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)

We have isolated a new class of mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that alter the transcription initiation properties of RNA polymerase holoenzyme. The rpoD(Lac) mutations increase expression of the lac operon in the absence of CAP-cAMP, allowing a strain lacking adenyl cyclase to grow on lactose. Four of the six alleles isolated have three- to fivefold increases in the amount of lac mRNA and beta-galactosidase per cell. We show that these four mutations increase transcription initiation from the same promoter used by wild-type RNA polymerase. The mutations were mapped and sequenced. One mutation occurs in the codon for amino acid 389 of the sigma 70 polypeptide. The remaining five mutations are clustered, affecting residues 570, 571 and 575. These five mutations are within or near a proposed helix-turn-helix motif in the C terminus of sigma 70.
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PMID:Mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that increase expression of the lac operon in the absence of CAP-cAMP. 284 53

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

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 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

Transcription from the major late promoter of adenovirus type 2 DNA (including DNA sequences from 56 nucleotides upstream to 33 nucleotides downstream of the CAP site) was reconstituted with transcription factors purified from HeLa cells. Five components, transcription factors (TF) IIA, -B, -E, -D and RNA polymerase II, were required for accurate initiation of transcription. Kinetic analyses combined with order of addition experiments suggested that TFIIA acted first during the initiation reaction and that this interaction was followed by the action of TFIID. In agreement with these conclusions, both TFIIA and TFIID were required to render a transcription reaction partially resistant to concentrations of Sarkosyl previously shown to inhibit an early step in the formation of a preinitiation complex. Related Sarkosyl studies indicated that the inferred complex was subsequently recognized by RNA polymerase II, which resulted in an increased level of Sarkosyl-resistant transcription (in the presence of TFIIA and TFIID), and that this interaction occurred independently of TFIIB and TFIIE. However, TFIIB and TFIIE were implicated, along with the other factors and RNA polymerase II, in the subsequent formation of a highly stable preinitiation complex, which was inferred from its ability to initiate (with added nucleotides) in the presence of heparin concentrations which blocked unbound factors. The identification of a new transcription factor, which was required only when viral sequences 3' to the major late promoter were part of the transcription unit, is also reported.
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PMID:Factors involved in specific transcription in mammalian RNA polymerase II. Functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site. 381 43

S1 nuclease was used to generate a series of deletions which extend into the CAP-cAMP binding site from upstream of the Escherichia coli lactose operon promoter (lacP). Deletion and insertion mutations were also created which changed the spacing region between the CAP-cAMP binding site and the lacP -35 region. The promoter activities of these mutations were compared by measuring the levels of beta-galactosidase gene expression in vivo. The results show that sequence information prior to 74 base pairs (-74) upstream from the transcription start site (designated as +1) is not necessary for the full activation of the lac promoter by the CAP-cAMP complex. However, the deletion which extends to the -71 position retains only one third of the promoter activity in the presence of the CAP-cAMP complex. Removal of one symmetrical element from the two fold symmetry in the CAP-cAMP binding site abolished the CAP-cAMP stimulation of the lac promoter. Spacer mutations which increase by one base pair or decrease by two base pairs the length of the spacing region between the CAP-cAMP binding site and the lacP -35 region drastically reduced the CAP-cAMP stimulation of the lac promoter. This suggests that the distance between the lac promoter transcription start site and CAP-cAMP binding site is crucial for the function of the lac promoter, despite the fact that this distance varies in other E. coli promoters positively regulated by CAP-cAMP. A deletion which extends to the -59 position results in a two fold enhanced expression of lac in the absence of CAP-cAMP. This is consistent with the existance of a competitive RNA polymerase binding site in this region which would normally act to inhibit RNA polymerase binding.
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PMID:Deletion analysis of the CAP-cAMP binding site of the Escherichia coli lactose promoter. 608 87

Several general principles emerge from the studies of Cro, lambda repressor, and CAP. The DNA-binding sites are recognized in a form similar to B-DNA. They do not form cruciforms or other novel DNA structures. There seem to be proteins that bind left-handed Z-DNA (87) and DNA in other conformations, but it remains to be seen how these structures are recognized or how proteins recognize specific sequences in single-stranded DNA. Cro, repressor, and CAP use symmetrically related subunits to interact with two-fold related sites in the operator sequences. Many other DNA-binding proteins are dimers or tetramers and their operator sequences have approximate two-fold symmetry. It seems likely that these proteins will, like Cro, repressor, and CAP, form symmetric complexes. However, there is no requirement for symmetry in protein-DNA interactions. Some sequence-specific DNA-binding proteins, like RNA polymerase, do not have symmetrically related subunits and do not bind to symmetric recognition sequences. Cro, repressor, and CAP use alpha-helices for many of the contacts between side chains and bases in the major groove. An adjacent alpha-helical region contacts the DNA backbone and may help to orient the "recognition" helices. This use of alpha-helical regions for DNA binding appears to be a common mode of recognition. Most of the contacts made by Cro, repressor, and CAP occur on one side of the double helix. However, lambda repressor contacts both sides of the double helix by using a flexible region of protein to wrap around the DNA. Recognition of specific base sequences involves hydrogen bonds and van der Waals interactions between side chains and the edges of base pairs. These specific interactions, together with backbone interactions and electrostatic interactions, stabilize the protein-DNA complexes. The current models for the complexes of Cro, repressor, and CAP with operator DNA are probably fundamentally correct, but it should be emphasized that model building alone, even when coupled with genetic and biochemical studies, cannot be expected to provide a completely reliable "high-resolution" view of the protein-DNA complex. For example, the use of standard B-DNA geometry for the operator is clearly an approximation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Protein-DNA recognition. 623 44

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

Some unintegrated and all integrated forms of murine leukemia viral DNA contain long terminal repeats (LTRs). The entire nucleotide sequence of the LTR and adjacent cellular sequences at the 5' end of a cloned integrated proviral DNA obtained from BALB/Mo mouse has been determined. It was compared to the nucleotide sequence of the LTR at the 3' end. The results indicate: (i) a direct 517-nucleotide repeat at the 5' and 3' termini; (ii) 145 nucleotides out of 517 nucleotides represent sequences between the 5'-CAP nucleotide and 3' end of the primer tRNA (strong-stop DNA); (iii) an 11-nucleotide inverted repeat is present at the ends of the 5'-LTR and a total of 17 out of 21 nucleotides at the termini are inverted repeats; (iv) sequences CAATAAAAG (at positions -24 to -31) and CAATAAAC (at positions +46 to +53) resembling the hypothetical DNA-dependent RNA polymerase II promoter site can be identified in the 5'-LTR; (v) the sequence GAAA appears to be repeated on both sides of the junction of viral and cellular sequences; and (vi) in analogy with the bacterial transposons, the presence of an inverted repeat sequence at the termini of 5'-LTR suggests that M-MLV also has the integration properties of a transposon.
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PMID:Structure of Moloney murine leukemia viral DNA: nucleotide sequence of the 5' long terminal repeat and adjacent cellular sequences. 625 55

The B goes to A conformational transition caused by high ethanol concentrations was studied for seven DNA restriction fragments with overlapping and known sequences. Since the DNAs are homogeneous and range in GC content from 44-63%, they permit an evaluation of the influence of DNA sequence and base composition on the B goes to A transition. Moreover, their small size (80-301 bp) minimizes precipitation artifacts. The B- form spectra (in low salt) and the transition toward the C- form (in ethanol concentrations below the B goes to A transition) agree with prior measurements on chromosomal DNAs and are similar for all seven DNAs. At higher ethanol concentrations (80%), all fragments undergo a transition to the A- form as judged by the large increase of the positive CD band at 270 nm. Difference spectra among the fragments reveal minor differences between the A- form spectra. The ethanol concentration necessary to cause this transition is 72 +/- 2% for all fragments, thus excluding a preference of the CAP-, E. coli RNA polymerase-, or lac repressor-binding sequences for the A- form. The kinetics of the B goes to A transition in 80% ethanol are biphasic; the initial rapid transition is an intramolecular B goes to A form shift and the slower transition is an aggregation (but not precipitation) of the DNA
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PMID:Circular dichroism studies of the B goes to A conformational transition in seven small DNA restriction fragments containing the Escherichia coli lactose control region. 625 44


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