct Interactions with RNA Polymerase and Regulators

Abstract

NusG is a conserved regulatory protein that interacts with elongating complexes (ECs) of RNA polymerase (RNAP), DNA, and RNA to modulate transcription in multiple and sometimes opposite ways. In E. coli, NusG suppresses pausing and increases elongation rate, enhances termination by E. coli ρ and phage HK022 Nun protein, and promotes antitermination by λN and in ribosomal RNA operons. We report NMR studies that suggest E. coli NusG consists of two largely independent Nand C-terminal structural domains, NTD and CTD. Based on tests of the functions of the NTD and CTD and variants of NusG in vivo and in vitro, we find that NTD alone is sufficient to suppress pausing and enhance transcript elongation in vitro. However, neither domain alone can enhance ρ-dependent termination or support antitermination, indicating that interactions of both domains with ECs are required for these processes. We propose that the two domains of NusG mediate distinct interactions with ECs: the NTD interacts with RNAP and the CTD interacts with ρ and other regulators, providing NusG with different combinations of interactions to effect different regulatory outcomes.

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

“…The Rho-binding domain in NusG has also recently been shown to interact with the S10 protein subunit of the ribosome, which has implications for the mechanism by which translation-uncoupled nascent transcripts may be subject to termination of their synthesis (9). Although null mutations in rho or nusG confer inviability in wild-type E. coli (11), missense mutations in the two genes are known that confer a transcription termination-defective (that is, polarity relief) phenotype (1,12,13,27,36).In the present study, we found that overexpression of the protein YdgT restores transcriptional polarity in the rho and nusG mutants and, furthermore, that the combination of rho, ydgT, and hha mutations confers extreme sickness in E. coli. YdgT (also called Cnu [30]) and Hha belong to the H-NS family of proteins (33), whose prototypic member H-NS participates in both regulation of transcription initiation and nucleoid structure (reviewed in references 16, 20, 23, and 59).…”

“…The Rho-binding domain in NusG has also recently been shown to interact with the S10 protein subunit of the ribosome, which has implications for the mechanism by which translation-uncoupled nascent transcripts may be subject to termination of their synthesis (9). Although null mutations in rho or nusG confer inviability in wild-type E. coli (11), missense mutations in the two genes are known that confer a transcription termination-defective (that is, polarity relief) phenotype (1,12,13,27,36).…”

“…Recently, Rho has also been reported to bind RNA polymerase (21). NusG has two domains that bind Rho and RNA polymerase, respectively (13,36). The Rho-binding domain in NusG has also recently been shown to interact with the S10 protein subunit of the ribosome, which has implications for the mechanism by which translation-uncoupled nascent transcripts may be subject to termination of their synthesis (9).…”

Antisense Oligonucleotide-stimulated Transcriptional Pausing Reveals RNA Exit Channel Specificity of RNA Polymerase and Mechanistic Contributions of NusA and RfaH

“…Before testing for competition between RfaH and antisense RNA, we next asked if NusG might exhibit a similar effect. Although the NusG CTD does not inhibit NusG binding to RNAP (38) and full-length NusG has little effect on hairpin-stabilized pausing (12), we reasoned that high concentrations of NusG might compete for antisense RNA binding. Indeed, we found that 500 nM NusG partially suppressed pause stimulation by 1 M antisense RNA (Fig.…”

Compromised Factor-Dependent Transcription Termination in a nusA Mutant of Escherichia coli: Spectrum of Termination Efficiencies Generated by Perturbations of Rho, NusG, NusA, and H-NS Family Proteins

“…The mechanism is absolutely dependent on the essential Rho protein that binds the nascent transcript and is then believed to signal RNA polymerase to terminate transcription, but there is no consensus as yet on its details (reviewed in references 1,3,14,40,41,43, and 48; see also references 21 and 31). As with their effects on transcription elongation, NusA and NusG have been reported to function antagonistically for Rho-dependent transcription termination as well, with NusG increasing its efficiency (7,8,11,29,35,38,52) and NusA decreasing it (8,27,(32)(33)(34); indeed, it has been reported that mutations in rho can suppress the inviability associated with the loss of NusA (58). On the other hand, Gottesman and coworkers (9,(52)(53)(54) have suggested that both NusA and NusG act cooperatively to promote Rho-dependent termination.…”