Evidence for Contact between the Cyclic AMP Receptor Protein and the σ70 Subunit of Escherichia coli RNA Polymerase

Ruzhong Jin,Karim A Sharif,Joseph S Krakow

doi:10.1074/jbc.270.33.19213

Ruzhong Jin, Karim A Sharif + Show 1 more

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https://doi.org/10.1074/jbc.270.33.19213

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Abstract

The loop at the 52-position of the cAMP receptor protein (CRP) has been suggested as a potential site for contacting RNA polymerase on Class II promoters where the CRP binding site is located at position -41.5 (Bell, A., Gaston, K., Williams, R., Chapman, K., Kolb, A., Buc, H., Minchin, S., Williams, J., and Busby, S. (1990) Nucleic Acids Res. 18, 7243-7250). Using protein-protein photo-cross-linking, evidence is presented showing that the 52-loop of CRP is in physical proximity to the delta subunit of RNA polymerase holoenzyme. This interaction required the presence of a functional preinitiation complex. The CRP suppressor mutation, K52N, increased the efficiency of cross-linking, indicating an improved physical interaction between the CRP 52-loop and the delta subunit. Evidence for direct interaction between the CRP 156-162 loop and delta subunit of RNA polymerase on both gal and lac promoters are also provided. The data indicate that CRP bound to the gal promoter contacts both the alpha and delta 70 subunits of RNA polymerase.

Highlights

From the Department of Biological Sciences, Hunter College of the City University of New York, New York, New York 10021
Evidence indicates that contact between cAMP receptor protein (CRP) and RNA polymerase is involved in transcription activation at the Zac promoter
DNase I footprinting demonstrated that CRP and RNA polymerase cooperatively bind to the lac promoter (Li and Krakow, 1985; Ren et aZ., 1988; Straney et aZ., 1989)

Summary

Introduction

Evidence indicates that contact between CRP and RNA polymerase is involved in transcription activation at the Zac promoter. CRP "positive control" mutants which affect transcription activation without altering specific DNA binding and bending have been isolated; they map on an exposed loop spanning amino acid residues 156 to 162 in the C-terminal domain (Bell et aZ., 1990; Eschenlauer et aZ., 1991; Zhou et al, 1993). CRP activates transcription from Class I promoters by contact of a primary activation site in its C-terminal domain (amino acid residues 156-162) with the C terminus of the IX subunit of RNA polymerase. Protein-protein photo-cross-linking has provided direct evidence that the CRP 161 position contacts the C-terminal domain of the IX subunit of RNA polymerase (Chen et al, 1994)

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