Formation of termination-resistant transcription complex at phage lambda nut locus: effects of altered translation and a ribosomal mutation (original) (raw)
Cell, 1984
Employing specifically engineered plasmids in which the expression of E. coli galK cistron is regulated by transcription termination, we have analyzed the antitermination function of phage lambda N gene product in S30 extracts. Antitermination by N, dependent on its site of action, nutL, is defective in the extracts prepared from nusA, nusB, and nusE mutants. By complementation analysis, we demonstrate that none of the these nus mutations affects the synthesis of N or the other nus gene products to cause a defect in antitermination. Rather, these mutations have inactivated a set of specific host components, the Nus factors, which are essential for N activity. Curiously, an appreciable portion of N and Nus complementation activities of an S30 extract is ribosome-associated. The significance of this finding remains to be uncovered.
Proceedings of the National Academy of Sciences, 1985
Bacteriophage X N gene product acts to modify host RNA polymerase allowing the formation of a termination-resistant transcription apparatus. Previous studies have demonstrated that the nusE71 mutation that has altered the ribosomal protein S10 prevents N action in vivo. Using a coupled transcription-translation system, we demonstrate here that purified S10 protein as well as the 30S ribosomal subunit is sufficient to restore N activity in the nusE mutant extract, allowing antitermination of Rho-dependent and Rho-independent terminators. This provides direct biochemical evidence that the S10 protein itself is one of the cellular components necessary for the formation of an antitermination apparatus.
Bacteriophage X N gene product acts to modify host RNA polymerase allowing the formation of a termination-resistant transcription apparatus. Previous studies have demonstrated that the nusE71 mutation that has altered the ribosomal protein S10 prevents N action in vivo. Using a coupled transcription-translation system, we demonstrate here that purified S10 protein as well as the 30S ribosomal subunit is sufficient to restore N activity in the nusE mutant extract, allowing antitermination of Rho-dependent and Rho-independent terminators. This provides direct biochemical evidence that the S10 protein itself is one of the cellular components necessary for the formation of an antitermination apparatus. The N gene product of bacteriophage X acts to suppress transcription termination in Escherichia coli (1-5). The action ofN depends on a recognition site (nut) encoded by the phage genome (6-9). It is thought that N acts to modify RNA polymerase at the nut site, allowing the formation of a...
Proceedings of the National Academy of Sciences, 1984
We demonstrate that the protein product of the Escherichia coli nusB gene is essential for transcription antitermination in vitro by phage X N gene product. We recently have described a convenient 'biochemical assay for N protein activity in the S30-coupled transcription translation system and demonstrated that N action requires the 69-kDa L factor (nusA), the product of E. coli nusA gene. Using a complementation assay for the restoration of N activity specifically in the nusB mutant extract, we have purified the nusR complementing activity. This activity is due to a 15-kDa polypeptide that is overproduced in E. colicontaining multiple copies of the nusB gene. We find that pusA and nusB are required for N activity to suppress a rho-dependent as well as a rho-independent terminator. The requirement for nusB protein in antitermination could not be overcome by an excess of nusA or N protein, nor could an excess of nusB overcome the requirements for nusA in antitermination. Our results suggest that the formation of an antitermination apparatus by N requires nusA and nusB proteins in equimolar amounts.
Proceedings of the National Academy of Sciences, 1994
During transcription of phage A early operons, the N gene product alters host RNA polymerase (RNAP) so that transcription proceeds through multiple stop signals. Here, we reproduce the essence of N activity with purified components in synthetic transcription units that contain A pL promoter and the N-recognition site, nutL, followed by a variety ofintrinsic terminators. We show that three host factors (NusA, NusE, and NusG) are essential for N to allow appreciable transcription through multiple terminators and that this persistent antitermination is stimulated by a fourth factor, NusB. Remarkably, in the absence of all four factors, N suppresses various terminators placed near the nut site. This basal antitenation activity of N is enhanced by NusA and is diminished by high salt and temperature. We postulate that N interacts with RNAP directly, inducing the terminationresistant state. While NusA facilitates this interaction, the other factors strengthen it sufficiently over time and distance so that RNAP bypasses multiple terminators. The dispensability of NusB for persistent antitermination in vitro, but not in vivo, raises the possibility that NusB performs two functions: it increases the stability of N antitermination complex and also counteracts an inhibitory factor in the cell.
Journal of Molecular Biology, 1995
Phage HK022 Nun protein excludes phage l by terminating transcription 1 Department of Biochemistry near the l nut sites. We have established a purified in vitro system that and Molecular Biophysics reproduces the in vivo sequence and factor requirements of Nun. Nun arrests 2 Institute of Cancer Research transcription by E. coli RNA polymerase at or near elongation pause sites College of Physicians and Surgeons, Columbia distal to the nut sites. The boxB sequence of nut is required for optimal Nun University, New York activity; boxA plays a lesser role. The efficiency of transcription arrest is strongly enhanced by the four E. coli Nus factors. The factors increase the NY 10032, U.S.A. specific activity of Nun, and allow it to act at higher ribonucleoside triphosphate concentrations. A wild-type boxA is required for stimulation by Nus factors. Nun and the l N antitermination protein compete for their opposing reactions. This competition may be at the level of binding of boxB RNA.
Nucleic Acids Research, 2010
Phage j propagation in Escherichia coli host cells requires transcription antitermination on the j chromosome mediated by jN protein and four host Nus factors, NusA, B, E (ribosomal S10) and G. Interaction of E. coli NusB:NusE heterodimer with the single stranded BoxA motif of jnutL or jnutR RNA is crucial for this reaction. Similarly, binding of NusB:NusE to a BoxA motif is essential to suppress transcription termination in the ribosomal RNA (rrn) operons. We used fluorescence anisotropy to measure the binding properties of NusB and of NusB:NusE heterodimer to BoxA-containing RNAs differing in length and sequence. Our results demonstrate that BoxA is necessary and sufficient for binding. We also studied the gain-of-function D118N NusB mutant that allows j growth in nusA1 or nusE71 mutants. In vivo j burst-size determinations, CD thermal unfolding measurements and X-ray crystallography of this as well as various other NusB D118 mutants showed the importance of size and polarity of amino acid 118 for RNA binding and other interactions. Our work suggests that the affinity of the NusB:NusE complex to BoxA RNA is precisely tuned to maximize control of transcription termination.
Escherichia coli nusG mutations that block transcription termination by coliphage HK022 Nun protein
Molecular Microbiology, 1999
The Escherichia coli nusG gene product is required for transcription termination by phage HK022 Nun protein at the nutR site in vivo. We show that it is also essential for Nun termination at nutL. Three recessive missense nusG mutations have been isolated that inhibit termination by Nun at nutR. The mutations are ineffective in a pL nutL fusion, even when nutR replaces nutL. The mutant strains support growth, indicating that N antitermination activity is not impaired. Transcription arrest by Nun in vitro is stimulated by NusG protein at both nutR and nutL. Mutant NusG protein fails to enhance transcriptional arrest by Nun at either site. The mutant protein, like the wild-type protein, suppresses transcriptional pausing by RNA polymerase and stimulates Rho-dependent termination. These results imply that the role of NusG in Nun termination may be distinct from its roles in other transcription reactions.
Journal of Biological Chemistry, 2013
Background: The mode of interaction between the transcription factor, NusA, and the antiterminator, N, is unknown. Results: When bound to the transcription elongation complex (EC), NusA-NTD interacts with N. Conclusion: The EC-induced away-movement of NusA-C-terminal domain changed the interaction surface of NusA for N. Significance: N-NusA interaction converts NusA into an antiterminator by influencing the NusA-RNA polymerase interaction. The bacterial transcription elongation factor, NusA, functions as an antiterminator when it is bound to the lambdoid phage derived antiterminator protein, N. The mode of N-NusA interaction is unknown, knowledge of which is essential to understand the antitermination process. It was reported earlier that in the absence of the transcription elongation complex (EC), N interacts with the C-terminal AR1 domain of NusA. However, the functional significance of this interaction is obscure. Here we identified mutations in NusA N terminus (NTD) specifically defective for N-mediated antitermination. These are located at a convex surface of the NusA-NTD, situated opposite its concave RNA polymerase (RNAP) binding surface. These NusA mutants disrupt the N-nut site interactions on the nascent RNA emerging out of a stalled EC. In the N/NusA-modified EC, a Cys-53 (S53C) from the convex surface of the NusA-NTD forms a specific disulfide (S-S) bridge with a Cys-39 (S39C) of the NusA binding region of the N protein. We conclude that when bound to the EC, the N interaction surface of NusA shifts from the AR1 domain to its NTD domain. This occurred due to a massive away-movement of the adjacent AR2 domain of NusA upon binding to the EC. We propose that the close proximity of this altered N-interaction site of NusA to its RNAP binding surface, enables N to influence the NusA-RNAP interaction during transcription antitermination that in turn facilitates the conversion of NusA into an antiterminator. Escherichia coli RNAP terminates transcription at the intrinsic terminators composed of specific RNA sequences or when it is dislodged by the nascent RNA-binding protein, Rho (1, 2). Lambdoid phages encode proteins, like N and Q, that make the EC 3 termination resistant. This phenomenon is called transcription antitermination (Refs. 3 and 4; Fig. 1A).