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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)

A novel source of transcription has been detected in the replication region of plasmid R6K by using fusions involving the galK reporter gene. The -35 and -10 consensus RNA polymerase binding sites were identified in the region overlapping the binding sites for the R6K-encoded replication protein pi. Transcription from this promoter, designated P2, is repressed in vivo by pi-protein levels that are inhibitory for replication. Promoter-down mutations in P2 induced in vitro by bisulfite mutagenesis result in a reduced copy number of a beta-replicon but not of a gamma-replicon. Implications of the role of P2 in R6K replication are discussed.
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PMID:Identification of a novel promoter in the replication control region of plasmid R6K. 162 64

The structural genes of the ilvGMEDA operon of Escherichia coli are preceded by two promoters, ilvPG1 and ilvPG2, and a leader-attenuator region. Alkylation protection and hydroxyl radical footprinting techniques have been used to demonstrate that integration host factor (IHF) interacts with the nucleotides in a consensus-like DNA sequence located immediately downstream of the RNA polymerase transcriptional pause site in the leader-attenuator region. In the presence of purified IHF protein, in vitro transcriptional pausing of RNA polymerase at the leader-attenuator pause site is increased 2-fold and, concomitantly, a 2-fold increase in transcriptional termination at the attenuator is observed. Strains containing chromosomal transcriptional fusions of various segments of the ilvGMEDA promoter-attenuator region to the galK gene were used to show that IHF also decreases the in vivo basal level of transcriptional readthrough at the attenuator 2-fold. The binding of IHF to another target site in the ilvPG1 promoter region represses transcription from this promoter and causes a 4-fold stimulation of transcription initiation from the downstream ilvPG2 promoter 4-fold. This IHF-mediated control of transcription initiation from the upstream promoter region is independent of the regulation of transcription termination effected by IHF interaction at the attenuator site. Thus, IHF is capable of regulating the expression of the ilvGMEDA operon in opposing manners; it can activate transcription initiation of this operon from the ilvPG2 promoter 4-fold and increase the termination of this transcription at the downstream attenuator 2-fold.
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PMID:Integration host factor-mediated expression of the ilvGMEDA operon of Escherichia coli. 170 60

In the active orientation the DnaA protein/dnaA box complex blocks transcribing RNA polymerase. The extent of transcription termination at different dnaA boxes was used to determine in vivo their various affinities to the DnaA protein. The rate of transcription distal to the respective dnaA box was monitored by the expression of the reporter gene galK. The dnaA boxes (5'-TTATACACA and 5'-TTATCCAAA), present in oriC, showed the strongest binding affinity. The dnaA box 5'-TTTTCCACA was mutated at eight positions such that the boxes differed from the consensus sequence, 5'-TT(A/T)T(A/C)CA(A/C)A, defined so far. Based on the different properties of the dnaA boxes, a new consensus sequence was derived: 5'-(T/C)(T/C)(A/T/C)T(A/C)C(A/G)(A/C/T(A/C).
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PMID:DnaA protein/DNA interaction. Modulation of the recognition sequence. 203 27

We used two different approaches to study the requirement for Escherichia coli Nus factors for the activity of bacteriophage lambda late antiterminator Q. Using an in vitro coupled transcription-translation assay, based on Q-dependent synthesis of galactokinase from a pR'-tR'-galK template, we showed that mutations in the host nusB and nusE genes do not affect Q activity. A mutation in nusA (nusA1) only partially affects Q action at all temperatures tested. Defective Q function in the nusA1 mutant extract could be restored by the addition of pure NusA but not by excess Q. In a pure transcription system, measurement of the run-off transcript produced by Q-mediated suppression of tR' revealed that NusA is greatly stimulatory to Q activity, whereas NusB and S10, in the presence or absence of NusA, have no effect. Unidentified E. coli factor(s) present in an S30 extract efficiently suppress the natural pausing by RNA polymerase at +15, +16 of pR' without affecting Q activity. These results show that NusA is the only host protein that directly participates in Q function.
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PMID:An analysis of the role of host factors in transcription antitermination in vitro by the Q protein of coliphage lambda. 214 85

Using modified Saccharomyces cerevisiae Ty1 elements located on a 2 mu plasmid, reverse-transcriptase-mediated transposition into yeast chromosomes of expression cassettes containing a foreign gene can be induced. These expression cassettes consist of the yeast ARG3 and CUP1 promoter sequences fused to the Escherichia coli galK structural gene. Expression cassettes as large as 2 kb can be inserted into Ty elements and transposed efficiently to various sites in the yeast genome. A third yeast promoter (from the yeast CAR1 gene) seems to be unsuitable for use in the expression cassette. This may be because it does not allow the transcription run-through necessary for Ty1 transposition. Ways of improving this vector system are discussed, as are its advantages over episomal vector systems.
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PMID:Retrovirus-like vectors for Saccharomyces cerevisiae: integration of foreign genes controlled by efficient promoters into yeast chromosomal DNA. 284 30

Initiation of DNA replication from the Escherichia coli origin, oriC, is dependent on an RNA polymerase-mediated transcription event. The function of this RNA synthetic event in initiation, however, remains obscure. Since control of the synthesis of this RNA could serve a key role in the overall initiation process, transcription regulatory sites within and near oriC were identified using the galK fusion vector system. Our results confirm the existence of a transcription termination signal within oriC, first identified by Hansen et al. (1981), for the 16 kd transcript that is transcribed counterclockwise towards oriC. Termination is shown to be 92% efficient. A similar approach led to the detection of transcription termination within the chromosomal replication origin of Klebsiella pneumoniae. Approximately 50% of the E. coli 16 kd transcripts appear to terminate before reaching oriC between the XhoI (+416 bp) and the HindIII (+243 bp) sites. The predominant 3' ends of RNA that enter oriC, as determined by SI nuclease mapping, were located at positions +20 +/- 2, +23 +/- 2, +37, +39, +52, +66, +92, and +107. These termination sites, which map cl to RNA . DNA junctions identified by Kohara et al. (1985), appear as triplets and quadruplets. The E. coli oriC Pori-L promoter described in in vitro transcription studies by Lother and Messer (1981) was not detected in this study in either wildtype cells or isogenic dnaA mutants at the nonpermissive temperature. A new promoter activity, Pori-R1, was identified within the E. coli origin in the clockwise direction.
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PMID:Transcription termination within the Escherichia coli origin of DNA replication, oriC. 301 76

The ilvC gene encodes acetohydroxy acid isomeroreductase (EC 1.1.1.89), the second enzyme in the parallel isoleucine-valine biosynthetic pathway. Expression of the ilvC gene is induced by acetohydroxy acid isomeroreductase substrates, acetohydroxybutyrate or acetolactate. This substrate induction is mediated by a positive activator encoded by an adjacent gene, ilvY. The ilvY and ilvC genes are transcribed in opposite directions from promoters that are overlapping. In this paper we characterize the in vitro DNA binding properties of the ilvY-encoded activator protein. The ilvY product binds to two adjacent operator sites located in the divergent-overlapping ilvY and ilvC promoter region. One of these operators, designated O1 contains regions of dyad symmetry centered at position +17 relative to the ilvY transcriptional start site, and the second site, designated O2, contains an homologous inverted repeat sequence centered about the -35 region of the ilvC promoter. Binding of the ilvY product at the O1 and O2 operator sites is co-operative and this ilvY protein-DNA complex in the presence of acetohydroxy acid isomeroreductase substrate is a prerequisite for RNA polymerase binding to the ilvC promoter as detected by DNase I protection experiments. Additionally, chromosomal galK transcriptional fusion assays were performed to characterize the regulation of the ilvY and ilvC promoters in vivo. Transcription of the ilvC gene is maintained at a basal level of activity which is elevated as much as 15-fold in the presence of ilvY product and acetohydroxybutyrate. The ilvY product represses ilvY transcription in a manner that does not appear to be dependent on acetohydroxy acid isomeroreductase substrate. We discuss models in which activation of ilvC transcription results from a direct interaction of ilvY protein with RNA polymerase or an ilvY-mediated alteration of the DNA conformation of the ilvC -35 promoter region. Additionally, we discuss the role of acetohydroxybutyrate and acetolactate in ilvY transcriptional regulation.
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PMID:Transcriptional activation at adjacent operators in the divergent-overlapping ilvY and ilvC promoters of Escherichia coli. 306 77

We have characterized the functional attributes of a 211-base pair region containing the Escherichia coli tryptophan operon rho-dependent terminator trp t', utilizing a series of constructs that alter the orientation, location, or extent of the trp t' sequences with respect to the trp promoter. In each instance, the extent of the rho-dependent response was monitored in vivo by read-through expression into a distal galactokinase gene and was compared with the results of transcription assays in vitro. As expected, transcription termination in vivo is dependent on the proper orientation of the terminator, and a tandem repeat of the terminator increases termination proportionally. Placing the terminator fragment only 14 nucleotides from the promoter does not affect termination significantly, supporting the belief that sequences outside of the 211-base pair fragment itself are dispensable. One construct, which lacks 116 base pairs, including the region encoding the normal RNA end points, still reduces galK activity in vivo and terminates transcription in vitro. Our results indicate that the rho response depends primarily on sequences in this 95-base pair segment, causing RNA polymerase to terminate transcription in a region 15-45 nucleotides further downstream.
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PMID:Signals sufficient for rho-dependent transcription termination at trp t' span a region centered 60 base pairs upstream of the earliest 3' end point. 327 76

We have used the galK gene, minus its promoter, to quantitate transcription of the orfE--pyrE operon of Escherichia coli in front of and after the intercistronic attenuator. Expression of the hybrid genes was studied in a bacterium with mutations that permit changes in the UTP and GTP pools during exponential growth. It was found that the greater part of pyrE gene regulation by the nucleotides takes place at the intercistronic attenuator and that promoter control contributes only little, ca. twofold. When pools of both UTP and GTP were high only 5%-6% of the mRNA chains were continued into the pyrE gene. However, when the UTP pool was reduced (from 1.3 to 0.2 mumol/g dry weight) nearly 100% of transcription passed the attenuator. Likewise, a reduction in the GTP pool (from 3.2 to 0.8 mumol/g dry weight) resulted in 25%-30% escape of attenuation. Regulation by attenuation disappeared when a premature stop-codon was introduced near the end of orfE such that translational coupling to transcription was prevented in the attenuator area. Therefore, we attribute the modulation of attenuation to nucleotide-induced variations in the kinetics of mRNA chain elongation. In support for this it was found that an RNA polymerase mutant with reduced RNA chain growth rate transcribed past the pyrE attenuator at a high frequency in the presence of a high UTP pool, but only when coupling of translation to transcription was allowed at the end of orfE.
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PMID:Effect of UTP and GTP pools on attenuation at the pyrE gene of Escherichia coli. 330 6

Transcription in vitro of the regulatory region of the ilvGMEDA operon yields two attenuated RNAs initiated from the tandem promoters ilvGp1 and ilvGp2. Both S1 nuclease analysis and the fusion of ilvGp1 to galK indicate that transcription is not initiated in vivo from ilvGp1. However deletion of DNA sequences 150 to 100 bp upstream of ilvGp2 drastically reduces expression in vivo from ilvGp2. Both the distance separating ilvGp2 from the upstream DNA sequences and their relative orientation to each other on the DNA helix affect expression from ilvGp2. Deletion of DNA sequences approximately 400 bp upstream of ilvGp2 increases expression in vivo from this promoter. Analysis of products of transcription in vitro indicates that the effects observed in vivo are probably not due to DNA conformation or interactions of RNA polymerase.
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PMID:Effect of the deletion of upstream DNA sequences on expression from the ilvGp2 promoter of the ilvGMEDA operon of Escherichia coli K-12. 354 37


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