Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both α and σ70 subunits of Escherichia coli RNA polymerase

Irina Artsimovitch, Katsuhiko Murakami, Akira Ishihama, Martha M. Howe

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, P(m), has a -10 hexamer but lacks a recognizable -35 hexamer. Interactions between Mor and RNA polymerase were studied using in vitro transcription, DNase I footprinting, and the yeast interaction trap system. We observed reduced promoter activity in vitro using reconstituted RNA polymerases with C-terminal deletions in α or σ70. As predicted if α were binding to P(m), we detected a polymerase-dependent footprint in the - 60 region. Reconstituted RNA polymerases containing Ala substitutions in the α C-terminal domain were used to assay Mor-dependent transcription from P(m) in vitro. The D258A substitution and α deletion gave large reductions in activation, whereas the L262A, R265A, and N268A substitutions caused smaller reductions. The interaction trap assay revealed weak interactions between Mor and both α and σ70; consistent with a key role of α-D258, the D258A substitution abolished interaction, whereas the R265A substitution did not. We propose that: (i) α-D258 is a Mor 'contact site'; and (ii) residues Leu- 262, Arg-265, and Asn-268 indirectly affect Mor-polymerase interaction by stabilizing the ternary complex via α-DNA contact.

Original languageEnglish (US)
Pages (from-to)32343-32348
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number50
DOIs
StatePublished - Dec 30 1996

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DNA-Directed RNA Polymerases
Transcription
Protein C
Escherichia coli
Transcriptional Activation
Substitution reactions
Chemical activation
Bacteriophage mu
RNA Polymerase III
Deoxyribonuclease I
Assays
Yeasts
Bacteriophages
Yeast
DNA
Enterobacteria phage Mu Mor protein
In Vitro Techniques
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both α and σ70 subunits of Escherichia coli RNA polymerase",
abstract = "Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, P(m), has a -10 hexamer but lacks a recognizable -35 hexamer. Interactions between Mor and RNA polymerase were studied using in vitro transcription, DNase I footprinting, and the yeast interaction trap system. We observed reduced promoter activity in vitro using reconstituted RNA polymerases with C-terminal deletions in α or σ70. As predicted if α were binding to P(m), we detected a polymerase-dependent footprint in the - 60 region. Reconstituted RNA polymerases containing Ala substitutions in the α C-terminal domain were used to assay Mor-dependent transcription from P(m) in vitro. The D258A substitution and α deletion gave large reductions in activation, whereas the L262A, R265A, and N268A substitutions caused smaller reductions. The interaction trap assay revealed weak interactions between Mor and both α and σ70; consistent with a key role of α-D258, the D258A substitution abolished interaction, whereas the R265A substitution did not. We propose that: (i) α-D258 is a Mor 'contact site'; and (ii) residues Leu- 262, Arg-265, and Asn-268 indirectly affect Mor-polymerase interaction by stabilizing the ternary complex via α-DNA contact.",
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Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both α and σ70 subunits of Escherichia coli RNA polymerase. / Artsimovitch, Irina; Murakami, Katsuhiko; Ishihama, Akira; Howe, Martha M.

In: Journal of Biological Chemistry, Vol. 271, No. 50, 30.12.1996, p. 32343-32348.

Research output: Contribution to journalArticle

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T1 - Transcription activation by the bacteriophage Mu Mor protein requires the C-terminal regions of both α and σ70 subunits of Escherichia coli RNA polymerase

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AU - Howe, Martha M.

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