Home LiteratureArticle Details
PMID: 10747024 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

The Escherichia coli RNA polymerase alpha subunit linker: length requirements for transcription activation at CRP-dependent promoters.

The EMBO journal ·Vol. 19 ·No. 7 ·2000-04-03 ·Pages 1555-66

Meng W, Savery NJ, Busby SJ, Thomas MS

Abstract

The C-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alphaCTD) plays a key role in transcription initiation at many activator-dependent promoters. This domain is connected to the N-terminal domain by an unstructured linker, which is proposed to confer a high degree of mobility on alphaCTD. To investigate the role of this linker in transcription activation we tested the effect of altering the linker length on promoters dependent on the cyclic AMP receptor protein (CRP). Short deletions within the alpha linker decrease CRP-dependent transcription at a Class I promoter while increasing the activity of a Class II promoter. Linker extension impairs CRP-dependent transcription from both promoters, with short extensions exerting a more marked effect on the Class II promoter. Activation at both classes of promoter was shown to remain dependent upon activating region 1 of CRP. These results show that the response to CRP of RNA polymerase containing linker-modified alpha subunits is class specific. These observations have important implications for the architecture of transcription initiation complexes at CRP-dependent promoters.

MeSH Terms
Amino Acid Sequence Base Sequence Cyclic AMP Receptor Protein/genetics,metabolism DNA, Bacterial/genetics DNA-Directed RNA Polymerases/chemistry,genetics,metabolism Escherichia coli/enzymology,genetics,metabolism Genes, Bacterial Models, Biological Molecular Sequence Data Mutagenesis Promoter Regions, Genetic Protein Structure, Tertiary Sequence Homology, Amino Acid Transcriptional Activation
Chemicals
Cyclic AMP Receptor Protein DNA, Bacterial DNA-Directed RNA Polymerases RNA polymerase alpha subunit
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Meng W
Division of Molecular and Genetic Medicine, University of Sheffield Medical School, Sheffield S10 2RX, UK.
Savery N J
Busby S J
Thomas M S
References (62)
62 references, click to expand
  1. Transcription activation by catabolite activator protein (CAP).
    J Mol Biol. 1999 Oct 22;293(2):199-213 PMID: 10550204
  2. The Escherichia coli RNA polymerase alpha subunit: structure and function.
    Curr Opin Genet Dev. 1995 Apr;5(2):197-203 PMID: 7613089
  3. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system.
    Gene. 1977;2(2):95-113 PMID: 344137
  4. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli.
    J Mol Biol. 1980 Apr;138(2):179-207 PMID: 6997493
  5. Deletion mutagenesis of the Escherichia coli galactose operon promoter region.
    J Mol Biol. 1983 Jun 25;167(2):259-74 PMID: 6408264
  6. The locus of sequence-directed and protein-induced DNA bending.
    Nature. 1984 Apr 5-11;308(5959):509-13 PMID: 6323997
  7. Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoter.
    J Mol Biol. 1984 Dec 25;180(4):881-909 PMID: 6098691
  8. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.
    J Mol Biol. 1986 May 5;189(1):113-30 PMID: 3537305
  9. Transcription activation at Escherichia coli promoters dependent on the cyclic AMP receptor protein: effects of binding sequences for the RNA polymerase alpha-subunit.
    Biochem J. 1995 Jul 1;309 ( Pt 1):77-83 PMID: 7619086
  10. Spacing requirements for transcription activation by Escherichia coli FNR protein.
    J Bacteriol. 1995 Dec;177(23):6704-10 PMID: 7592457
  11. Solution structure of the activator contact domain of the RNA polymerase alpha subunit.
    Science. 1995 Dec 1;270(5241):1495-7 PMID: 7491496
  12. 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
  13. DNA-binding determinants of the alpha subunit of RNA polymerase: novel DNA-binding domain architecture.
    Genes Dev. 1996 Jan 1;10(1):16-26 PMID: 8557191
  14. Location of the C-terminal domain of the RNA polymerase alpha subunit in different open complexes at the Escherichia coli galactose operon regulatory region.
    Nucleic Acids Res. 1996 Jun 15;24(12):2242-51 PMID: 8710492
  15. Identification of an UP element within the IHF binding site at the PL1-PL2 tandem promoter of bacteriophage lambda.
    J Mol Biol. 1996 Jul 26;260(4):484-91 PMID: 8759315
  16. Transcription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase.
    Cell. 1996 Dec 13;87(6):1123-34 PMID: 8978616
  17. Transcription activation at class II CRP-dependent promoters: the role of different activating regions.
    Nucleic Acids Res. 1997 Jan 15;25(2):326-32 PMID: 9016561
  18. Transcription activation at class II CAP-dependent promoters.
    Mol Microbiol. 1997 Mar;23(5):853-9 PMID: 9076723
  19. A nested set of C-terminal deletions of the alpha subunit of Escherichia coli RNA polymerase define regions concerned with assembly, proteolysis, stabilization and transcriptional activation in vivo.
    Genes Cells. 1997 Jan;2(1):81-94 PMID: 9112442
  20. Flexible linker in the RNA polymerase alpha subunit facilitates the independent motion of the C-terminal activator contact domain.
    J Mol Biol. 1997 Apr 11;267(4):953-62 PMID: 9135123
  21. Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors.
    Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11274-8 PMID: 9326599
  22. Spacing requirements for interactions between the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase and the cAMP receptor protein.
    Biochem J. 1998 Feb 15;330 ( Pt 1):413-20 PMID: 9461538
  23. Transcription activation at promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein: organisation of the RNA polymerase alpha subunits.
    J Mol Biol. 1998 Apr 10;277(4):789-804 PMID: 9545373
  24. Transcription activation at Class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase alpha subunit.
    EMBO J. 1998 Jun 15;17(12):3439-47 PMID: 9628879
  25. Structure of the Escherichia coli RNA polymerase alpha subunit amino-terminal domain.
    Science. 1998 Jul 10;281(5374):262-6 PMID: 9657722
  26. Identification of an UP element consensus sequence for bacterial promoters.
    Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):9761-6 PMID: 9707549
  27. The C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase is required for efficient rho-dependent transcription termination.
    J Mol Biol. 1998 Dec 18;284(5):1379-90 PMID: 9878357
  28. Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the alpha subunit of RNA polymerase at class I promoters.
    Biochem J. 1999 Feb 1;337 ( Pt 3):415-23 PMID: 9895284
  29. An inactive open complex mediated by an UP element at Escherichia coli promoters.
    Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7202-7 PMID: 10377392
  30. 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
  31. Scanning calorimetric study of the thermal unfolding of catabolite activator protein from Escherichia coli in the absence and presence of cyclic mononucleotides.
    Biochemistry. 1988 Jul 12;27(14):5257-61 PMID: 2844254
  32. Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products.
    Gene. 1990 Apr 30;89(1):7-12 PMID: 2197183
  33. Stringent spacing requirements for transcription activation by CRP.
    Cell. 1990 Aug 24;62(4):733-43 PMID: 2167178
  34. Helical phase dependent action of CRP: effect of the distance between the CRP site and the -35 region on promoter activity.
    Nucleic Acids Res. 1990 Nov 11;18(21):6325-30 PMID: 2173826
  35. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription.
    Nucleic Acids Res. 1990 Dec 25;18(24):7243-50 PMID: 2259621
  36. 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
  37. 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
  38. Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees.
    Science. 1991 Aug 30;253(5023):1001-7 PMID: 1653449
  39. Allosteric changes in the cAMP receptor protein of Escherichia coli: hinge reorientation.
    Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9700-4 PMID: 1409686
  40. Mapping the cAMP receptor protein contact site on the alpha subunit of Escherichia coli RNA polymerase.
    Mol Microbiol. 1992 Sep;6(18):2599-605 PMID: 1333035
  41. E. coli RNA polymerase, deleted in the C-terminal part of its alpha-subunit, interacts differently with the cAMP-CRP complex at the lacP1 and at the galP1 promoter.
    Nucleic Acids Res. 1993 Jan 25;21(2):319-26 PMID: 8382795
  42. Identification of the functional subunit of a dimeric transcription activator protein by use of oriented heterodimers.
    Cell. 1993 Apr 23;73(2):375-9 PMID: 8477449
  43. Protein-protein communication within the transcription apparatus.
    J Bacteriol. 1993 May;175(9):2483-9 PMID: 8478317
  44. Rifampicin region revisited. New rifampicin-resistant and streptolydigin-resistant mutants in the beta subunit of Escherichia coli RNA polymerase.
    J Biol Chem. 1993 Jul 15;268(20):14820-5 PMID: 8325861
  45. Identification of the activating region of catabolite gene activator protein (CAP): isolation and characterization of mutants of CAP specifically defective in transcription activation.
    Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6081-5 PMID: 8392187
  46. Transcriptional regulation by cAMP and its receptor protein.
    Annu Rev Biochem. 1993;62:749-95 PMID: 8394684
  47. Transcription activation at Class I CAP-dependent promoters.
    Mol Microbiol. 1993 May;8(5):797-802 PMID: 8394979
  48. Improved single and multicopy lac-based cloning vectors for protein and operon fusions.
    Gene. 1987;53(1):85-96 PMID: 3596251
  49. Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites.
    J Mol Biol. 1988 Apr 20;200(4):709-23 PMID: 3045325
  50. 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
  51. Factor independent activation of rrnB P1. An "extended" promoter with an upstream element that dramatically increases promoter strength.
    J Mol Biol. 1994 Feb 4;235(5):1421-35 PMID: 8107083
  52. 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
  53. The functional subunit of a dimeric transcription activator protein depends on promoter architecture.
    EMBO J. 1994 Oct 3;13(19):4549-57 PMID: 7925296
  54. Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). I. Saturation and alanine-scanning mutagenesis.
    J Mol Biol. 1994 Nov 4;243(4):595-602 PMID: 7966284
  55. Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). II. Role at Class I and class II CAP-dependent promoters.
    J Mol Biol. 1994 Nov 4;243(4):603-10 PMID: 7966285
  56. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.
    Nucleic Acids Res. 1994 Oct 25;22(21):4375-80 PMID: 7971267
  57. Location, structure, and function of the target of a transcriptional activator protein.
    Genes Dev. 1994 Dec 15;8(24):3058-67 PMID: 8001824
  58. Selectivity of the Escherichia coli RNA polymerase E sigma 38 for overlapping promoters and ability to support CRP activation.
    Nucleic Acids Res. 1995 Mar 11;23(5):819-26 PMID: 7708498
  59. 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
  60. 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
  61. 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
  62. Structural requirements for the interdomain linker of alpha subunit of Escherichia coli RNA polymerase.
    Biochemistry. 2000 May 23;39(20):6243-9 PMID: 10821700
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1093/emboj/19.7.1555
Published
2000-04-03
Pages
1555-66
Language
English
Region
England
NLM ID
8208664
PMCID
PMC310225
Subset
IM
External Links
PubMed source DOI: 10.1093/emboj/19.7.1555 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2