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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism by which the cyclic AMP receptor protein, CRP, stimulates transcription of the Escherichia coli araBAD promoter was studied in vitro. Under one set of conditions, CRP stimulated by eightfold the rate of RNA polymerase open complex formation on supercoiled DNA template containing the normal wild-type araBAD regulatory region. Since previous studies in vivo had identified an upstream site termed araO2 that is involved in both repression and in the CRP requirement for PBAD induction, we performed similar experiments in vitro. Deletion of araO2 or alterations of its orientation with respect to the araI site by half integral numbers of turns greatly reduced the CRP requirement for induction of PBAD. Linearizing the DNA has the same effect as deleting araO2 from the supercoiled DNA template. The similarity of conditions that relieve the classical repression of PBAD in vivo and the conditions that eliminate the requirement for CRP for maximal activity in vitro suggest a close relationship between repression in the ara system and the role of CRP. At lower concentrations of AraC protein and slightly different conditions than those used in the above-mentioned experiments, CRP does stimulate transcription from linear or supercoiled templates lacking araO2. On linear DNA under these conditions, one dimer of AraC protein binds to linear araPBAD DNA, but is incapable of stimulating transcription without the additional binding of CRP. The responses of the ara system under the second set of conditions are unlike its behavior in vivo.
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PMID:Transcription of Escherichia coli ara in vitro. The cyclic AMP receptor protein requirement for PBAD induction that depends on the presence and orientation of the araO2 site. 301 84

A G:C to T:A transversion at bp position -19 in the gal operon promoter region relieves the dependence of galP1 promoter activity on the cAMP-CRP complex. Deletion analysis shows that expression from the promoter is decreased on replacement of the sequence between 49 and 54 bp upstream from the P1 start point. Moreover, protection experiments show that RNA polymerase interacts with this region in open complexes at P1. We propose that this contact is necessary for optimal P1 activity; point mutations in the gal promoter region can alter DNA flexibility and hence the strength of this contact; CRP factor activates P1 transcription by favouring formation of this contact; and the gal repressor blocks P1 activity by binding to this zone.
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PMID:RNA polymerase makes important contacts upstream from base pair -49 at the Escherichia coli galactose operon P1 promoter. 303 92

Expression of the glucitol (gut) operon in Escherichia coli is regulated by an unusual, complex system which consists of an activator (encoded by the gutM gene) and a repressor (encoded by the gutR gene) in addition to the cAMP-CRP complex (CRP, cAMP receptor protein). The activator and repressor are predicted to possess 119 (Mr = 12,955) and 257 (Mr = 28,240) aminoacyl residues, respectively, as deduced from the nucleotide sequences of their structural genes. Both of the genes encoding the two regulators are located downstream from the other known gut structural genes. Reverse transcriptase mapping revealed that the gutM gene is a promoter-distal constituent of the gut operon. The gutR gene has its own promoter, but expression of this gene is primarily due to readthrough from the gut operon operator-promoter. Thus, the gut operon consists of at least five structural genes and has the following gene order: gutOPABDMR. Interestingly, synthesis of the mRNA, which initiates at the promoter specific to the gutR gene, occurs within the gutM gene. Expressional control of the gut operon appears to occur as a consequence of the antagonistic action of the products of the autogenously regulated gutM and gutR genes. An additional cistron of the gut operon, of unknown function, may follow the gutR gene.
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PMID:Positive and negative regulators for glucitol (gut) operon expression in Escherichia coli. 306 73

We show, using dot matrix comparisons and statistical analysis of sequence alignments, that seven sequenced sigma factors, E. coli sigma-70 and sigma-32, B. subtilis sigma-43 and sigma-29, phage SP01 gene products 28 and 34, and phage T4 gene product 55, comprise a homologous family of proteins. Sigma-70, sigma-32, and sigma-43 each have two copies of a sequence similar to the helix-turn-helix DNA binding motif seen in CRP, and lambda repressor and cro proteins. B. subtilis sigma-29, SP01 gp28, and SP01 gp34 have at least one copy similar to this sequence. We propose that a second sequence, conserved in all seven proteins is the core RNA polymerase binding site. A third region, present only in sigma-70 and sigma-43, may also be involved in interaction with core. Available mutational evidence supports our model for sigma factor structure.
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PMID:Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. 309 89

The gal operon regulatory region contains two overlapping promoters, P1 and P2, regulated by cyclic AMP and the cyclic AMP receptor protein (cAMP X CRP). Starting with a mutation that eliminated P1, the promoter that is usually dependent on cAMP X CRP, we constructed a series of deletions that substituted increasing amounts of DNA sequence from upstream of P2, the promoter that usually functions in the absence of cAMP X CRP. Expression from P2 in vivo was halved by deletions that replace the -35 region with unrelated sequences, showing that the -35 sequence participates in promoter function, but is not essential. In vitro studies show that replacement of the -35 sequence increases the time for open complex formation at P2, but does not alter the transcription start point. We examined the effects of the same deletions at the wild type gal promoter region: again, the deletion that replaces the -35 region halves expression in vivo. However, in this case, in the absence of cAMP X CRP, the deletion switches expression from the P2 promoter to P1, the promoter that is usually dependent on cAMP X CRP. Moreover, although the deletion also removes the specific cAMP X CRP binding site, this P1 activity is sharply inhibited in a crp+ background. We argue that this is due to a direct contact between CRP and RNA polymerase bound at the P1 Pribnow box, and we discuss the role of the -35 sequence at these and other promoters.
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PMID:Transcription initiation at the Escherichia coli galactose operon promoters in the absence of the normal -35 region sequences. 309 98

We report in vitro studies of the interactions between purified E. coli RNA polymerase and DNA from the regulatory region of the E. coli galactose operon which carries a point mutation that simultaneously stops transcription initiation at the two normal start points, S1 and S2. In the presence of this point mutation, transcription initiates at a third start point 14/15 bp downstream of S1, showing that inactivation of the two normally active promoters, P1 and P2, unmasks a third weaker promoter, P3. Transcription initiation in the gal operon is normally regulated by the cyclic AMP receptor protein, CRP, that binds to the gal regulatory region and switches transcription from P2 to P1. With the point mutation, CRP binding switches transcription from P3 to P1, although the formation of transcriptionally competent complexes at P1 is very slow. The results are discussed with respect to the mechanism of transcription activation by the CRP factor and the similarities between the regulatory regions of the galactose and lactose operons.
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PMID:Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters. Interactions with RNA polymerase and the cyclic AMP receptor protein. 329 89

Two Escherichia coli control regions have been compared in their ability to be unwound by RNA polymerase during formation of the transcriptionally active ("open") complex: the wild-type control region, consisting of two overlapping binding sites P1 and P2, both weakly transcribed, and an "up" P1 mutant, the strong lac L8UV5 promoter. The final complexes were characterized by their topological unwinding, by DNase I and orthophenanthroline footprints, as well as by methylation of unpaired cytosine residues. At the wild-type control region, the RNA polymerase footprint is weak, and single-strand formation is incomplete and slow. The same signals are strong, complete and quickly established at lac L8UV5; yet the final complexes were found to be equally unwound (by 1.7 turns) in the absence of nucleotide substrates as well as during an abortive initiation cycle. At the lac wild-type region, open complex formation occurs slowly enough to permit the measurement of the extent of a single-stranded region and of topological unwinding during the latency period. Not all the final species are active and unwinding appears to precede, in time, full open-complex formation. At the lac UV5 promoter the same conclusion was reached by a different method involving those changes in the various parameters that characterize open-complex formation monitored by an abortive initiation assay, conducted at increasing levels of template superhelicity. From both approaches we conclude that, at these promoters, the formation of the single-stranded region occurs at the expense of a negative change in linking number, initially stored in a closed intermediate, perhaps as negative writhing. Furthermore, abortive transcription assays indicate that the specific initiation efficiency of the species stored at both promoters, P1 and P2, on the wild-type template is increased as a whole with increasing superhelicity (conversion of inactive species to active ones, increased efficiency of active ones). We conclude that negative supercoiling is not an extra-regulatory element of the lactose system, allowing modulation of expression of the wild-type promoter to the profit of P1. Instead, P2 and P1, in the absence of active catabolite receptor protein (CRP-cAMP), appear to be equally weak and to be equally affected by negative supercoiling in the range of superhelical densities examined. The physiological importance of the P1-P2 competition in the regulation of expression in this region is thus questioned. The major effect of CRP-cAMP stimulation appears to be the direct activation at the P1 promoter.
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PMID:Topological unwinding of strong and weak promoters by RNA polymerase. A comparison between the lac wild-type and the UV5 sites of Escherichia coli. 330 41

The control of transcription initiation at the lactose operon promoter was investigated in vitro. We found that an upstream promoter (termed lac P2) interfered with RNA polymerase binding at the principal promoter (termed lac P1). The start site for lac P2 was located at base pair position -22 relative to the P1 start site. The addition of cAMP receptor protein and cAMP was shown to repress lac P2 and to activate lac P1. Abortive initiation reactions for both promoters were used to investigate the coordinate repression-activation elicited by CRP-cAMP. The effects of lac promoter mutations (L8, Ps, and UV5) were consistent with an important RNA polymerase positioning role for CRP-cAMP in the activation of lac operon expression.
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PMID:Dual promoter control of the Escherichia coli lactose operon. 609 9

CRP-cAMP was shown to activate transcription initiation at the Escherichia coli lac promoter in vitro as a result of two separate effects. An indirect component of the activation resulted from an enhancement of the fraction of promoters productively bound by RNA polymerase. This effect was due largely to CRP-cAMP repression of RNA polymerase binding to an overlapping site (lac P2) within the promoter region. In addition, a direct enhancement of RNA polymerase binding at the principal lac promoter (lac P1) was found. The combination of indirect and direct activation by CRP-cAMP was suggested to be responsible for the large activation observed in vivo. Promoter strength parameters were also determined for the L8, UV5 and Ps promoters. The effect of CRP-cAMP on these mutant promoters was shown to be consistent with the activation mechanism deduced for the lac wild-type promoter. DNA supercoiling enhanced the promoter strength of the lac wild-type and UV5 promoters. The combination of supercoiling and CRP-cAMP was necessary for optimal promoter strength for the lac wild-type promoter.
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PMID:Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoter. 609 91

The regulatory protein CRP (or CAP) from E. coli is shown to display two distinct patterns of binding interactions with DNA-dependent RNA polymerase. The free core enzyme, and both the core and the holo polymerase when bound to single-stranded DNA, can bind CRP in a cAMP-independent association reaction. Instead, the binding of CRP to free holoenzyme and to holo or core polymerase bound to native DNA was undetectable in the absence of cAMP. The specific ligand of CRP (cAMP) strengthens distinctively this class of interactions. In no case could any release of sigma-factor be demonstrated. Estimates of the dissociation constants were obtained for the various binding reactions which were investigated under quasi-physiological ionic conditions. These, together with the known values of the in vivo concentrations of CRP and RNA polymerase, suggest that the interactions described may have a functional significance.
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PMID:Binding of CRP to DNA-dependent RNA polymerase from E. coli: modulation by cAMP of the interactions with free and DNA-bound holo and core enzyme. 624 68


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