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PMID: 8816769 Published · ppublish English Comparative Study Journal Article Research Support, U.S. Gov't, P.H.S.

Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 93 ·No. 19 ·1996-09-17 ·Pages 10162-6

Heyduk T, Heyduk E, Severinov K, Tang H, Ebright RH

Abstract

Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta (i.e., in alpha 2 beta, alpha 2 beta beta', and alpha 2 beta beta' sigma), and amino acids 175-210 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta' (i.e., in alpha 2 beta beta' and alpha 2 beta beta' sigma). The protected regions are conserved in the alpha homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of alpha for direct functional interaction with beta and beta'. The observed maximal magnitude of protection upon interaction with beta and the observed maximal magnitude of protection upon interaction with beta' both correspond to the expected value for complete protection of one of the two alpha protomers of RNAP (i.e., 50% protection). We propose that only one of the two alpha protomers of RNAP interacts with beta and that only one of the two alpha protomers of RNAP interacts with beta'.

MeSH Terms
Amino Acid Sequence Archaea/enzymology Binding Sites Chloroplasts/enzymology DNA-Directed RNA Polymerases/chemistry,metabolism Escherichia coli/enzymology Histidine Hydrolysis Hydroxyl Radical Macromolecular Substances Molecular Sequence Data Protein Multimerization Recombinant Proteins/chemistry,metabolism Saccharomyces cerevisiae/enzymology Sequence Homology, Amino Acid Sequence Tagged Sites Species Specificity
Chemicals
Macromolecular Substances Recombinant Proteins Hydroxyl Radical Histidine DNA-Directed RNA Polymerases
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Heyduk T
Department of Biochemistry and Molecular Biology, St. Louis University Medical School, MO 63104, USA.
Heyduk E
Severinov K
Tang H
Ebright R H
References (32)
32 references, click to expand
  1. Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase.
    Proc Natl Acad Sci U S A. 1995 May 23;92(11):4902-6 PMID: 7761421
  2. Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments.
    Proc Natl Acad Sci U S A. 1995 May 9;92(10):4591-5 PMID: 7753849
  3. Repression and activation of transcription by Gal and Lac repressors: involvement of alpha subunit of RNA polymerase.
    EMBO J. 1995 Sep 15;14(18):4523-9 PMID: 7556095
  4. Functional map of the alpha subunit of Escherichia coli RNA polymerase: amino acid substitution within the amino-terminal assembly domain.
    J Mol Biol. 1995 Dec 1;254(3):342-9 PMID: 7490753
  5. Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting.
    Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):71-5 PMID: 8552678
  6. Role of Escherichia coli RNA polymerase alpha subunit in modulation of pausing, termination and anti-termination by the transcription elongation factor NusA.
    EMBO J. 1996 Jan 2;15(1):150-61 PMID: 8598198
  7. Defective assembly of ribonucleic acid polymerase subunits in a temperature-sensitive alpha-subunit mutant of Escherichia coli.
    Biochemistry. 1980 Jul 22;19(15):3491-5 PMID: 6996704
  8. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.
    Anal Biochem. 1987 Nov 1;166(2):368-79 PMID: 2449095
  9. Creation of phosphorylation sites in proteins: construction of a phosphorylatable human interferon alpha.
    Proc Natl Acad Sci U S A. 1989 Jan;86(2):558-62 PMID: 2911594
  10. Monoclonal antibodies as probes of the topological arrangement of the alpha subunits of Escherichia coli RNA polymerase.
    Biochemistry. 1989 Apr 18;28(8):3299-305 PMID: 2472834
  11. Sequence analysis of two temperature-sensitive mutations in the alpha subunit gene (rpoA) of Escherichia coli RNA polymerase.
    Nucleic Acids Res. 1990 Oct 25;18(20):5945-8 PMID: 2235479
  12. Identification of a subunit assembly domain in the alpha subunit of Escherichia coli RNA polymerase.
    J Mol Biol. 1991 Mar 5;218(1):1-6 PMID: 2002495
  13. Bipartite functional map of the E. coli RNA polymerase alpha subunit: involvement of the C-terminal region in transcription activation by cAMP-CRP.
    Cell. 1991 Jun 14;65(6):1015-22 PMID: 1646077
  14. Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.
    Mol Cell Biol. 1991 Sep;11(9):4669-78 PMID: 1715023
  15. Functional specialization within the alpha-subunit of Escherichia coli RNA polymerase.
    J Mol Biol. 1991 Sep 5;221(1):23-9 PMID: 1920407
  16. Efficient site-directed mutagenesis using uracil-containing DNA.
    Methods Enzymol. 1991;204:125-39 PMID: 1943776
  17. Transfer of oxygen from an artificial protease to peptide carbon during proteolysis.
    Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10578-82 PMID: 1961724
  18. Interactions between three common subunits of yeast RNA polymerases I and III.
    Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5524-8 PMID: 8516295
  19. Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.
    Nucleic Acids Res. 1993 Aug 11;21(16):3749-54 PMID: 8367291
  20. A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase.
    Science. 1993 Nov 26;262(5138):1407-13 PMID: 8248780
  21. Oxidative polypeptide cleavage mediated by EDTA-Fe covalently linked to cysteine residues.
    Biochemistry. 1993 Nov 30;32(47):12761-7 PMID: 8251497
  22. Recombinant Escherichia coli RNA polymerase: purification of individually overexpressed subunits and in vitro assembly.
    Protein Expr Purif. 1993 Dec;4(6):503-11 PMID: 8286946
  23. Identification of the target of a transcription activator protein by protein-protein photocrosslinking.
    Science. 1994 Jul 1;265(5168):90-2 PMID: 8016656
  24. Mapping protein domains involved in macromolecular interactions: a novel protein footprinting approach.
    Biochemistry. 1994 Aug 16;33(32):9643-50 PMID: 8068641
  25. Domain organization of RNA polymerase alpha subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding.
    Cell. 1994 Sep 9;78(5):889-96 PMID: 8087855
  26. Functional map of the alpha subunit of Escherichia coli RNA polymerase. Deletion analysis of the amino-terminal assembly domain.
    J Mol Biol. 1994 Sep 16;242(2):107-15 PMID: 8089834
  27. Use of aryl azide cross-linkers to investigate protein-protein interactions: an optimization of important conditions as applied to Escherichia coli RNA polymerase and localization of a sigma 70-alpha cross-link to the C-terminal region of alpha.
    Biochemistry. 1994 Oct 11;33(40):12092-9 PMID: 7918430
  28. Mapping staphylococcal nuclease conformation using an EDTA-Fe derivative attached to genetically engineered cysteine residues.
    Biochemistry. 1994 Nov 22;33(46):13625-41 PMID: 7947771
  29. Promoter structure, promoter recognition, and transcription activation in prokaryotes.
    Cell. 1994 Dec 2;79(5):743-6 PMID: 8001112
  30. Structural map of the alpha subunit of Escherichia coli RNA polymerase: structural domains identified by proteolytic cleavage.
    J Mol Biol. 1995 May 12;248(4):723-8 PMID: 7752234
  31. Functional map of the alpha subunit of Escherichia coli RNA polymerase: insertion analysis of the amino-terminal assembly domain.
    J Mol Biol. 1995 May 12;248(4):756-67 PMID: 7752238
  32. The Escherichia coli RNA polymerase alpha subunit: structure and function.
    Curr Opin Genet Dev. 1995 Apr;5(2):197-203 PMID: 7613089
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.93.19.10162
Published
1996-09-17
Pages
10162-6
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC38354
Subset
IM
Grants
NIGMS NIH HHS · GM50514 · United States
NIGMS NIH HHS · GM51527 · United States
External Links
PubMed source DOI: 10.1073/pnas.93.19.10162 PMC Full Text View journal details
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