Analysis of regions within the bacteriophage T4 AsiA protein involved in its binding to the sigma70 subunit of E. coli RNA polymerase and its role as a transcriptional inhibitor and co-activator - PubMed (original) (raw)
Analysis of regions within the bacteriophage T4 AsiA protein involved in its binding to the sigma70 subunit of E. coli RNA polymerase and its role as a transcriptional inhibitor and co-activator
Debashis Pal et al. J Mol Biol. 2003.
Abstract
Bacteriophage T4 AsiA, a protein of 90 amino acid residues, binds to the sigma(70) subunit of Escherichia coli RNA polymerase and inhibits host or T4 early transcription or, together with the T4 MotA protein, activates T4 middle transcription. To investigate which regions within AsiA are involved in forming a complex with sigma(70) and in providing transcriptional functions we generated random mutations throughout AsiA and targeted mutations within the C-terminal region. We tested mutant proteins for their ability to complement the growth of T4 asiA am phage under non-suppressing conditions, to inhibit E. coli growth, to interact with sigma(70) region 4 in a two-hybrid assay, to bind to sigma(70) in a native protein gel, and to inhibit or activate transcription in vitro using a T4 middle promoter that is active with RNA polymerase alone, is inhibited by AsiA, and is activated by MotA/AsiA. We find that substitutions within the N-terminal half of AsiA, at amino acid residues V14, L18, and I40, rendered the protein defective for binding to sigma(70). These residues reside at the monomer-monomer interface in recent NMR structures of the AsiA dimer. In contrast, AsiA missing the C-terminal 44 amino acid residues interacted well with sigma(70) region 4 in the two-hybrid assay, and AsiA missing the C-terminal 17 amino acid residues (Delta74-90) bound to sigma(70) and was fully competent in standard in vitro transcription assays. However, the presence of the C-terminal region delayed formation of transcriptionally competent species when the AsiA/polymerase complex was pre-incubated with the promoter in the absence of MotA. Our results suggest that amino acid residues within the N-terminal half of AsiA are involved in forming or maintaining the AsiA/sigma(70) complex. The C-terminal region of AsiA, while not absolutely required for inhibition or co-activation, aids inhibition by slowing the formation of transcription complexes between a promoter and the AsiA/polymerase complex.
Similar articles
- A family of anti-sigma70 proteins in T4-type phages and bacteria that are similar to AsiA, a Transcription inhibitor and co-activator of bacteriophage T4.
Pineda M, Gregory BD, Szczypinski B, Baxter KR, Hochschild A, Miller ES, Hinton DM. Pineda M, et al. J Mol Biol. 2004 Dec 10;344(5):1183-97. doi: 10.1016/j.jmb.2004.10.003. J Mol Biol. 2004. PMID: 15561138 - Bacteriophage T4 MotA activator and the β-flap tip of RNA polymerase target the same set of σ70 carboxyl-terminal residues.
Bonocora RP, Decker PK, Glass S, Knipling L, Hinton DM. Bonocora RP, et al. J Biol Chem. 2011 Nov 11;286(45):39290-6. doi: 10.1074/jbc.M111.278762. Epub 2011 Sep 12. J Biol Chem. 2011. PMID: 21911499 Free PMC article. - A basic/hydrophobic cleft of the T4 activator MotA interacts with the C-terminus of E.coli sigma70 to activate middle gene transcription.
Bonocora RP, Caignan G, Woodrell C, Werner MH, Hinton DM. Bonocora RP, et al. Mol Microbiol. 2008 Jul;69(2):331-43. doi: 10.1111/j.1365-2958.2008.06276.x. Mol Microbiol. 2008. PMID: 18485078 Free PMC article. - Transcriptional takeover by sigma appropriation: remodelling of the sigma70 subunit of Escherichia coli RNA polymerase by the bacteriophage T4 activator MotA and co-activator AsiA.
Hinton DM, Pande S, Wais N, Johnson XB, Vuthoori M, Makela A, Hook-Barnard I. Hinton DM, et al. Microbiology (Reading). 2005 Jun;151(Pt 6):1729-1740. doi: 10.1099/mic.0.27972-0. Microbiology (Reading). 2005. PMID: 15941982 Review. - Transcriptional control in the prereplicative phase of T4 development.
Hinton DM. Hinton DM. Virol J. 2010 Oct 28;7:289. doi: 10.1186/1743-422X-7-289. Virol J. 2010. PMID: 21029433 Free PMC article. Review.
Cited by
- Direct activator/co-activator interaction is essential for bacteriophage T4 middle gene expression.
Yuan AH, Hochschild A. Yuan AH, et al. Mol Microbiol. 2009 Nov;74(4):1018-30. doi: 10.1111/j.1365-2958.2009.06916.x. Epub 2009 Oct 15. Mol Microbiol. 2009. PMID: 19843221 Free PMC article. - Determinants of affinity and activity of the anti-sigma factor AsiA.
Gilmore JM, Bieber Urbauer RJ, Minakhin L, Akoyev V, Zolkiewski M, Severinov K, Urbauer JL. Gilmore JM, et al. Biochemistry. 2010 Jul 27;49(29):6143-54. doi: 10.1021/bi1002635. Biochemistry. 2010. PMID: 20545305 Free PMC article. - Structural basis of σ appropriation.
Shi J, Wen A, Zhao M, You L, Zhang Y, Feng Y. Shi J, et al. Nucleic Acids Res. 2019 Sep 26;47(17):9423-9432. doi: 10.1093/nar/gkz682. Nucleic Acids Res. 2019. PMID: 31392983 Free PMC article. - Visualizing the phage T4 activated transcription complex of DNA and E. coli RNA polymerase.
James TD, Cardozo T, Abell LE, Hsieh ML, Jenkins LM, Jha SS, Hinton DM. James TD, et al. Nucleic Acids Res. 2016 Sep 19;44(16):7974-88. doi: 10.1093/nar/gkw656. Epub 2016 Jul 25. Nucleic Acids Res. 2016. PMID: 27458207 Free PMC article. - Mutational analysis of sigma70 region 4 needed for appropriation by the bacteriophage T4 transcription factors AsiA and MotA.
Baxter K, Lee J, Minakhin L, Severinov K, Hinton DM. Baxter K, et al. J Mol Biol. 2006 Nov 10;363(5):931-44. doi: 10.1016/j.jmb.2006.08.074. Epub 2006 Aug 30. J Mol Biol. 2006. PMID: 16996538 Free PMC article.