A hybrid sigma subunit directs RNA polymerase to a hybrid promoter in Escherichia coli

Ashok Kumar, Brenda Grimes, Mary Logan, Stephen Wedgwood, Helen Williamson, Richard S. Hayward

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Most of the sigma (transcriptional initiation specificity) subunits of RNA polymerase, from a wide range of eubacteria, show strong elements of amino acid sequence similarity. There is evidence that two of the “conserved” regions, 2.4 and 4.2, are involved in recognition of the consensus DNA sequences centred near -10 and -35, respectively, which define promoter sites for the initiation of transcription. Since all the alternative sigma subunits of the above type function by binding to a common core polymerase enzyme in a given bacterium, it can be predicted that a hybrid sigma might be functional, and if so should permit RNA polymerase to initiate only at a correspondingly hybrid promoter. To test these predictions, a hybrid gene encoding the amino-proximal 529 amino acids of the major Escherichia coli σ protein, σ70 (including region 2.4) followed by the last 82 amino acids of the heat-shock σ protein, σ32 (including region 4.2) was constructed and fused to Plac on a plasmid. Major-consensus, heat-shock and hybrid promoters were fused to a chloramphenicol acetyl transferase (CAT) reporter gene on a compatible plasmid. CAT assays showed that, as predicted, a promoter with a “heat-shock” -35 consensus and a “major” -10 consensus sequence (PHM) required Plac-dependent production of the hybrid sigma (σ70-32) for activity in vivo. PHM then became a strong promoter. The hybrid sigma gene has potential advantages over its parents for structure-function studies.

Original languageEnglish (US)
Pages (from-to)563-571
Number of pages9
JournalJournal of molecular biology
Volume246
Issue number5
DOIs
StatePublished - Jan 1 1995

Fingerprint

Sigma Factor
DNA-Directed RNA Polymerases
Escherichia coli
Consensus Sequence
Chloramphenicol
Transferases
Shock
Plasmids
Hot Temperature
Bacteria
Amino Acids
Escherichia coli Proteins
Transcription Initiation Site
Heat-Shock Proteins
Reporter Genes
Genes
Amino Acid Sequence
Enzymes

Keywords

  • E. coli
  • Hybrid promoter
  • Hybrid sigma
  • In vivo
  • Transcription

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

A hybrid sigma subunit directs RNA polymerase to a hybrid promoter in Escherichia coli. / Kumar, Ashok; Grimes, Brenda; Logan, Mary; Wedgwood, Stephen; Williamson, Helen; Hayward, Richard S.

In: Journal of molecular biology, Vol. 246, No. 5, 01.01.1995, p. 563-571.

Research output: Contribution to journalArticle

Kumar, Ashok ; Grimes, Brenda ; Logan, Mary ; Wedgwood, Stephen ; Williamson, Helen ; Hayward, Richard S. / A hybrid sigma subunit directs RNA polymerase to a hybrid promoter in Escherichia coli. In: Journal of molecular biology. 1995 ; Vol. 246, No. 5. pp. 563-571.
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