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. 1983 May 11;11(9):2701–2716. doi: 10.1093/nar/11.9.2701

Chemical modifications of the sigma subunit of the E. coli RNA polymerase.

C S Narayanan, J S Krakow
PMCID: PMC325918  PMID: 6344020

Abstract

The function of arginine, cysteine and carboxylic amino acid (glutamic and aspartic) residues of sigma was studied using chemical modification by group specific reagents. Following modification of 3 arginine residues with phenylglyoxal or 3 cysteine residues with N-ethylmaleimide (NEM) sigma activity was lost. Analysis of the kinetic data for inactivation indicated that one arginine or cysteine residue is essential for sigma activity. At low NEM concentration alkylation was limited to a non-critical cysteine which was identified as cysteine-132. Modification of arginine or cysteine residues had no observable effect on the binding of the inactivated sigma to the core polymerase. Modification of aspartic and/or glutamic acid residues with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) or 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMC) resulted in loss of sigma activity. The inactivation data indicated that one carboxylic amino acid residue is essential for sigma activity. Sigma modified with EDC, CMC or EDC in the presence of glycine was inactive in supporting promoter binding and initiation by core polymerase. Reaction with EDC plus (3H)glycine resulted in the incorporation of glycine into sigma. The (3H)glycine-sigma was unable to form a stable holoenzyme complex.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Borders C. L., Jr, Riordan J. F. An essential arginyl residue at the nucleotide binding site of creatine kinase. Biochemistry. 1975 Oct 21;14(21):4699–4704. doi: 10.1021/bi00692a021. [DOI] [PubMed] [Google Scholar]
  2. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  3. Burton Z., Burgess R. R., Lin J., Moore D., Holder S., Gross C. A. The nucleotide sequence of the cloned rpoD gene for the RNA polymerase sigma subunit from E coli K12. Nucleic Acids Res. 1981 Jun 25;9(12):2889–2903. doi: 10.1093/nar/9.12.2889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chenchick A., Beabealashvilli R., Mirzabekov A. Topography of interaction of Escherichia coli RNA polymerase subunits with lac UV5 promoter. FEBS Lett. 1981 Jun 1;128(1):46–50. doi: 10.1016/0014-5793(81)81076-9. [DOI] [PubMed] [Google Scholar]
  5. Colman R. F. A glutamyl residue in the active site of triphosphopyridine nucleotide-dependent isocitrate dehydrogenase of pig heart. J Biol Chem. 1973 Dec 10;248(23):8137–8143. [PubMed] [Google Scholar]
  6. Hansen U. M., McClure W. R. A noncycling activity assay for the omega subunit of Escherichia coli RNA polymerase. J Biol Chem. 1979 Jul 10;254(13):5713–5717. [PubMed] [Google Scholar]
  7. Harris J. D., Heilig J. S., Martinez I. I., Calendar R., Isaksson L. A. Temperature-sensitive Escherichia coli mutant producing a temperature-sensitive sigma subunit of DNA-dependent RNA polymerase. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6177–6181. doi: 10.1073/pnas.75.12.6177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hillel Z., Wu C. W. Subunit topography of RNA polymerase from Escherichia coli. A cross-linking study with bifunctional reagents. Biochemistry. 1977 Jul 26;16(15):3334–3342. doi: 10.1021/bi00634a008. [DOI] [PubMed] [Google Scholar]
  9. Kantrowitz E. R., Lipscomb W. N. An essential residue at the active site of aspartate transcarbamylase. J Biol Chem. 1976 May 10;251(9):2688–2695. [PubMed] [Google Scholar]
  10. Krakow J. S. Inhibition of Azotobacter vinelandii ribonucleic acid polymerase by glutamyl, tyrosyl copolymers. Biochemistry. 1974 Mar 12;13(6):1101–1105. doi: 10.1021/bi00703a007. [DOI] [PubMed] [Google Scholar]
  11. Kudo T., Doi R. H. Free sigma factor of Escherichia coli RNA polymerase can bind to DNA. J Biol Chem. 1981 Oct 10;256(19):9778–9781. [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lowe P. A., Aebi U., Gross C., Burgess R. R. In vitro thermal inactivation of a temperature-sensitive sigma subunit mutant (rpoD800) of Escherichia coli RNA polymerase proceeds by aggregation. J Biol Chem. 1981 Feb 25;256(4):2010–2015. [PubMed] [Google Scholar]
  14. Lowe P. A., Hager D. A., Burgess R. R. Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. Biochemistry. 1979 Apr 3;18(7):1344–1352. doi: 10.1021/bi00574a034. [DOI] [PubMed] [Google Scholar]
  15. MarSchel A. H., Bodley J. W. Inactivation of Escherichia coli elongation factor Ts by the arginine-specific reagent butanedione. J Biol Chem. 1979 Mar 25;254(6):1816–1820. [PubMed] [Google Scholar]
  16. Marshall M., Cohen P. P. Evidence for an exceptionally reactive arginyl residue at the binding site for carbamyl phosphate in bovine ornithine transcarbamylase. J Biol Chem. 1980 Aug 10;255(15):7301–7305. [PubMed] [Google Scholar]
  17. Narayanan C. S., Krakow J. S. Effect of lysine modification on the activity of the sigma subunit of Escherichia coli RNA polymerase. Biochemistry. 1982 Nov 23;21(24):6103–6111. doi: 10.1021/bi00267a012. [DOI] [PubMed] [Google Scholar]
  18. Osawa T., Yura T. Amber mutations in the structural gene for RNA polymerase sigma factor of Escherichia coli. Mol Gen Genet. 1980;180(2):293–300. doi: 10.1007/BF00425841. [DOI] [PubMed] [Google Scholar]
  19. Park C. S., Hillel Z., Wu C. W. DNA strand specificity in promoter recognition by RNA polymerase. Nucleic Acids Res. 1980 Dec 11;8(23):5895–5912. doi: 10.1093/nar/8.23.5895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ramachandran N., Colman R. F. Evidence for a critical glutamyl and an aspartyl residue in the function of pig heart diphosphopyridine nucleotide dependent isocitrate dehydrogenase. Biochemistry. 1977 Apr 19;16(8):1564–1573. doi: 10.1021/bi00627a006. [DOI] [PubMed] [Google Scholar]
  21. Rohrbach M. S., Bodley J. W. Selective chemical modification of Escherichia coli elongation factor G: butanedione modification of an arginine essential for nucleotide binding. Biochemistry. 1977 Apr 5;16(7):1360–1363. doi: 10.1021/bi00626a019. [DOI] [PubMed] [Google Scholar]
  22. Satre M., Lunardi J., Pougeois R., Vignais P. V. Inactivation of Escherichia coli BF1-ATPase by dicyclohexylcarbodiimide. Chemical modification of the beta subunit. Biochemistry. 1979 Jul 10;18(14):3134–3140. doi: 10.1021/bi00581a034. [DOI] [PubMed] [Google Scholar]
  23. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  24. Simpson R. B. The molecular topography of RNA polymerase-promoter interaction. Cell. 1979 Oct;18(2):277–285. doi: 10.1016/0092-8674(79)90047-3. [DOI] [PubMed] [Google Scholar]
  25. Srivastava A., Modak M. J. Phenylglyoxal as a template site-specific reagent for DNA and RNA polymerases. Selective inhibition of initiation. J Biol Chem. 1980 Feb 10;255(3):917–921. [PubMed] [Google Scholar]
  26. Stöckel P., May R., Strell I., Cejka Z., Hoppe W., Heumann H., Zillig W., Crespi H. L. The subunit positions within RNA polymerase holoenzyme determined by triangulation of centre-to-centre distances. Eur J Biochem. 1980 Nov;112(2):419–423. doi: 10.1111/j.1432-1033.1980.tb07221.x. [DOI] [PubMed] [Google Scholar]
  27. Takahashi K. The reaction of phenylglyoxal with arginine residues in proteins. J Biol Chem. 1968 Dec 10;243(23):6171–6179. [PubMed] [Google Scholar]
  28. Wu F. Y., Yarbrough L. R., Wu C. W. Conformational transition of Escherichia coli RNA polymerase induced by the interaction of sigma subunit with core enzyme. Biochemistry. 1976 Jul 27;15(15):3254–3258. doi: 10.1021/bi00660a014. [DOI] [PubMed] [Google Scholar]
  29. Yarbrough L. R., Wu C. W. Role of sulfhydryl residues of Escherichia coli ribonucleic acid polymerase in template recognition and specific initiation. J Biol Chem. 1974 Jul 10;249(13):4079–4085. [PubMed] [Google Scholar]
  30. von Gabain A., Bujard H. Interaction of E. coli RNA polymerase with promotors of coliphage T5: the rates of complex formation and decay and their correlation with in vitro and in vivo transcriptional activity. Mol Gen Genet. 1977 Dec 9;157(3):301–311. doi: 10.1007/BF00268667. [DOI] [PubMed] [Google Scholar]
  31. von Gabain A., Hayward G. S., Bujard H. Physical mapping of the HindIII, EcoRI, Sal and Sma restriction endonuclease cleavage fragments from bacteriophage T5 DNA. Mol Gen Genet. 1976 Feb 2;143(3):279–290. doi: 10.1007/BF00269404. [DOI] [PubMed] [Google Scholar]

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