Influence of the Location of the cAMP Receptor Protein Binding Site on the Geometry of a Transcriptional Activation Complex in Escherichia coli (original) (raw)
Related papers
Biochemistry, 1996
The interactions between the cAMP receptor protein (CRP) and RNA polymerase during transcriptional activation at the Escherichia coli malT promoter have been analyzed using a combination of footprinting methods. We show that a closed complex is formed at this promoter in the absence of activator and that CRP merely stabilizes the open complex. The R-subunits of the RNA polymerase are involved in this effect as shown by KMnO 4 footprinting. The open complex formed in the presence of CRP is structurally identical to the one found at a CRP-independent promoter up-mutant. UV-laser footprinting yields distinct signals for the different protein-DNA interactions within the complex and for interactions between CRP and RNA polymerase. We monitor these signals in promoter variants that place the CRP binding site at different distances upstream of the start site of transcription. Signals within the core promoter region, as well as those located just upstream of the-35 hexamer, are unaffected by the position of the CRP binding site. Contacts of RNA polymerase with the upstream promoter region change in a mutant RNA polymerase containing a truncated R-subunit. We conclude that at least one of the R-subunits of RNA polymerase binds to DNA upstream of the-35 hexamer and that this interaction is unaffected by the position of the CRP binding site. We discuss models that account for the different activities of CRP in transcriptional activation as a function of promoter geometry.
Influence of DNA geometry on transcriptional activation in Escherichia coli
The EMBO Journal
Transcription from many Escherichia coli promoters can be activated by the cAMP-CRP complex bound at different locations upstream of the promoter. At some locations the mechanism of activation involves direct protein-protein contacts between CRP and the RNA polymerase. We positioned the CRP binding site at various distances from the transcription start site of the malT promoter and measured the in vivo activities of these promoter variants. From the activation profiles we deduce that the protein-protein interactions involved in transcriptional activation are rather rigid. A heterologous protein (IHF) that bends the DNA to a similar degree as does CRP activates transcription when bound at sites equivalent to activating positions for CRP. DNA geometry makes a major contribution to the process of transcriptional activation and DNA upstream of the activator binding site participates in this process. Removal of this DNA decreases the capacity of the malT promoter to be activated by CRP in vitro. We conclude that both DNA topology and direct protein-protein contacts contribute to transcriptional activation and that the relative importance of these two modes of activation depends on the nature of the activator and on the location of the activator binding site.
Proceedings of the National Academy of Sciences, 1997
Interactions between the cAMP receptor protein (CRP) and the carboxy-terminal regulatory domain (CTD) of Escherichia coli RNA polymerase ␣ subunit were analyzed at promoters carrying tandem DNA sites for CRP binding using a chemical nuclease covalently attached to ␣. Each CRP dimer was found to direct the positioning of one of the two ␣ subunit CTDs. Thus, the function of RNA polymerase may be subject to regulation through protein-protein interactions between the two ␣ subunits and two different species of transcription factors.
Proceedings of the National Academy of Sciences of the United States of America, 1997
We have studied the kinetics of transcriptional initiation and activation at the malT and malTp1 promoters of Escherichia coli using UV laser footprinting. Contrary to previous studies and because of the very rapid signal acquisition by this technique, we can obtain structural information about true reaction intermediates of transcription initiation. The consequences of adding a transcriptional activator, the cAMP receptor protein/cAMP complex (CRP), are monitored in real time, permitting us to assign specific interactions to the activation of discrete steps in transcription initiation. Direct protein–protein contacts between CRP and the RNA polymerase appeared very rapidly, followed by DNA melting around the −10 hexamer. CRP slightly increased the rate of this isomerization reaction but, more importantly, favored the establishment of additional contacts between the DNA upstream of the CRP binding site and RNA polymerase subsequent to open complex formation. These contacts make a major contribution to transcriptional activation by stabilizing open forms of the promoter complex, thereby indirectly accelerating promoter escape. The ensemble of the kinetic, structural signals demonstrated directly that CRP exerts most of its activating effects on the late stages of transcriptional initiation at the malT promoter.
J Mol Biol, 1998
We have constructed a family of promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein (CRP), with one of the sites centred between base-pairs 41 and 42 upstream from the transcription start site, and the second site located further upstream. In vivo activity measurements show that the activity of these promoters is completely dependent on CRP and that, depending on the precise location, CRP bound at the upstream site increases transcription activation. Hydroxyl radical footprinting was exploited to investigate the binding of CRP and RNA polymerase holoenzyme (RNAP) to these promoters. The study shows that the C-terminal domains of the RNAP a subunits bind adjacent to the upstream CRP and that their precise positioning depends on the location of upstream-bound CRP. The C-terminal domains of the RNAP a subunits interact with both the upstream and downstreambound CRP via activating region 1 of CRP.
Nucleic Acids Research, 1993
A deletion of the C-terminal part of the a-subunit of RNA polymerase is known to affect differently promoters activated by CRP depending on the location of the CRP binding site at the promoter. When the CRP binding site is located at-61.5, as at lacPl (a type I promoter), activation is strongly Impaired while it is not significantly affected at gaIPl where CRP binds 41.5 bp upstream of the start of the message (type 11 promoter). We have investigated the differences In the architecture of the corresponding open complexes by comparing the positioning of holoenzymes reconstituted respectively with native or with truncated c-subunits (containing the first 235 or 256 residues of a) at two 'up' promoter mutants of the lacPl and gaIP1 promoters (respectively lacUV5 and gaI9Al 6C). First, the affinity of wild-type RNA polymerase for both promoters is increased by the presence of CRP and cAMP. By contrast, holoenzymes reconstituted with truncated a-subunits, show cooperative binding at the gaIPl promoter only. Second, footprinting data confirm these observations and indicate that the truncated holoenzymes are unable to recognize regions of the promoter upstream from position-40. The absence of contacts between the truncated enzymes and CRP at the lacPl promoter can explain the deficiency in activation. At the gaIP1 promoter, where the CRP site is closer to the initiation site, protein-protein contacts can still occur with the truncated polymerases, showing that the C-terminal part of the at-subunit is not involved in activation.
Spacing requirements for Class I transcription activation in bacteria are set by promoter elements
Nucleic acids research, 2014
The Escherichia coli cAMP receptor protein (CRP) activates transcription initiation at many promoters by binding upstream of core promoter elements and interacting with the C-terminal domain of the RNA polymerase α subunit. Previous studies have shown stringent spacing is required for transcription activation by CRP. Here we report that this stringency can be altered by the nature of different promoter elements at target promoters. Several series of CRP-dependent promoters were constructed with CRP moved to different upstream locations, and their activities were measured. The results show that (i) a full UP element, located immediately downstream of the DNA site for CRP, relaxes the spacing requirements for activation and increases the recruitment of RNAP and open complex formation; (ii) the distal UP subsite plays the key role in this relaxation; (iii) modification of the extended -10 element also affects the spacing requirements for CRP-dependent activation. From these results, we...
Journal of Biological Chemistry, 2003
The C-terminal domain of the ␣ subunit (␣CTD) of bacterial RNA polymerase plays an important role in promoter recognition. It is known that ␣CTD binds to the DNA minor groove at different locations at different promoters via a surface-exposed determinant, the 265 determinant. Here we describe experiments that permit us to determine the location and orientation of binding of ␣CTD at any promoter. In these experiments, a DNA cleavage reagent is attached to specific locations on opposite faces of the RNA polymerase ␣ subunit. After incorporation of the tagged ␣ subunits into holo-RNA polymerase, patterns of DNA cleavage due to the reagent are determined in open complexes. The locations of DNA cleavage due to the reagent attached at different positions allow the position and orientation of ␣CTD to be deduced. Here we present data from experiments with simple Escherichia coli promoters that are activated by the cyclic AMP receptor protein.
Genes & Development, 2002
Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at different promoters has been studied using RNA polymerase holoenzyme derivatives containing two full-length ␣ subunits, or containing one full-length ␣ subunit and one truncated ␣ subunit lacking the ␣ C-terminal domain (␣CTD). At a promoter having a single DNA site for CRP, activation requires only one full-length ␣ subunit. Likewise, at a promoter having a single DNA site for CRP and one adjacent UP-element subsite (high-affinity DNA site for ␣CTD), activation requires only one full-length ␣ subunit. In contrast, at promoters having two DNA sites for CRP, or one DNA site for CRP and two UP-element subsites, activation requires two full-length ␣ subunits. We conclude that a single copy of ␣CTD is sufficient to interact with one CRP molecule and one adjacent UP-element subsite, but two copies of ␣CTD are required to interact with two CRP molecules or with one CRP molecule and two UP-element subsites. 4 Corresponding author. E-MAIL s.j.w.busby@bham.ac.uk; FAX 44-121-414-7366 Article and publication are at http://www.genesdev.org/cgi/
Journal of Bacteriology, 2000
A DNA cleavage reagent, specifically tethered to residue 581 of the Escherichia coli RNA polymerase 70 subunit, has been used to investigate the location of 70 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 the galp 1 promoter by the cyclic AMP receptor protein does not involve major conformation changes in or repositioning of 70 region 4.