Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the α subunit of RNA polymerase at Class I promoters

The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters. At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase α subunit (αCTD). Since αCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions. We previously showed that, at these promoters, the CRP–αCTD interaction and the CRP–UP element interaction contribute independently and additively to transcription initiation. In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5. This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two αCTDs of RNA polymerase are not optimally positioned. Two experiments to test this hypothesis are presented.

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Spacing requirements for interactions between the C-terminal domain of the α subunit of Escherichia coli RNA polymerase and the cAMP receptor protein

Biochemical Journal ◽

10.1042/bj3300413 ◽

1998 ◽

Vol 330 (1) ◽

pp. 413-420 ◽

Cited By ~ 8

Author(s):

S. Georgina LLOYD ◽

J. W. Stephen BUSBY ◽

J. Nigel SAVERY

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Binding Site ◽

Transcription Initiation ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Α Subunit ◽

Synthetic Promoters ◽

Terminal Domain ◽

Camp Receptor

During transcription initiation at bacterial promoters, the C-terminal domain of the RNA polymerase α subunit (αCTD) can interact with DNA-sequence elements (known as UP elements) and with activator proteins. We have constructed a series of semi-synthetic promoters carrying both an UP element and a consensus DNA-binding site for the Escherichia coli cAMP receptor protein (CRP; a factor that activates transcription by making direct contacts with αCTD). At these promoters, the UP element was located at a variety of distances upstream of the CRP-binding site, which was fixed at position -41.5 bp upstream of the transcript start. At some positions, the UP element caused enhanced promoter activity whereas, at other positions, it had very little effect. In no case was the CRP-dependence of the promoter relieved. DNase I and hydroxyl-radical footprinting were used to study ternary RNA polymerase-CRP-promoter complexes formed at two of the most active of these promoters, and co-operativity between the binding of CRP and purified α subunits was studied. The footprints show that αCTD binds to the UP element as it is displaced upstream but that this displacement does not prevent αCTD from being contacted by CRP. Models to account for this are discussed.

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Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the α subunit of RNA polymerase at Class I promoters

Biochemical Journal ◽

10.1042/0264-6021:3370415 ◽

1999 ◽

Vol 337 (3) ◽

pp. 415 ◽

Cited By ~ 6

Author(s):

Emma C. LAW ◽

Nigel J. SAVERY ◽

Stephen J.W. BUSBY

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Class I ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Α Subunit ◽

Terminal Domain ◽

Camp Receptor

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Determinants of the C-Terminal Domain of the Escherichia coli RNA Polymerase α Subunit Important for Transcription at Class I Cyclic AMP Receptor Protein-Dependent Promoters

Journal of Bacteriology ◽

10.1128/jb.184.8.2273-2280.2002 ◽

2002 ◽

Vol 184 (8) ◽

pp. 2273-2280 ◽

Cited By ~ 50

Author(s):

Nigel J. Savery ◽

Georgina S. Lloyd ◽

Stephen J. W. Busby ◽

Mark S. Thomas ◽

Richard H. Ebright ◽

...

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Rna Polymerase ◽

Class Ii ◽

Class I ◽

Receptor Protein ◽

Α Subunit ◽

Cyclic Amp Receptor Protein ◽

Terminal Domain

ABSTRACT Alanine scanning of the Escherichia coli RNA polymerase α subunit C-terminal domain (αCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of αCTD affected class I CRP-dependent transcription from the CC(−61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for αCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in αCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.

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The Escherichia coli cAMP receptor protein bound at a single target can activate transcription initiation at divergent promoters: a systematic study that exploits new promoter probe plasmids

Biochemical Journal ◽

10.1042/bj20021003 ◽

2002 ◽

Vol 368 (3) ◽

pp. 835-843 ◽

Cited By ~ 26

Author(s):

Mohamed Samir EL-ROBH ◽

Stephen J.W. BUSBY

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Transcription Initiation ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Divergent Promoters ◽

Single Target ◽

Promoter Probe ◽

Camp Receptor

We report the first detailed quantitative study of divergent promoters dependent on the Escherichia coli cAMP receptor protein (CRP), a factor known to activate transcription initiation at target promoters by making direct interactions with the RNA polymerase holoenzyme. In this work, we show that CRP bound at a single target site is able to activate transcription at two divergently organized promoters. Experiments using promoter probe plasmids, designed to study divergent promoters in vivo and in vitro, show that the divergent promoters function independently. Further in vitro experiments show that two holo RNA polymerase molecules cannot be accommodated simultaneously at the divergent promoters.

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Cyclic AMP Receptor Protein-Dependent Activation of the Escherichia coli acsP2 Promoter by a Synergistic Class III Mechanism

Journal of Bacteriology ◽

10.1128/jb.185.17.5148-5157.2003 ◽

2003 ◽

Vol 185 (17) ◽

pp. 5148-5157 ◽

Cited By ~ 62

Author(s):

Christine M. Beatty ◽

Douglas F. Browning ◽

Stephen J. W. Busby ◽

Alan J. Wolfe

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Rna Polymerase ◽

Class I ◽

Receptor Protein ◽

Class Iii ◽

Α Subunit ◽

Cyclic Amp Receptor Protein ◽

Rna Polymerase Holoenzyme

ABSTRACT The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by “focusing” RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions −69.5 and −122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the α subunit of RNA polymerase holoenzyme (α-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the α-CTD. Other surface-exposed residues in the α-CTD contributed to acs transcription, suggesting that the α-CTD may interact with at least one protein other than CRP.

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