1887

Abstract

The conventional model of Rho-dependent transcription termination in bacteria requires RNA-dependent translocase activity of the termination factor Rho as well as many kinetically controlled steps to execute efficient RNA release from the transcription elongation complex (EC). The involvement of the kinetically controlled steps, such as RNA binding, translocation and RNA release from the EC, means that this termination process must be kinetically coupled to the transcription elongation process. The existence of these steps has not previously been delineated in detail. Moreover, the requirement for translocase activity in Rho-dependent termination has recently been questioned by a radical view, wherein Rho binds to the elongating RNA polymerase (RNAP) prior to loading onto the mRNA. Using growth assays, microarray analyses and reporter-based transcription termination assays , we showed that slowing of the transcription elongation rate by using RNAP mutants ( and ) and growth of the strains in minimal medium suppressed the termination defects of five Rho mutants, three NusG mutants defective for Rho binding and the defects caused by two Rho inhibitors, Psu and bicyclomycin. These results established the existence of kinetically controlled steps in the Rho-dependent termination process and further reinforced the importance of ‘kinetic coupling’ between the two molecular motors, Rho and RNAP, and also argue strongly that the Rho translocation model is an accurate representation of the situation. Finally, these results indicated that one of the major roles of NusG in Rho-dependent termination is to enhance the speed of RNA release from the EC.

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2012-06-01
2019-10-22
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References

  1. Banerjee S. , Chalissery J. , Bandey I. , Sen R. . ( 2006; ). Rho-dependent transcription termination: more questions than answers. . J Microbiol 44:, 11–22.[PubMed]
    [Google Scholar]
  2. Boudvillain M. , Nollmann M. , Margeat E. . ( 2010; ). Keeping up to speed with the transcription termination factor Rho motor. . Transcription 1:, 70–75. [CrossRef] [PubMed]
    [Google Scholar]
  3. Burns C. M. , Nowatzke W. L. , Richardson J. P. . ( 1999; ). Activation of Rho-dependent transcription termination by NusG. Dependence on terminator location and acceleration of RNA release. . J Biol Chem 274:, 5245–5251. [CrossRef] [PubMed]
    [Google Scholar]
  4. Cardinale C. J. , Washburn R. S. , Tadigotla V. R. , Brown L. M. , Gottesman M. E. , Nudler E. . ( 2008; ). Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli . . Science 320:, 935–938. [CrossRef] [PubMed]
    [Google Scholar]
  5. Chalissery J. , Banerjee S. , Bandey I. , Sen R. . ( 2007; ). Transcription termination defective mutants of Rho: role of different functions of Rho in releasing RNA from the elongation complex. . J Mol Biol 371:, 855–872. [CrossRef] [PubMed]
    [Google Scholar]
  6. Chalissery J. , Muteeb G. , Kalarickal N. C. , Mohan S. , Jisha V. , Sen R. . ( 2011; ). Interaction surface of the transcription terminator Rho required to form a complex with the C-terminal domain of the antiterminator NusG. . J Mol Biol 405:, 49–64. [CrossRef] [PubMed]
    [Google Scholar]
  7. Epshtein V. , Dutta D. , Wade J. , Nudler E. . ( 2010; ). An allosteric mechanism of Rho-dependent transcription termination. . Nature 463:, 245–249. [CrossRef] [PubMed]
    [Google Scholar]
  8. Galloway J. L. , Platt T. . ( 1988; ). Signals sufficient for ρ-dependent transcription termination at trp t′ span a region centered 60 base pairs upstream of the earliest 3′ end point. . J Biol Chem 263:, 1761–1767.[PubMed]
    [Google Scholar]
  9. Jin D. J. , Gross C. A. . ( 1991; ). RpoB8, a rifampicin-resistant termination-proficient RNA polymerase, has an increased K m for purine nucleotides during transcription elongation. . J Biol Chem 266:, 14478–14485.[PubMed]
    [Google Scholar]
  10. Jin D. J. , Burgess R. R. , Richardson J. P. , Gross C. A. . ( 1992; ). Termination efficiency at rho-dependent terminators depends on kinetic coupling between RNA polymerase and rho. . Proc Natl Acad Sci U S A 89:, 1453–1457. [CrossRef] [PubMed]
    [Google Scholar]
  11. Kalarickal N. C. , Ranjan A. , Kalyani B. S. , Wal M. , Sen R. . ( 2010; ). A bacterial transcription terminator with inefficient molecular motor action but with a robust transcription termination function. . J Mol Biol 395:, 966–982. [CrossRef] [PubMed]
    [Google Scholar]
  12. Kalayani B. S. , Muteeb G. , Qayyum M. Z. , Sen R. . ( 2011; ). Interaction with the nascent RNA is a prerequisite for the recruitment of Rho to the transcription elongation complex in vitro . . J Mol Biol 413:, 548–560. [CrossRef] [PubMed]
    [Google Scholar]
  13. Li J. , Mason S. W. , Greenblatt J. . ( 1993; ). Elongation factor NusG interacts with termination factor rho to regulate termination and antitermination of transcription. . Genes Dev 7:, 161–172. [CrossRef] [PubMed]
    [Google Scholar]
  14. Linderoth N. A. , Calendar R. L. . ( 1991; ). The Psu protein of bacteriophage P4 is an antitermination factor for rho-dependent transcription termination. . J Bacteriol 173:, 6722–6731.[PubMed]
    [Google Scholar]
  15. Magyar A. , Zhang X. , Kohn H. , Widger W. R. . ( 1996; ). The antibiotic bicyclomycin affects the secondary RNA binding site of Escherichia coli transcription termination factor Rho. . J Biol Chem 271:, 25369–25374. [CrossRef] [PubMed]
    [Google Scholar]
  16. Magyar A. , Zhang X. , Abdi F. , Kohn H. , Widger W. R. . ( 1999; ). Identifying the bicyclomycin binding domain through biochemical analysis of antibiotic-resistant rho proteins. . J Biol Chem 274:, 7316–7324. [CrossRef] [PubMed]
    [Google Scholar]
  17. McDowell J. C. , Roberts J. W. , Jin D. J. , Gross C. . ( 1994; ). Determination of intrinsic transcription termination efficiency by RNA polymerase elongation rate. . Science 266:, 822–825. [CrossRef] [PubMed]
    [Google Scholar]
  18. Mooney R. A. , Schweimer K. , Rösch P. , Gottesman M. , Landick R. . ( 2009; ). Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators. . J Mol Biol 391:, 341–358. [CrossRef] [PubMed]
    [Google Scholar]
  19. Pani B. , Banerjee S. , Chalissery J. , Muralimohan A. , Loganathan R. M. , Suganthan R. B. , Sen R. . ( 2006; ). Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu. . J Biol Chem 281:, 26491–26500. [CrossRef] [PubMed]
    [Google Scholar]
  20. Pani B. , Ranjan A. , Sen R. . ( 2009; ). Interaction surface of bacteriophage P4 protein Psu required for complex formation with the transcription terminator Rho. . J Mol Biol 389:, 647–660. [CrossRef] [PubMed]
    [Google Scholar]
  21. Pasman Z. , von Hippel P. H. . ( 2000; ). Regulation of rho-dependent transcription termination by NusG is specific to the Escherichia coli elongation complex. . Biochemistry 39:, 5573–5585. [CrossRef] [PubMed]
    [Google Scholar]
  22. Peters J. M. , Vangeloff A. D. , Landick R. . ( 2011; ). Bacterial transcription terminators: the RNA 3′-end chronicles. . J Mol Biol 412:, 793–813. [CrossRef] [PubMed]
    [Google Scholar]
  23. Richardson J. P. . ( 2002; ). Rho-dependent termination and ATPases in transcript termination. . Biochim Biophys Acta 1577:, 251–260.[PubMed] [CrossRef]
    [Google Scholar]
  24. Schwartz A. , Rabhi M. , Jacquinot F. , Margeat E. , Rahmouni A. R. , Boudvillain M. . ( 2009; ). A stepwise 2′-hydroxyl activation mechanism for the bacterial transcription termination factor Rho helicase. . Nat Struct Mol Biol 16:, 1309–1316. [CrossRef] [PubMed]
    [Google Scholar]
  25. Sen R. , Challissery J. , Muteeb G. . ( 2008; ). Nus factors of Escherichia coli . . In EcoSal-Escherichia coli and Salmonella; Cellular and Molecular Biology. Edited by Böck A. , Curtiss R. III , Kaper J. B. , Karp P. D. , Neidhardt F. C. , Nyström T. , Slauch J. M. , Squires C. L. . . Washington, DC:: American Society for Microbiology;. http://www.ecosal.org.
    [Google Scholar]
  26. Sullivan S. L. , Gottesman M. E. . ( 1992; ). Requirement for E. coli NusG protein in factor-dependent transcription termination. . Cell 68:, 989–994. [CrossRef] [PubMed]
    [Google Scholar]
  27. Vogel U. , Jensen K. F. . ( 1994; ). The RNA chain elongation rate in Escherichia coli depends on the growth rate. . J Bacteriol 176:, 2807–2813.[PubMed]
    [Google Scholar]
  28. von Hippel P. H. , Pasman Z. . ( 2002; ). Reaction pathways in transcript elongation. . Biophys Chem 101–102:, 401–423. [CrossRef] [PubMed]
    [Google Scholar]
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