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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Replacement of the CRP-binding site of the gal control region by curved sequences can lead to the restoration of promoter strength in vivo. One curved sequence called 5A6A, however, failed to do so. The gene hns exerts a strong negative control on the resulting 5A6A gal promoter as well as on the distant bla promoter, specifically in a 5A6A gal context. The product of this gene, H-NS, displays a better affinity for this particular insert compared to other curved sequences. Mechanisms by which H-NS may repress promoters both at short and long distances from a favoured binding site are discussed.
Mol Microbiol 1994 Apr
PMID:Modulated expression of promoters containing upstream curved DNA sequences by the Escherichia coli nucleoid protein H-NS. 805 48

The alpha subunit of Escherichia coli RNA polymerase plays a major role in the assembly of the core enzyme. The amino-terminal two-thirds of the alpha subunit, as far as position 235, are involved in this assembly. To define the site(s) within this region required for core enzyme assembly, we constructed a set of amino-terminal and internal deletion mutants of the rpoA gene. The overexpressed alpha derivatives were purified to apparent homogeneity and examined for their abilities to assemble beta and beta' subunits into active core enzymes in vitro. Among a total of 22 alpha derivatives tested, only four mutants retained the activity form active core enzyme. These mutants had deletions of the extreme amino-terminal residues as far as amino acid residue 30. The minimum fragment with full activity of the core assembly was alpha(21 to 235), with deletions of 20 amino-terminal and 94 carboxy-terminal amino acid residues. Most of the other mutants appeared to be defective in the formation of stable alpha dimers as analyzed by high-pressure liquid chromatography gel filtration, although some formed self-aggregates. These results, taken together, suggest that the amino-terminal region of the alpha subunit with the core assembly activity is highly structured, and any deletion within this domain disrupts its ordered conformation. Deletions of the extreme amino-terminal region did not affect transcription activation by CRP at the lacP1 promoter or by OmpR at the ompC promoter.
J Mol Biol 1994 Sep 16
PMID:Functional map of the alpha subunit of Escherichia coli RNA polymerase. Deletion analysis of the amino-terminal assembly domain. 808 34

The transcriptional organization of the gene cluster encoding the F1845 fimbrial adhesin of a diarrhoea-associated Escherichia coli was investigated. Genes daaA to daaE were determined to constitute a single transcriptional unit under the control of the daaA promoter. The nucleotide sequence of daaA and that of an upstream open reading frame encoded on the opposite strand, designated daaF, were determined to share limited homology with the papB and papI genes of the P fimbrial adhesin, respectively. The 5' termini of the daaF and daaABCDE transcripts were mapped by primer extension and nuclease protection analyses. The promoters for these transcripts were associated with potential regulatory sequences including two consensus leucine-responsive regulatory protein (Lrp)-binding sites which contained differentially methylated GATC sequences, a cAMP-CRP-binding site, and an integration host factor (IHF)-binding site. Expression of the daa locus was determined to be dependent on Lrp, subject to catabolite repression, and dependent on IHF.
Mol Microbiol 1993 Mar
PMID:Transcriptional organization of the F1845 fimbrial adhesin determinant of Escherichia coli. 809 64

The RhaS and RhaR regulatory proteins are encoded in the Escherichia coli L-rhamnose gene cluster. We used complementation analysis and DNA mobility shift assays to show that RhaR is not the direct activator of the L-rhamnose catabolic operon, rhaBAD. An in-frame deletion of rhaS (rhaS-rhaR+) eliminated expression from the rhaBAD promoter, pBAD, while overexpression of rhaS greatly speeded the normally slow induction of transcription from pBAD. Expression from pBAD in a coupled transcription-translation assay was only detected when rhaS+ DNA was added to allow synthesis of RhaS protein. RhaS thus appears to be the direct L-rhamnose-specific activator of rhaBAD expression. Deletion mapping located the binding site for the L-rhamnose-specific regulator to a region overlapping position -70 relative to the rhaBAD transcription start site. Deletion mapping and DNA mobility shift assays located a CRP binding site just upstream from the binding site for the L-rhamnose-specific regulator. Quantitative primer extension analysis showed that induction of both the rhaBAD and rhaSR messages was unusually slow, requiring 40 to 50 minutes to reach a steady-state level. Induction of rhaBAD apparently involves a regulatory cascade in which RhaR first induces rhaSR expression, then RhaS accumulates and induces rhaBAD expression.
J Mol Biol 1993 Nov 05
PMID:A regulatory cascade in the induction of rhaBAD. 823 Feb 10

FNR is a transcriptional regulator controlling the expression of a number of Escherichia coli genes in response to anoxia. It is structurally-related to CRP (the cyclic AMP receptor protein) except for the presence of a cysteine-rich N-terminal extension, which may form part of an iron-binding, redox-sensing domain in FNR. Site-directed substitution has previously shown that four of the cysteine residues (C20, C23, C29 and C122) are essential for FNR function, whereas the fifth (C16) is not. The FNR protein exists in two forms separable by non-reducing SDS-PAGE, and in studies with altered FNR proteins containing single substitutions at each of the five cysteine residues it was concluded that the faster-migrating form (FNR(27)), possesses an intramolecular disulphide bond linking C122 to one of the cysteines near the N-terminus. FNR(27) was more abundant in aerobic cells but the physiological significance of this was not established. Footprint studies indicated that FNR proteins lacking essential cysteine residues are impaired in their ability to protect FNR sites in the ndh promoter. The non-essential cysteine residue (C16) was identified as the source of the most reactive sulphydryl group and all of the inactive proteins exhibited different sulphydryl reactivities. The iron content of the C122A-substituted protein was much reduced but those of the other proteins were less affected. Electrospray mass spectrometry confirmed the accuracy of the gene-derived amino acid composition of FNR with a mutant protein and it showed that a fraction of the wild-type protein may carry a 78 Da substituent which could not be removed with dithiothreitol or beta-mercaptoethanol.
Mol Microbiol 1993 Apr
PMID:Properties of FNR proteins substituted at each of the five cysteine residues. 849 98

Repression by CytR depends on the formation of nucleoprotein complexes in which the CytR repressor and the cAMP-CRP activator complex bind co-operatively to the DNA. Transcription initiation from CytR-regulated promoters requires cAMP-CRP; therefore, the cAMP-CRP complex functions both as an activator and as a co-repressor in these promoters. Another interesting aspect of the CytR regulon is that each promoter appears to have individual features. Therefore, structural and functional rules governing the formation of repression and activation complexes in one promoter may not be valid for other promoters of the CytR regulon. Here we show that the Escherichia coli nupG gene contains one CytR- and four CRP-binding sites in the control region. Notably, the architecture of the CytR binding site is different from previously described targets. In addition, the CytR repressor triggers a DNA repositioning of a cAMP-CRP complex in the -35 region upon binding to its operator. Thus, formation of the repression and activation complexes at the nupG promoter involves different subsets of CRP-binding sites. These findings show that the bacterium uses positive and negative regulatory modules to differentially control the expression of CytR- and cAMP-CRP-regulated genes.
Mol Microbiol 1995 Sep
PMID:Gene-regulatory modules in Escherichia coli: nucleoprotein complexes formed by cAMP-CRP and CytR at the nupG promoter. 859 34

Sigma-S and the cAMP-CRP complex are global regulatory factors involved in stationary-phase induction of large groups of genes in Escherichia coli. csiE, a gene located at 57.25 min (co-ordinate 2674) of the physical map of the E. coli chromosome, is under the control of both of these factors. Sigma-S plays a positive, though not absolutely essential, role in the expression of csiE. Regulation by cAMP-CRP has both positive and negative elements, with the latter being dependent on the presence of sigma s, whose expression is negatively influenced by cAMP-CRP. csiE has a single transcriptional start site located 33 bp upstream of the initiation codon. By a 5'-deletion approach, we show that 72 bp upstream of the csiE transcriptional start site are sufficient for regulation by sigma s and cAMP-CRP. A deletion upstream of nucleotide -38 with respect to the start site eliminates positive cAMP-CRP control and makes the remaining expression fully dependent on sigma S. Our results indicate that transcription at the csiE promoter can be initiated in vivo by sigma S-containing RNA polymerase alone as well as by sigma 70-containing RNA polymerase in conjunction with cAMP-CRP or a cAMP-CRP-dependent secondary regulator. The promoter region of poxB, the structural gene for pyruvate oxidase, which is also under the control of sigma S and cAMP-CRP, is very similar to the corresponding region of csiE, suggesting a similar regulatory mechanism also for poxB.
Mol Microbiol 1995 Oct
PMID:Regulatory characteristics and promoter analysis of csiE, a stationary phase-inducible gene under the control of sigma S and the cAMP-CRP complex in Escherichia coli. 859 57

LIM domains are Zn-binding arrays found in a number of proteins involved in the control of cell differentiation, including several developmentally regulated transcription factors and a human proto-oncogene product. The rat cysteine-rich intestinal protein, CRIP, is a 76-residue polypeptide which contains a LIM motif. The solution structure of CRIP has been determined by homonuclear and 1H-15N heteronuclear correlated nuclear magnetic resonance spectroscopy. Structures with individual distance violations of < or = 0.03 angstrom and penalties (squared sum of distance violations) of < or = 0.06 angstrom2 were generated with a total of 500 nuclear Overhauser effect (NOE)-derived distance restraints (averaging 15.6 restraints per refined residue). Superposition of backbone heavy atoms of ordered residues relative to mean atom positions is achieved with pairwise rms deviations of 0.54(+/-0.14) angstrom. As observed previously for a peptide with the sequence of the C-terminal LIM domain from the avian cysteine-rich protein, CRP (cCRP-LIM2), CRIP binds two equivalents of zinc, forming N-terminal CCHC (Cys3, Cys6, His24, Cys27) and C-terminal CCCC (Cys30, Cys33, Cys51, Cys55) modules. The CCHC and CCCC modules in CRIP contain two orthogonally-arrayed antiparallel beta-sheets. The C-terminal end of the CCHC module contains a tight turn and the C terminus of the CCCC module forms an alpha-helix. The modules pack via hydrophobic interactions, forming a compact structure that is similar to that observed for cCRP-LIM2. The most significant differences between the structures occur at the CCHC module-CCCC module interface, which results in a difference in the relative orientations of the modules, and at the C terminus where the alpha-helix appears to be packed more tightly against the preceding antiparallel beta-sheet. The greater abundance of NOE information obtained for CRIP relative to cCRP-LIM2, combined with the analysis of J-coupling and proton chemical shift data, have allowed a more detailed evaluation of the molecular level interactions that stabilize the fold of the LIM motif.
J Mol Biol 1996 Mar 22
PMID:Structure of the cysteine-rich intestinal protein, CRIP. 863 52

Previous work with semi-synthetic promoters containing a single CRP binding site centred at 41.5 bp from the transcription start site has demonstrated enhanced transcription (synergism) when a second binding site, for CRP or FNR, is placed upstream at around -91 bp. The ansB promoter in Escherichia coli is co-activated in a co-dependent manner by one dimer each of CRP and FNR protein whose binding sites are at around -91 and -41 bp, respectively, from the transcription start site. Similarly, the homologous ansB promoter in Salmonella is co-activated by two dimers of CRP which function synergistically. The binding sites at the E. coli promoter have been changed by mutation to provide a number of active promoter derivatives carrying other combinations of FNR and CRP binding sites. The co-dependent versus synergistic interaction of these activators and their requirement for known activating regions have been examined. The results demonstrate that FNR can co-activate when located upstream at around -91 bp in combination with either FNR or CRP downstream. When FNR occupies the downstream site the promoter is co-dependent on an upstream activator, but not when CRP occupies this site. Activating region 1 in CRP (defined by substitutions at residue H159) and its putative equivalent in FNR (defined by substitutions at S73) are mainly required in the upstream activator; the putative equivalent in FNR of activating region 3 of CRP (defined by substitutions at G85 and K52, respectively) is mainly required in the dimer which binds downstream. Activating region 1 of FNR is required only in the downstream subunit of the upstream activator in a promoter which is co-dependent on two FNR dimers. These data suggest that both bound upstream and downstream activators interact with RNA polymerase to promote transcription, and that co-dependence is determined by the nature of the activator plus the promoter context.
Mol Microbiol 1995 Nov
PMID:Transcriptional co-activation at the ansB promoters: involvement of the activating regions of CRP and FNR when bound in tandem. 874 35

The CytR repressor protein relies on protein-protein interactions to the cAMP-CRP complex to bind its operators with sufficiently high affinity to repress transcription. Here, the quaternary structure of CytR and the mechanism underlying the cooperative binding of CytR and cAMP-CRP have been analyzed. Using a modified Ferguson analysis in which protein-DNA complexes are separated in a non-denaturing gel system, we show that CytR binds its operators as a dimer alone as well as in a ternary complex with cAMP-CRP. Analyses of DNA binding of CytR at low protein concentrations indicate that CytR is a dimer in solution at physiological concentrations. Moreover, the CytR inducer cytidine was found not to have any effect on the oligomerization of free CytR or DNA bound CytR. Thus, these data rule out the possibility that the cooperative DNA binding of CytR and cAMP-CRP involves induced dimerization of CytR, and they suggest that cytidine interrupts the cooperative binding of CytR and cAMP-CRP solely by perturbing the protein-protein interactions between the two proteins.
J Mol Biol 1996 Jul 12
PMID:CytR/cAMP-CRP nucleoprotein formation in E. coli: the CytR repressor binds its operator as a stable dimer in a ternary complex with cAMP-CRP. 876 93


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