SUMMARY NusG homologs regulate transcription and coupled processes in all living organisms. The Escherichia coli (E. coli) two-domain paralogs NusG and RfaH have conformationally identical N-terminal domains (NTDs) but dramatically different carboxy-terminal domains (CTDs), a β-barrel in NusG and an α-hairpin in RfaH. Both NTDs interact with elongating RNA polymerase (RNAP) to reduce pausing. In NusG, NTD and CTD are completely independent, and NusG-CTD interacts with termination factor Rho or ribosomal protein S10. In contrast, RfaH-CTD makes extensive contacts with RfaH-NTD to mask an RNAP-binding site therein. Upon RfaH interaction with its DNA target, the operon polarity suppressor (ops) DNA, RfaH-CTD is released, allowing RfaH-NTD to bind to RNAP. Here we show that the released RfaH-CTD completely refolds from an all-α to an all-β conformation identical to that of NusG-CTD. As a consequence, RfaH-CTD binding to S10 is enabled and translation of RfaH-controlled operons is strongly potentiated.

SUMMARY In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the β gate loop as an RNAP element required for antipausing activity of a bacterial virulence factor RfaH, a member of the universally conserved NusG family. Interactions with the gate loop are necessary for suppression of pausing and termination by RfaH, but are dispensable for RfaH binding to RNAP mediated by the β′ clamp helices. We hypothesize that, upon binding to the clamp helices and the gate loop, RfaH bridges the gap across the DNA channel, stabilizing RNAP/nucleic acid contacts and disfavoring isomerization into a paused state. We show that contacts with the gate loop are also required for antipausing by NusG and propose that most NusG homologs use similar mechanisms to increase RNAP processivity.

RfaH is a bacterial elongation factor that increases expression of distal genes in several long, horizontally acquired operons. RfaH is recruited to the transcription complex during RNA chain elongation through specific interactions with a DNA element called ops. Following recruitment, RfaH remains bound to RNA polymerase (RNAP) and acts as an antiterminator by reducing RNAP pausing and termination at some factor-independent and Rho-dependent signals. RfaH consists of two domains connected by a flexible linker. The N-terminal RfaH domain (RfaHN) recognizes the ops element, binds to the RNAP and reduces pausing and termination in vitro. Functional analysis of single substitutions in this domain reported here suggests that three separate RfaHN regions mediate these functions. We propose that a polar patch on one side of RfaHN interacts with the non-template DNA strand during recruitment, whereas a hydrophobic surface on the opposite side of RfaHN remains bound to the β′ subunit clamp helices domain throughout transcription of the entire operon. The third region is apparently dispensable for RfaH binding to the transcription complex but is required for the antitermination modification of RNAP.

Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF-UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage.cysteine cross-linking | transcription | ATPase stimulation | UvrB R NA polymerase (RNAP) stalled at DNA lesions on the transcribed strand elicits a preferential pathway for nucleotide excision repair (NER) called transcription-coupled repair (TCR), which is present in Bacteria and Eukarya (1). Bacterial transcription-repair coupling factor (TRCF; also known as Mfd) orchestrates this process by specific recognition of the transcription and NER assemblies, which reflects its twofold role. First, TRCF relieves transcription-dependent NER inhibition due to occlusion of the DNA lesion by RNAP (2). TRCF, an SF2 ATPase with dsDNA translocase but no helicase activity (3), approaches the stalled RNAP from behind and induces its forward translocation by stepping on dsDNA using ATP hydrolysis (4, 5). The consequent collapse of the upstream end of the transcription bubble leads to massive destabilization of the otherwise stable ternary elongation complex (TEC) and transcription termination (4-7). Rho, the only other known bacterial enzymatic terminator, induces termination by a similar forward-translocation mechanism, but translocates along the nascent RNA (8). Second, TRCF recruits the Uvr(A)BC endonuclease to the unmasked lesion by binding to UvrA (4, 9). This initiates a cascade of events resulting in lesion excision and gap filling (4, 10). TRCF also has roles beyond TCR-in the rescue of replication forks stalled by head-on collisions with RNAPs (11), in the development of antibiotic resistance (12, 13), recombination (14, 15), and transcriptional regulation (16, 17).The crystal structure of apo TRCF (18) revealed a multimodular enzyme with eight domains connected by flexible linkers (Fig. 1A), an architecture that appears primed for large conformational changes, which are believed to be critical for coupling RNAP recognition to recruitment of NER enzymes. Domains D1 and D2 of TRCF are similar to the NER protein UvrB, which also binds UvrA (18,19), suggesting that these domains serve as a pl...

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo-tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNASer. Ser-allo-tRNA was converted into Sec-allo-tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec-allo-tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDHH with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo-tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80%). Thus, our allo-tRNAUTu system offers a new selenoprotein engineering platform.