Faculty Opinions recommendation of Exploitation of a chemical nuclease to investigate the location and orientation of the Escherichia coli RNA polymerase alpha subunit C-terminal domains at simple promoters that are activated by cyclic AMP receptor protein.

2003 ◽  
Author(s):  
Ann Stock
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Alpha Subunit ◽  
Receptor Protein ◽  
Subunit C ◽  
Cyclic Amp Receptor Protein ◽  
Chemical Nuclease ◽  
Terminal Domains

scholarly journals Interactions between the cyclic AMP receptor protein and the Alpha subunit of RNA polymerase at the Escherichia Coli galactose operon P1 Promoter

1994 ◽  
Vol 22 (21) ◽  
pp. 4375-4380 ◽  
Author(s):  
Adrian Attey ◽  
Tamara Belyaeva ◽  
Nigel savery ◽  
Jim Hoggett ◽  
Nobuyuki Fujita ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Alpha Subunit ◽  
Receptor Protein ◽  
Cyclic Amp Receptor Protein

scholarly journals Interdependence of Activation at rhaSR by Cyclic AMP Receptor Protein, the RNA Polymerase Alpha Subunit C-Terminal Domain, and RhaR

2000 ◽  
Vol 182 (23) ◽  
pp. 6774-6782 ◽  
Author(s):  
Carolyn C. Holcroft ◽  
Susan M. Egan
Keyword(s):  
Cyclic Amp ◽  
Rna Polymerase ◽  
Synergistic Effects ◽  
Receptor Protein ◽  
Mobility Shift ◽  
Α Subunit ◽  
Subunit C ◽  
Cyclic Amp Receptor Protein ◽  
Terminal Domain ◽  
Full Activation

ABSTRACT The Escherichia coli rhaSR operon encodes two AraC family transcription activators, RhaS and RhaR, and is activated by RhaR in the presence of l-rhamnose. β-Galactosidase assays of various rhaS-lacZ promoter fusions combined with mobility shift assays indicated that a cyclic AMP receptor protein (CRP) site located at −111.5 is also required for full activation of rhaSR expression. To address the mechanisms of activation by CRP and the RNA polymerase α-subunit C-terminal domain (α-CTD) at rhaSR, we tested the effects of alanine substitutions in CRP activating regions 1 and 2, overexpression of a truncated version of α (α-Δ235), and alanine substitutions throughout α-CTD. We found that DNA-contacting residues in α-CTD are required for full activation, and for simplicity, we discuss α-CTD as a third activator of rhaSR. CRP and RhaR could each partially activate transcription in the absence of the other two activators, and α-CTD was not capable of activation alone. In the case of CRP, this suggests that this activation involves neither an α-CTD interaction nor cooperative binding with RhaR, while in the case of RhaR, this suggests the likelihood of direct interactions with core RNA polymerase. We also found that CRP, RhaR, and α-CTD each have synergistic effects on activation by the others, suggesting direct or indirect interactions among all three. We have some evidence that the α-CTD–CRP and α-CTD–RhaR interactions might be direct. The magnitude of the synergistic effects was usually greater with just two activators than with all three, suggesting possible redundancies in the mechanisms of activation by CRP, α-CTD, and RhaR.

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

2003 ◽  
Vol 185 (17) ◽  
pp. 5148-5157 ◽  
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.

scholarly journals 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

2002 ◽  
Vol 184 (8) ◽  
pp. 2273-2280 ◽  
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.

Interactions between the Escherichia coli cyclic AMP receptor protein and RNA polymerase at Class II promoters

1993 ◽  
Vol 10 (4) ◽  
pp. 789-797 ◽  
Author(s):  
David West ◽  
Roy Williams ◽  
Virgil Rhodius ◽  
Andrew Bell ◽  
Naveen Sharma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class Ii ◽  
Receptor Protein ◽  
Cyclic Amp Receptor Protein

scholarly journals Positioning of Region 4 of the Escherichia coli RNA Polymerase ς70 Subunit by a Transcription Activator

2000 ◽  
Vol 182 (10) ◽  
pp. 2982-2984 ◽  
Author(s):  
Jonathan A. Bown ◽  
Annie Kolb ◽  
Claude F. Meares ◽  
Akira Ishihama ◽  
Stephen D. Minchin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Dna Cleavage ◽  
Transcription Activation ◽  
Receptor Protein ◽  
Transcription Activator ◽  
Cyclic Amp Receptor Protein ◽  
Conformation Changes ◽  
Major Conformation

ABSTRACT A DNA cleavage reagent, specifically tethered to residue 581 of theEscherichia coli RNA polymerase ς70 subunit, has been used to investigate the location of ς70 region 4 in different complexes at the galp 1 promoter and the effect of the cyclic AMP receptor protein. The positions of DNA cleavage by the reagent are not affected by the cyclic AMP receptor protein. We conclude that transcription activation at thegalp 1 promoter by the cyclic AMP receptor protein does not involve major conformation changes in or repositioning of ς70 region 4.

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