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C L Turnbough

Publications and source records attributed to C L Turnbough.

At least 19 recordsLinked to original sources
PubMed2001

A long T. A tract in the upp initially transcribed region is required for regulation of upp expression by UTP-dependent reiterative transcription in Escherichia coli.

In Escherichia coli, pyrimidine-mediated regulation of upp expression occurs by UTP-sensitive selection of alternative transcriptional start sites, which produces transcripts that differ in the ability to be elongated. The upp initially transcribed region contains the sequence GATTTTTTTTG (nontemplate strand). Initiation can occur at either the first or the second base in this sequence (designated G6 and A7, with numbering from the promoter -10 region). High intracellular UTP levels favor initiation at position A7; however, the resulting transcripts are subject to reiterative transcription (i.e., repetitive UMP addition) within the 8-bp T. A tract in the initially transcribed region and are aborted. In contrast, low intracellular UTP levels favor initiation at position G6, which results in transcripts that can, in part, avoid reiterative transcription and be elongated normally. In this study, we examined the regulatory requirement for the long T. A tract in the upp initially transcribed region. We constructed upp promoter mutations that shorten the T. A tract to 7, 6, 5, 4, 3, or 2 bp and examined the effects of these mutations on upp expression and regulation. The results indicate that pyrimidine-mediated regulation is gradually reduced as the T. A tract is shortened from 7 to 3 bp; at which point regulation ceases. This reduction in regulation is due to large-percentage increases in upp expression in cells grown under conditions of pyrimidine excess. Quantitation of cellular transcripts and in vitro transcription studies indicate that the observed effects of a shortened T. A tract on upp expression and regulation are due to increases in the fraction of both G6- and A7-initiated transcripts that avoid reiterative transcription and are elongated normally.

AT Rich Sequence↗
PubMed1999

Multiple mechanisms are used for growth rate and stringent control of leuV transcriptional initiation in Escherichia coli.

Expression of the Escherichia coli leuV operon, which contains three tRNA(1)(Leu) genes, is regulated by several mechanisms including growth-rate-dependent control (GRDC) and stringent control (SC). Structural variants of the leuV promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be different and that GRDC of the leuV promoter occurs in the absence of guanosine 3', 5'-bisdiphosphate. To determine the mechanisms of the leuV promoter regulation, we have examined the stability of promoter open complexes and the effects of nucleotide triphosphate (NTP) concentration on the efficiency of the leuV promoter and its structural variants in vitro and in vivo. The leuV promoter open complexes were an order of magnitude more stable to heparin challenge than those of rrnBp(1). The major initiating nucleotide GTP as well as other NTPs increased the stability of the leuV promoter open complexes. When the cellular level of purine triphosphates was increased at slower growth rates by pyrimidine limitation, a 10% reduction in leuV promoter activity was seen. It therefore appears that transcription initiation from the leuV promoter is less sensitive to changes in intracellular NTP concentration than that from rrnBp(1). Comparative analysis of regulation of the leuV promoter with and without upstream activating sequences (UAS) demonstrated that the binding site for factor of inversion stimulation (FIS) located in UAS is essential for maximal GRDC. Moreover, the presence of UAS overcame the effects of leuV promoter mutations, which abolished GRDC of the leuV core promoter. However, although the presence of putative FIS binding site was essential for optimal GRDC, both mutant and wild-type leuV promoters containing UAS showed improved GRDC in a fis mutant background, suggesting that FIS protein is an important but not unique participant in the regulation of the leuV promoter.

Base Sequence↗
PubMed1998

Regulation of carAB expression in Escherichia coli occurs in part through UTP-sensitive reiterative transcription.

In Escherichia coli, expression of the carAB operon is subject to cumulative repression, which occurs by ArgR-mediated repression at a downstream promoter, P2, and by pyrimidine-mediated regulation at an upstream promoter, P1. In this study, we show that pyrimidine-mediated regulation occurs in part through a mechanism involving UTP-sensitive reiterative transcription (i.e., repetitive addition of U residues to the 3' end of a nascent transcript due to transcript-template slippage). In this case, reiterative transcription occurs at the end of a run of three T x A base pairs in the initially transcribed region of the carAB P1 promoter. The sequence of this region is 5'-GTTTGC (nontemplate strand). In the proposed regulatory mechanism, increased intracellular levels of UTP promote reiterative transcription, which results in the synthesis of transcripts with the sequence GUUUU(n) (where n = 1 to >30). These transcripts are not extended downstream to include structural gene sequences. In contrast, lower levels of UTP enhance normal template-directed addition of a G residue at position 5 of the nascent transcript. This addition precludes reiterative transcription and permits normal transcript elongation capable of producing translatable carAB transcripts. Thus, carAB expression, which is necessary for pyrimidine nucleotide (and arginine) biosynthesis, increases in proportion to the cellular need for UTP. The proposed mechanism appears to function independently of a second pyrimidine-mediated control mechanism that involves the regulatory proteins CarP and integration host factor.

Artificial Gene Fusion↗
PubMed1997

Transcription regulation by initiating NTP concentration: rRNA synthesis in bacteria.

The sequence of a promoter determines not only the efficiency with which it forms a complex with RNA polymerase, but also the concentration of nucleoside triphosphate (NTP) required for initiating transcription. Escherichia coli ribosomal RNA (rrn P1) promoters require high initiating NTP concentrations for efficient transcription because they form unusually short-lived complexes with RNA polymerase; high initiating NTP concentrations [adenosine or guanosine triphosphate (ATP or GTP), depending on the rrn P1 promoter] are needed to bind to and stabilize the open complex. ATP and GTP concentrations, and therefore rrn P1 promoter activity, increase with growth rate. Because ribosomal RNA transcription determines the rate of ribosome synthesis, the control of ribosomal RNA transcription by NTP concentration provides a molecular explanation for the growth rate-dependent control and homeostatic regulation of ribosome synthesis.

Adenosine Triphosphate↗
PubMed1997

Regulation of upp expression in Escherichia coli by UTP-sensitive selection of transcriptional start sites coupled with UTP-dependent reiterative transcription.

Expression of the upp gene of Escherichia coli, which encodes the pyrimidine salvage enzyme uracil phosphoribosyltransferase, is negatively regulated by pyrimidine availability. In this study, we demonstrate that this regulation occurs mainly by UTP-sensitive selection of alternative transcriptional start sites, which produces transcripts that differ in the ability to be productively elongated. The upp initially transcribed region contains the sequence GATTTTTTTTG (nontemplate strand). Transcription is initiated primarily at the first two bases in this sequence, designated G6 and A7 (counting from the promoter -10 region). High intracellular levels of UTP favor initiation at position A7; however, the resulting transcripts are subject to reiterative transcription (i.e., repetitive nucleotide addition) within the run of T residues in the initially transcribed region. The resulting AUUUUn (where n = 1 to >50) transcripts are not extended to include downstream upp sequences. In contrast, low intracellular levels of UTP strongly favor initiation at position G6, which results in transcripts that generally do not engage in reiterative transcription and thus can be normally elongated. This mechanism ensures that high levels of uracil phosphoribosyltransferase are produced only under conditions of pyrimidine limitation. The mechanisms that account for UTP-sensitive start site selection and different fates of upp transcripts, as well as the general use of UTP-dependent reiterative transcription in gene regulation, are discussed in detail.

Escherichia coli↗
PubMed1995

Regulation of codBA operon expression in Escherichia coli by UTP-dependent reiterative transcription and UTP-sensitive transcriptional start site switching.

Reiterative transcription is the repetitive addition of nucleotides to the 3' end of a nascent transcript due to slippage between the transcript and DNA template. Recently, we showed that pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli occurs, in part, through a mechanism in which induction of UTP-dependent reiterative transcription within the initially transcribed region prevents downstream extension of the nascent transcript to include structural gene sequences. In this study we demonstrate that pyrimidine-mediated regulation of codBA operon expression in E. coli also involves UTP-dependent reiterative transcription during initiation; however, the mechanism is different from that of the pyrBI operon. The initially transcribed region of the codBA promoter contains the sequence GATTTTTTG (non-template strand). Our results show that transcription is initiated primarily at the first two bases designated G7 and A8 (counting from the -10 region). When transcripts are initiated at position A8, UTP-dependent reiterative transcription always occurs within the run of T residues in the initially transcribed region. The AUUUUn (where n = 1 to > 15) transcripts produced by this reaction are not extended productively to include downstream codBA sequences. In contrast, most transcripts initiated at position G7 do not engage in reiterative transcription and can be elongated normally. Characterization of a codBA promoter mutation that prevents reiterative transcription showed that this reaction is required for virtually all pyrimidine-mediated regulation of operon expression and that UTP levels control the selection of the G7 and A8 transcriptional start sites. These results suggest a model for regulation in which high intracellular levels of UTP favor transcriptional initiation at position A8 and thus the accompanying reiterative transcription, which together preclude initiation at position G7. Low levels of UTP inhibit initiation at position A8 and the associated reiterative transcription, thereby allowing high levels of initiation at position G7 and operon expression. Our results also indicate critical sequence requirements for reiterative transcription, which are important for understanding the mechanism of this reaction as well as for identifying other promoters at which this reaction may occur. Of particular interest is the indication that an RNA:DNA hybrid forms during transcriptional initiation and the strength of this hybrid controls the extent of reiterative transcription.

Bacterial Outer Membrane Proteins↗
PubMed1994

Regulation of pyrBI operon expression in Escherichia coli by UTP-sensitive reiterative RNA synthesis during transcriptional initiation.

Pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli K-12 occurs through UTP-sensitive transcriptional attenuation and through a second mechanism that functions at the level of transcriptional initiation. In this study we demonstrate that this second control mechanism is based on UTP-sensitive reiterative RNA synthesis within a run of three T-A base pairs in the pyrBI initially transcribed region. Our results show that high UTP levels induce the synthesis in vitro of nascent transcripts with the sequence AAUUUUn (where n = 1 to > 30), which are not extended downstream to include pyrBI sequences. Synthesis of these transcripts, which are initiated at the predominant in vivo transcriptional start site, inhibits the production of full-length pyrBI transcripts. A TTT to GTA mutation in the pyrBI initially transcribed region eliminates reiterative transcription and stimulates productive transcription in vitro. When introduced into the E. coli chromosome, this mutation causes a sevenfold increase in pyrBI expression in cells grown under conditions of pyrimidine excess and nearly abolishes pyrimidine-mediated regulation of pyrBI expression when coupled with a mutation that eliminates attenuation control. Additional experiments indicate that the context of the three T-A base pairs within the pyrBI initially transcribed region is important for reiterative transcription. A possible mechanism for reiterative transcription and the likely involvement of this process in the regulation of other genes are discussed.

Base Composition↗
PubMed1994

Nucleotide-specific transcriptional pausing in the pyrBI leader region of Escherichia coli K-12.

Expression of the pyrBI operon, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is regulated primarily through a UTP-sensitive transcriptional attenuation control mechanism in Escherichia coli. In this mechanism, the extent of coupling between transcription and translation within the pyrBI leader region determines the level of p-independent transcriptional termination at an attenuator preceding the pyrB gene. A key feature in this mechanism is transcriptional pausing that occurs before the attenuator in the pyrBI leader region when UTP concentrations are low. In this study, we characterized in detail this UTP-sensitive transcriptional pausing in vitro. Our results show that pausing occurs, to different extents, before the addition of every uridine residue in the leader transcript. This pausing occurred only at low UTP concentrations (20-200 microM) that cause high levels of pyrBI expression in vivo. Transcriptional pausing did not occur in the pyrBI leader region at 20 microM CTP or ATP and only occurred strongly at one site at 20 microM GTP. We also demonstrated that NusA functions as a general enhancer of UTP-sensitive transcriptional pausing in the pyrBI leader region. Finally, we identified at the 3' end of the pyrBI attenuator a 3-base region at which termination occurs. Selection of termination sites within this region was shifted by varying the UTP concentration or including NusA in the transcription reaction mixture.

Adenosine Triphosphate↗
PubMed1994

An Escherichia coli RNA polymerase defective in transcription due to its overproduction of abortive initiation products.

One of the potential regulatory steps in procaryotic transcription is promoter clearance, a transition step in transcription initiation at which an RNA polymerase (RNAP) switches from the initial transcribing stage to the elongation stage. The biological significance of promoter clearance and the role of RNAP in this process are not understood. One approach to address these questions is to study mutant RNAPs that have altered promoter clearance. Because the antibiotic rifampicin inhibits transcription by preventing an initial transcribing complex from entering the elongation mode, mutant RNAPs which confer rifampicin (Rifr) are likely to be altered in promoter clearance. To test this hypothesis, we studied the effects of Rifr RNAPs on the pyrBI promoter, which is subject to control of promoter clearance in response to the availability of UTP. Two Rifr alleles that carry a different altered amino acid residue at position 529 of the beta subunit appeared to be defective in transcription from the pyrBI promoter in vivo. Biochemical analysis of one of these mutant RNAPs, RpoB3401 with a R529C change in the beta subunit, showed that it overproduces aborted initiation products from the pyrBI promoter and thus is defective in promoter clearance leading to reduced productive initiation. The severity of overproducing the aborted initiation products is an inverse function of the UTP concentration indicating that RpoB3401 has reduced affinity for UTP and thus is subject to a high Km barrier during promoter clearance. The defect of RpoB3401 in abortive initiation in vitro could account fully for its reduced initiation activity in vivo demonstrating the biological significance of abortive synthesis in transcription initiation. Our results indicate that at least part of the "rif region" is important for the process of abortive initiation and that promoter clearance can be regulated in part by modulating the Km of RNAP for nucleotides during initiation. The mutant enzyme is not altered in stuttering RNA synthesis at the pyrBI promoter, previously observed with wild-type RNAP. Our results also show that the mechanisms underlying the two non-productive initiation events (abortive synthesis and stuttering synthesis) at the pyrBI promoter are distinct.

Amino Acid Sequence↗
PubMed1994

Effects of transcriptional start site sequence and position on nucleotide-sensitive selection of alternative start sites at the pyrC promoter in Escherichia coli.

In Escherichia coli, expression of the pyrC gene is regulated primarily by a translational control mechanism based on nucleotide-sensitive selection of transcriptional start sites at the pyrC promoter. When intracellular levels of CTP are high, pyrC transcripts are initiated predominantly with CTP at a site 7 bases downstream of the Pribnow box. These transcripts form a stable hairpin at their 5' ends that blocks ribosome binding. When the CTP level is low and the GTP level is high, conditions found in pyrimidine-limited cells, transcripts are initiated primarily with GTP at a site 9 bases downstream of the Pribnow box. These shorter transcripts are unable to form a hairpin at their 5' ends and are readily translated. In this study, we examined the effects of nucleotide sequence and position on the selection of transcriptional start sites at the pyrC promoter. We characterized promoter mutations that systematically alter the sequence at position 7 or 9 downstream of the Pribnow box or vary the spacing between the Pribnow box and wild-type transcriptional initiation region. The results reveal preferences for particular initiating nucleotides (ATP > or = GTP > UTP >> CTP) and for starting positions downstream of the Pribnow box (7 >> 6 and 8 > 9 > 10). The results indicate that optimal nucleotide-sensitive start site switching at the wild-type pyrC promoter is the result of competition between the preferred start site (position 7) that uses the poorest initiating nucleotide (CTP) and a weak start site (position 9) that uses a good initiating nucleotide (GTP). The sequence of the pyrC promoter also minimizes the synthesis of untranslatable transcripts and provides for maximum stability of the regulatory transcript hairpin. In addition, the results show that the effects of the mutations on pyrC expression and regulation are consistent with the current model for translational control. Possible effects of preferences for initiating nucleotides and start sites on the expression and regulation of other genes are discussed.

Base Sequence↗
PubMed1994

Identification of the Shine-Dalgarno sequence required for expression and translational control of the pyrC gene in Escherichia coli K-12.

Expression of the pyrC gene in Escherichia coli K-12 is regulated by a translational control mechanism in which CTP (and perhaps GTP) pool sizes determine the selection of alternative transcriptional start sites at the pyrC promoter. High CTP levels cause transcription to start primarily at a site that directs the synthesis of untranslatable pyrC transcripts. These transcripts form a hairpin at their 5' ends that blocks ribosome binding to the Shine-Dalgarno (SD) sequence. The pyrC ribosome binding site is unusual in that it contains two potential SD sequences, designated SD1 and SD2, which are located 11 and 4 nucleotides upstream of the translational initiation codon, respectively. In this study, we examined the functions of these two SD sequences in translational initiation. Mutations that inactivate either SD1 or SD2 were constructed and incorporated separately into a pyrC::lacZ protein fusion. The effects of the mutations on pyrC::lacZ expression, regulation, and transcript levels were determined. The results indicate that SD1 is the only functional pyrC SD sequence. The SD2 mutation did cause a small reduction in expression, but this effect appeared to be due to a decrease in transcript stability. In addition, we constructed a mutation that introduces a long spacer region between the hairpin at the 5' end of the pyrC transcript and a new pyrC SD sequence. As predicted by the model for translational control, this mutation caused constitutive expression of a pyrC::lacZ protein fusion.

Base Sequence↗
PubMed1993

Genetic evidence that promoter P2 is the physiologically significant promoter for the pyrBI operon of Escherichia coli K-12.

The pyrBI operon of Escherichia coli K-12 encodes the two nonidentical subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase (ATCase). Expression of this operon is negatively regulated by pyrimidine availability primarily through UTP-sensitive transcriptional attenuation and, to a lesser extent, at the level of transcriptional initiation. Previous studies indicated that the pyrBI operon was transcribed from tandem sigma 70 promoters designated P1 and P2, with the large majority of transcription initiated at the more downstream promoter P2. To more clearly define the roles of these promoters, mutations that severely impair or inactivate individual promoters were constructed in the chromosomal pyrBI operon, and their effects on ATCase synthesis were measured. In cells grown under conditions of either pyrimidine excess or pyrimidine limitation, more than 99% of all ATCase synthesis was directed by transcripts initiated at promoter P2, indicating that it is the only physiologically significant pyrBI promoter. However, mutations that effectively inactivate promoter P1 caused a 15% reduction in ATCase levels, apparently by inhibiting transcription from promoter P2 by an unknown mechanism. Support for this explanation was provided by the demonstration that little, if any, transcriptional initiation occurred at promoter P1 in a transcriptional fusion vector whereas a high level of transcription was initiated at promoter P2 in an equivalent construction. Our results also provide evidence for pyrimidine-mediated regulation of transcriptional initiation at promoter P2 over a severalfold range and show that cells can grow reasonably well with very low levels of ATCase, apparently because of changes in the concentration of allosteric effectors that increase the specific activity of the enzyme.

Aspartate Carbamoyltransferase↗
PubMed1992

Translational control of pyrC expression mediated by nucleotide-sensitive selection of transcriptional start sites in Escherichia coli.

Expression of the pyrC gene, which encodes the pyrimidine biosynthetic enzyme dihydroorotase, is negatively regulated by pyrimidine availability in Escherichia coli. To define the mechanism of this regulation, an essential regulatory region between the pyrC promoter and the initial codons of the pyrC structural gene was identified. Mutational analysis of this regulatory region showed that the formation of a hairpin at the 5' end of the pyrC transcript, which overlaps the pyrC ribosome binding site, is required for repression of pyrC expression. Formation of the hairpin appears to be controlled by nucleotide-sensitive selection of the site of pyrC transcriptional initiation. When the CTP level is high, the major pyrC transcript is initiated with this nucleotide at a position seven bases downstream of the pyrC -10 region. This transcript is capable of forming a stable hairpin at its 5' end. When the CTP level is low and the GTP level is high, conditions found in cells limited for pyrimidines, the major pyrC transcript is initiated with GTP at a position two bases further downstream. This shorter transcript appears to be unable to form a stable hairpin at its 5' end. These results suggest a model for regulation in which the longer pyrC transcripts are synthesized predominantly under conditions of pyrimidine excess and form the regulatory hairpin, which blocks pyrC translational initiation. In contrast, the shorter pyrC transcripts are synthesized primarily under conditions of pyrimidine limitation, and they are readily translated, resulting in a high level of dihydroorotase synthesis. The data also indicate that a low level of pyrimidine-mediated regulation may occur at the level of transcriptional initiation.

Base Sequence↗
PubMed1990

Characterization of transcriptional initiation from promoters P1 and P2 of the pyrBI operon of Escherichia coli K12.

Expression of the pyrBI operon of Escherichia coli K12, which encodes the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated by pyrimidine availability, primarily through an attenuation control mechanism, and also appears to be subject to stringent control. Previous in vitro transcription studies indicated that the pyrBI operon is transcribed from tandem promoters designated P1 and P2, which appeared to be of similar strength. In this study, we characterized these promoters in detail and examined their role in pyrBI expression. Our results show that although transcription is initiated at both promoters in vivo, greater than 99% of the pyrBI transcripts are initiated at promoter P2, indicating that this is the only physiologically significant promoter. The level of transcripts initiated at promoter P2 was found to be higher in cells grown under pyrimidine-limiting conditions compared to that in cells grown under conditions of pyrimidine excess, indicating pyrimidine-mediated regulation at the level of transcriptional initiation. In vitro characterization of transcription from the pyrBI promoter region showed that non-physiological reaction conditions used in the original identification of the two promoters greatly overestimated the strength of promoter P1. Further in vitro characterization of the two promoters showed that transcription from promoter P2, but not from promoter P1, is inhibited by guanosine tetraphosphate and exhibits salt and heparin sensitivity typical of a stringently controlled promoter. In addition, heparin-challenge experiments revealed a UTP-induced instability of transcriptional initiation complexes at promoter P2 which may be of regulatory significance.

Amino Acid Sequence↗
PubMed1990

Role of the purine repressor in the regulation of pyrimidine gene expression in Escherichia coli K-12.

The pyrC and pyrD genes of Escherichia coli K-12 encode the pyrimidine biosynthetic enzymes dihydroorotase and dihydroorotate dehydrogenase, respectively. A highly conserved sequence in the promoter regions of these two genes is similar to the pur operator, which is the binding site for the purine repressor (PurR). In this study, we examined the role of PurR in the regulation of pyrC and pyrD expression. Our results show that pyrC and pyrD expression was repressed approximately twofold in cells grown in the presence of adenine [corrected] through a mechanism requiring PurR. A mutation, designated pyrCp926, which alters a 6-base-pair region within the conserved sequence in the pyrC promoter eliminated PurR-mediated repression of pyrC expression. This result indicates that PurR binds to the pyrC (and presumably to the pyrD) conserved sequence and inhibits transcriptional initiation. We also demonstrated that the pyrCp926 mutation had no effect on pyrimidine-mediated regulation of pyrC expression, indicating that pyrimidine and purine effectors act through independent mechanisms to control the expression of the pyrC and pyrD genes.

Amidohydrolases↗
PubMed1989

Multiple control mechanisms for pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli K-12.

Expression of the pyrBI operon of Escherichia coli K-12, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase, is negatively regulated over a several-hundredfold range by pyrimidine availability. This regulation occurs, at least in large part, through a UTP-sensitive attenuation control mechanism in which transcriptional termination at the pyrBI attenuator, a rho-independent transcriptional terminator located immediately upstream of the pyrB structural gene, is regulated by the relative rates of transcription and translation within the pyrBI leader region. There is suggestive evidence that an additional, attenuator-independent control mechanism also contributes to this regulation. To measure the level of regulation that occurs through the attenuation and attenuator-independent control mechanisms, we constructed a mutant strain in which a 9-base-pair deletion was introduced into the attenuator of the chromosomal pyrBI operon. This deletion, which removes the run of thymidine residues at the end of the attenuator, completely abolishes rho-independent transcriptional termination activity. When the mutant strain was grown under conditions of pyrimidine excess, the level of operon expression was 51-fold greater than that of an isogenic pyrBI+ strain. Under conditions of pyrimidine limitation, operon expression was increased an additional 6.5-fold in the mutant. These results demonstrate that the attenuation control mechanism is primarily responsible for pyrimidine-mediated regulation but that there is a significant contribution by an attenuator-independent control mechanism.

Aspartate Carbamoyltransferase↗