Regulation of carAB Expression in Escherichia coli Occurs in Part through UTP-Sensitive Reiterative Transcription (original) (raw)
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Journal of Bacteriology, 1998
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 ⅐ 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.
Journal of Molecular Biology, 1995
Reiterative transcription is the repetitive addition of nucleotides to the 3' University of Alabama at end of a nascent transcript due to slippage between the transcript and DNA template. Recently, we showed that pyrimidine-mediated regulation Birmingham, Birmingham of pyrBI operon expression in Escherichia coli occurs, in part, through a AL 35294, USA 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 AUUUU n (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.
Journal of Bacteriology, 2014
Reiterative transcription is a reaction catalyzed by RNA polymerase, in which nucleotides are repetitively added to the 3′ end of a nascent transcript due to upstream slippage of the transcript without movement of the DNA template. In Escherichia coli , the expression of several operons is regulated through mechanisms in which high intracellular levels of UTP promote reiterative transcription that adds extra U residues to the 3′ end of a nascent transcript during transcription initiation. Immediately following the addition of one or more extra U residues, the nascent transcripts are released from the transcription initiation complex, thereby reducing the level of gene expression. Therefore, gene expression can be regulated by internal UTP levels, which reflect the availability of external pyrimidine sources. The magnitude of gene regulation by these mechanisms varies considerably, even when control mechanisms are analogous. These variations apparently are due to differences in promo...
Journal of Bacteriology, 2001
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 tran...
1997
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 AUUUU n (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.
Journal of Bacteriology, 2001
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 ⅐ 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 ⅐ A tract in the upp initially transcribed region. We constructed upp promoter mutations that shorten the T ⅐ 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 ⅐ 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 ⅐ 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.
Journal of Biological Chemistry
The rrnB P1 promoter of Escherichia coli (starting sequence C"4-A"S-C"2-C"1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Rea. 16,9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3+ " mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1+" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-ternary complexes carry u factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding. A model is proposed explaining the stability of the -3+ ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.
Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli
The Journal of biological chemistry, 1993
The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R. L. (1988) Nucleic Acids Res. 16, 9789-9809). We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes. By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex. The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed wi...
The Embo Journal, 1984
Protein synthesis in 'minicells' showed that the DNA immediately preceding the pyrE gene of Escherichia coli directs the formation of considerable amounts of a polypeptide (mol. wt. -30 000) of unknown function. The nucleotide sequence of this DNA revealed the existence of an open reading frame (ORF) of 238 codons that ends 68 nucleotide residues upstream to the structure start ofpyrE, just prior to the GC-rich symmetry region of a sequence with features characteristic of a rho-independent transcription terminator. Deletion of the start of this 238 codons long ORF gene resulted in a dramatic fall in the level of pyrE expression, indicating that the two genes (ORF and pyrE) constitute an operon. Sl-nuclease digestion of RNA-DNA hybrids revealed that both genes are transcribed from two promoters (Pl and P2) located in front of the ORF start. Furthermore, when the RNA used in these experiments was prepared from cells with different levels of pyrE expression, created by manipulations in their pyrimidine nucleotide supply, the frequency of transcription initiations at P1 and P2 was found to be constitutive, while only a pyrimidine regulated fraction of the mRNA chains reached into the pyrE gene. In vitro transcription of isolated DNA fragments showed that the mRNA chains are terminated between the ORF gene and pyrE. From these observations we conclude that pyrE expression is controlled by a UTP modulated intercistronic attentuation.