Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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 Escherichia coli lactose (lac) operon transcription control region includes at least two sequences which are recognized by RNA polymerase holoenzyme in vitro, the normal lac promoter (termed P1) and an overlapping upstream promoter (termed P2). The structure of the P2 and the effect of RNA polymerase interaction at P2 on the association of RNA polymerase with P1 was analyzed by the isolation and characterization of various mutations at P2. A set of deletions with varying lengths of DNA between the lac P2 -10 region and a "-35 region" contributed by the vector DNA were constructed. In vitro studies indicate that as the spacing between the -10 region and "-35 region" is increased from 16 to 22 base pairs (bp), the steady state occupancy as measured by exonuclease III protection experiments and the ability to initiate transcripts from P2 decrease. Studies were also conducted using a single base pair insertion and a two base pair deletion between the natural -35 and -10 regions of P2. The mutation which decreases the in vitro occupancy and transcription initiation potential of P2 does not significantly affect the steady state in vitro occupancy of P1 nor the in vivo expression of the lac operon. These results are not consistent with the model that RNA polymerase occupancy at P2 competes with the P1 expression and therefore that this competition plays a role in cAMP bound catabolite gene activator protein (CAP-cAMP) control of the lac operon.
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PMID:Deletion analysis of the Escherichia coli lactose promoter P2. 298 54

We have isolated a cAMP-binding protein from highly purified yeast mitochondria by affinity chromatography. It is a lipophilic protein of molecular mass 45 000 Da, which is tightly membrane-bound and localized on the outer surface of the inner membrane. It can be solubilized in active form under mild conditions. The cAMP receptor resembles mitochondrial RNA polymerase prepared as described by Levens et al. [(1981) J. Biol. Chem. 256, 1474] in a surprisingly large number of properties including molecular mass. Comparison of the two proteins revealed that the polypeptide previously considered as RNA polymerase is, in fact, a mitochondrial cAMP receptor protein.
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PMID:Isolation of a cAMP receptor protein from yeast mitochondria (Mr 45000) and comparison with mitochondrial RNA polymerase (Mr 45000). 299 38

The Escherichia coli lac promoter has been shown to contain an RNA polymerase binding site (P2) that overlaps with, and is shifted 22 base-pairs upstream from the normal lac promoter (P1). In this paper, we provide RNA polymerase protection data obtained in vitro that show that, in the absence of CAP-cAMP, in vitro P2 is the preferred polymerase binding site on the P+ template. In the presence of CAP-cAMP, polymerase binding to P2 is reduced and more polymerase is bound at P1. Two lac P1 "-35 region" mutations, L157 and 4, which increase the homology between this region and the consensus "-10 region" sequence, are both shown to have an increased affinity for polymerase binding at P2. CAP-cAMP is also able to decrease the amount of polymerase bound to P2 and to increase the amount bound to P1 on these mutant promoter fragments. P2 does not initiate transcription efficiently in vivo. Nuclease S1 mapping experiments detect only a low level of transcription from one of the P2 "up" mutations, but no beta-galactosidase synthesis is directed by this mutant. Mutations such as L157 and 4, which alter the P2-10 region, also alter lac P sensitivity to CAP-cAMP in vivo, suggesting that the P2 sequence plays a role in CAP-cAMP regulation of lac P. Possible roles for P2 in vivo are discussed.
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PMID:Properties of lac P2 in vivo and in vitro. An overlapping RNA polymerase binding site within the lactose promoter. 299 53

The Escherichia coli galactose and lactose promoter regions have been studied by alkylation interference experiments. The data reveal those bases or phosphate groups which, when modified, prevent the binding of the catabolite activator protein (CAP) or RNA polymerase and hence are presumably in contact with the proteins. Interference contacts made by CAP at its primary binding sites at gal and lac are quite similar, indicating that CAP-cAMP uses the same mode of binding at these two operons. RNA polymerase, when bound in the presence of CAP-cAMP, exhibits contacts at the gal and lac P1-10 regions very much like those of the lac UV5 and T7 A3 promoters (Siebenlist, U., Simpson, R. B., and Gilbert, W. (1980) Cell 20, 269-281). CAP, therefore, does not detectably alter the structure of the open complex. The binding sites for CAP and RNA polymerase at lac, as deduced from interference experiments, do not overlap. However, at gal a CAP molecule is found much closer to the enzyme, and there is competition for a set of mutual contacts. These experiments thus reveal both similarities and differences in the mechanisms whereby CAP activates transcription at catabolite-sensitive operons.
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PMID:The binding of catabolite activator protein and RNA polymerase to the Escherichia coli galactose and lactose promoters probed by alkylation interference studies. 301 46

The monoclonal antibody (mAb) 64D1 was found to inhibit cAMP binding by the cAMP receptor protein (CRP) from Escherichia coli (Li, X.-M., and Krakow, J. S. (1985) J. Biol. Chem. 260, 4378-4383). CRP is relatively resistant to attack by the Staphylococcus aureus V8 protease, chymotrypsin, trypsin, and subtilisin whereas both mAb 64D1-CRP and cAMP-CRP are attacked by these proteases yielding N-terminal core fragments. The fragment patterns resulting from proteolysis of mAb 64D1-CRP and cAMP-CRP differ indicating that the CRP in each complex is in a different conformation. The data presented indicate that the preferred conformation of the antigenic site for mAb 64D1 is present in unliganded CRP. Binding of mAb 64D1 to CRP is inhibited at high cAMP concentration. Formation of a stable cAMP-CRP-lac P+-RNA polymerase open promoter complex resistant to dissociation by mAb 64D1 occurs at a much lower cAMP concentration. The observed increase in resistance to mAb 64D1 may reflect a possible conformational change in CRP effected by contact with RNA polymerase in the open promoter complex.
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PMID:A monoclonal antibody that inhibits cyclic AMP binding by the Escherichia coli cyclic AMP receptor protein. 303 13

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

The sigma subunits of eubacterial RNA polymerases determine the site selectivity of initiation of transcription at promoters. Mutations in rpoD, the gene that encodes sigma 70, the major sigma factor in Escherichia coli, should be useful in determining the molecular details of the process of transcription initiation. However, such mutations are likely to be deleterious or lethal, since sigma70 is an essential gene product. We designed a system for the rapid isolation and fine structure mapping of mutations in rpoD, which allows selection of mutations that would otherwise be deleterious to the cell. We used this system to isolate a new class of mutations in rpoD, mutations that relieve the requirement for CAP-cAMP for initiation at promoters in the mal regulon. These mutations, which we designate rpoD(Mal) mutations, occur in two clusters in the rpoD gene within regions previously suggested by amino acid sequence comparisons to be important for sigma structure or function. We cannot distinguish whether the rpoD(Mal) mutations affect mal expression by altering interaction between RNA polymerase and mal promoters or between RNA polymerase and the accessory transcription factor MalT. However, the effects of the mutations on activator-independent transcription from the lac promoter (4 rpoD(Mal) mutations decrease CAP-independent expression of the lac promoter in vivo) suggest that the regions of sigma identified by our mutations may be directly involved in promoter recognition.
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PMID:Mutations in rpoD that increase expression of genes in the mal regulon of Escherichia coli K-12. 305 19

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

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

The rotation diffusion of DNA double helices and their complexes with the cAMP receptor protein (CRP) has been simulated by bead models, in order to derive information on their structure in solution by comparison with results obtained from dichroism decay measurements. Straight DNA double helices are simulated by linear, rigid strings of overlapping beads. The radius of the beads and the length of the string are increased simultaneously by the same increments from initial outer dimensions derived from crystallographic data to final values, which are fitted to experimental rotation time constants observed for short DNA fragments (less than 100 bp). The final values reflect the solvated structure with the same 'solvation layer' added in all three dimensions. The protein is simulated by overlapping beads, which are assembled to a structure very similar to that found by x-ray crystallography. Complexes of the protein with DNA are formed with the centres of palindromic DNA sites at the centre of the two helix-turn-helix-motifs of the protein with some overlap of the two components. Simulation of the experimental data obtained for CRP complexes with specific DNA in the presence of cAMP requires strong bending of the double helices. According to our simulation the DNA is almost completely wrapped around the protein both in the complexes with a 62 bp fragment containing the standard CRP site and with a 80 bp fragment containing the second binding site of the lac operon. Simulations of the data obtained for a 203 bp fragment with both binding sites suggest that the two bound CRP proteins are in contact with each other and that the DNA is wrapped around the two protein dimers. A stereochemical model is suggested with a tetrahedral arrangement of the four protein subunits, which provides the advantage that two binding sites of the protein formed by two subunits each are located favorable for tight contacts to two binding sites on bent DNA, provided that the DNA sites are separated by an integer number of helix turns. In summary, the simulations demonstrate strong bending, which can be reflected by an arc radius in the range around 50 A. According to these data the overall bending angle of our longest DNA fragment is approximately 180 degrees, and thus the protruding ends are sufficiently close to each other such that RNA polymerase, for example, could contact both helical segments.
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PMID:Turn of promotor DNA by cAMP receptor protein characterized by bead model simulation of rotational diffusion. 327 91


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