1887

Abstract

The -infecting bacteriophage T7 encodes a 7 kDa protein, called Gp2, which is a potent inhibitor of the host RNA polymerase (RNAp). Gp2 is essential for T7 phage development. The interaction site for Gp2 on the RNAp is the β′ jaw domain, which is part of the DNA binding channel. The binding of Gp2 to the β′ jaw antagonizes several steps associated with interactions between the RNAp and promoter DNA, leading to inhibition of transcription at the open promoter complex formation step. In the structure of the complex formed between Gp2 and a fragment of the β′ jaw, amino acid residues in the β3 strand of Gp2 contribute to the primary interaction interface with the β′ jaw. The 7009 strain is resistant to T7 because it carries a charge reversal point mutation in the β′ jaw that prevents Gp2 binding. However, a T7 phage encoding a mutant form of Gp2, called Gp2, which carries triple amino acid substitutions E24K, F27Y and R56C, can productively infect this strain. By studying the molecular basis of inhibition of RNAp from the 7009 strain by Gp2, we provide several lines of evidence that the E24K and F27Y substitutions facilitate an interaction with RNAp when the primary interaction interface with the β′ jaw is compromised. The proposed additional interaction interface between RNAp and Gp2 may contribute to the multipronged mechanism of transcription inhibition by Gp2.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.062547-0
2012-11-01
2019-12-05
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/11/2753.html?itemId=/content/journal/micro/10.1099/mic.0.062547-0&mimeType=html&fmt=ahah

References

  1. Arnold K., Bordoli L., Kopp J., Schwede T.. ( 2006;). The swiss-model workspace: a web-based environment for protein structure homology modelling. . Bioinformatics 22:, 195–201. [CrossRef][PubMed]
    [Google Scholar]
  2. Belogurov G. A., Vassylyeva M. N., Svetlov V., Klyuyev S., Grishin N. V., Vassylyev D. G., Artsimovitch I.. ( 2007;). Structural basis for converting a general transcription factor into an operon-specific virulence regulator. . Mol Cell 26:, 117–129. [CrossRef][PubMed]
    [Google Scholar]
  3. Browning D. F., Busby S. J.. ( 2004;). The regulation of bacterial transcription initiation. . Nat Rev Microbiol 2:, 57–65. [CrossRef][PubMed]
    [Google Scholar]
  4. Burck K. B., Miller R. C. Jr. ( 1978;). Marker rescue and partial replication of bacteriophage T7 DNA. . Proc Natl Acad Sci U S A 75:, 6144–6148. [CrossRef][PubMed]
    [Google Scholar]
  5. Cámara B., Liu M., Reynolds J., Shadrin A., Liu B., Kwok K., Simpson P., Weinzierl R., Severinov K.. & other authors ( 2010;). T7 phage protein Gp2 inhibits the Escherichia coli RNA polymerase by antagonizing stable DNA strand separation near the transcription start site. . Proc Natl Acad Sci U S A 107:, 2247–2252. [CrossRef][PubMed]
    [Google Scholar]
  6. Chakraborty A., Wang D., Ebright Y. W., Korlann Y., Kortkhonjia E., Kim T., Chowdhury S., Wigneshweraraj S., Irschik H.. & other authors ( 2012;). Opening and closing of the bacterial RNA polymerase clamp. . Science 337:, 591–595. [CrossRef][PubMed]
    [Google Scholar]
  7. Chamberlin M.. ( 1974;). Isolation and characterization of prototrophic mutants of Escherichia coli unable to support the intracellular growth of T7. . J Virol 14:, 509–516.[PubMed]
    [Google Scholar]
  8. Ederth J., Artsimovitch I., Isaksson L. A., Landick R.. ( 2002;). The downstream DNA jaw of bacterial RNA polymerase facilitates both transcriptional initiation and pausing. . J Biol Chem 277:, 37456–37463. [CrossRef][PubMed]
    [Google Scholar]
  9. Ederth J., Mooney R. A., Isaksson L. A., Landick R.. ( 2006;). Functional interplay between the jaw domain of bacterial RNA polymerase and allele-specific residues in the product RNA-binding pocket. . J Mol Biol 356:, 1163–1179. [CrossRef][PubMed]
    [Google Scholar]
  10. Haugen S. P., Ross W., Gourse R. L.. ( 2008;). Advances in bacterial promoter recognition and its control by factors that do not bind DNA. . Nat Rev Microbiol 6:, 507–519. [CrossRef][PubMed]
    [Google Scholar]
  11. Ishihama A.. ( 1999;). Modulation of the nucleoid, the transcription apparatus, and the translation machinery in bacteria for stationary phase survival. . Genes Cells 4:, 135–143. [CrossRef][PubMed]
    [Google Scholar]
  12. James E., Liu M., Sheppard C., Mekler V., Cámara B., Liu B., Simpson P., Cota E., Severinov K.. & other authors ( 2012;). Structural and mechanistic basis for the inhibition of Escherichia coli RNA polymerase by T7 Gp2. . Mol Cell 47:, 755–766. [CrossRef][PubMed]
    [Google Scholar]
  13. Mekler V., Kortkhonjia E., Mukhopadhyay J., Knight J., Revyakin A., Kapanidis A. N., Niu W., Ebright Y. W., Levy R., Ebright R. H.. ( 2002;). Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex. . Cell 108:, 599–614. [CrossRef][PubMed]
    [Google Scholar]
  14. Mekler V., Minakhin L., Sheppard C., Wigneshweraraj S., Severinov K.. ( 2011;). Molecular mechanism of transcription inhibition by phage T7 gp2 protein. . J Mol Biol 413:, 1016–1027. [CrossRef][PubMed]
    [Google Scholar]
  15. Nechaev S., Severinov K.. ( 1999;). Inhibition of Escherichia coli RNA polymerase by bacteriophage T7 gene 2 protein. . J Mol Biol 289:, 815–826. [CrossRef][PubMed]
    [Google Scholar]
  16. Nechaev S., Severinov K.. ( 2003;). Bacteriophage-induced modifications of host RNA polymerase. . Annu Rev Microbiol 57:, 301–322. [CrossRef][PubMed]
    [Google Scholar]
  17. Papadopoulos J. S., Agarwala R.. ( 2007;). cobalt: constraint-based alignment tool for multiple protein sequences. . Bioinformatics 23:, 1073–1079. [CrossRef][PubMed]
    [Google Scholar]
  18. Piper S. E., Mitchell J. E., Lee D. J., Busby S. J.. ( 2009;). A global view of Escherichia coli Rsd protein and its interactions. . Mol Biosyst 5:, 1943–1947. [CrossRef][PubMed]
    [Google Scholar]
  19. Rokyta D., Badgett M. R., Molineux I. J., Bull J. J.. ( 2002;). Experimental genomic evolution: extensive compensation for loss of DNA ligase activity in a virus. . Mol Biol Evol 19:, 230–238. [CrossRef][PubMed]
    [Google Scholar]
  20. Rutherford S. T., Villers C. L., Lee J. H., Ross W., Gourse R. L.. ( 2009;). Allosteric control of Escherichia coli rRNA promoter complexes by DksA. . Genes Dev 23:, 236–248. [CrossRef][PubMed]
    [Google Scholar]
  21. Saecker R. M., Record M. T. Jr, Dehaseth P. L.. ( 2011;). Mechanism of bacterial transcription initiation: RNA polymerase – promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis. . J Mol Biol 412:, 754–771. [CrossRef][PubMed]
    [Google Scholar]
  22. Savalia D., Robins W., Nechaev S., Molineux I., Severinov K.. ( 2010;). The role of the T7 Gp2 inhibitor of host RNA polymerase in phage development. . J Mol Biol 402:, 118–126. [CrossRef][PubMed]
    [Google Scholar]
  23. Schmitt M. P., Beck P. J., Kearney C. A., Spence J. L., DiGiovanni D., Condreay J. P., Molineux I. J.. ( 1987;). Sequence of a conditionally essential region of bacteriophage T3, including the primary origin of DNA replication. . J Mol Biol 193:, 479–495. [CrossRef][PubMed]
    [Google Scholar]
  24. Sevostyanova A., Belogurov G. A., Mooney R. A., Landick R., Artsimovitch I.. ( 2011;). The β subunit gate loop is required for RNA polymerase modification by RfaH and NusG. . Mol Cell 43:, 253–262. [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.062547-0
Loading
/content/journal/micro/10.1099/mic.0.062547-0
Loading

Data & Media loading...

Supplements

Table S1 

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error