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

Abstract

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.Reiterative transcription, which is also known as pseudotemplated transcription, transcriptional slippage, and RNA polymerase stuttering, is a reaction catalyzed by a number of RNA polymerases, including bacterial, phage, viral, and eukaryotic enzymes (14,15,20). In this reaction, nucleotides are repetitively added to the 3Ј end of a nascent transcript due to slippage between the transcript and template (either DNA or RNA). Typically, slippage occurs between a homopolymeric sequence in the transcript and at least three complementary bases in the template. The mechanism apparently involves one or more rounds of a one-base upstream shift of the transcript so that the same nucleotide in the template specifies multiple residues in the transcript (11). Reiterative transcription can occur during initiation or elongation, resulting in transcripts that can be immediately released from the transcription complex or (in some cases) extended by normal elongation after a switch to nonreiterative nucleotide addition. Although reiterative transcription can involve the addition of any nucleotide, at least under certain conditions, addition of U or A residues appears most efficient (42). This increased efficiency presumably reflects a requirement in the reaction for disruption of a hybrid between transcript and template, which should be easiest with U:A or A:T base pairing.Recent studies indicate that reiterative transcription plays an important role in the expression and regulation of a number of bact...

Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

“…In addition to these sequence determinants, DNA topology and NTP concentrations also influence TSS selection (6,8,9,11,(21)(22)(23)(24)(25)(26). Thus, TSS selection is a multifactorial process, in which the ultimate outcome for a given promoter reflects the contributions of multiple promoter sequence determinants and multiple reaction conditions.…”

The Escherichia coli fis Promoter Is Regulated by Changes in the Levels of Its Transcription Initiation Nucleotide CTP

“…These studies uncovered a variety of fascinating regulatory strategies resulting from changes in intracellular NTP pools, including alterations in the rate of transcription so as to control the coupling of translation and transcription attenuation (45), alterations in the predominant transcription start site position so as to regulate the synthesis mRNAs with differential capacity to control translation (30,49,50), and stimulation of high rates of reiterative transcription so as to control productive transcript synthesis (39,(51)(52)(53). An effect of the transcription initiation NTP concentration on the kinetics of transcription initiation complex formation was demonstrated in the ribosomal RNA promoter rrnB P1 (54).…”

Regulation of Pyrimidine Biosynthetic Gene Expression in Bacteria: Repression without Repressors

“…This transcript, with poly(U) at its 3Ј end, can no longer be productively elongated and is eventually released from the transcription initiation complex (97). Reiterative transcription was then found to participate in the regulation of carAB expression (53) and that of the pyrimidine salvage operons codBA (137) and upp-uraA (157). The latter two mechanisms provided additional surprises, integrating two of the newly discovered paradigms.…”

“…These genes are included in the upp-uraA operon (5), which hereafter will be referred to as the upp operon for simplicity. Expression of the upp operon is negatively regulated over an approximately sixfold range by pyrimidine availability (7,157). The sequence of the upp promoter region containing the ITR is 5Ј-TATAATCCGTCGAT TTTTTTTGTG, with the Ϫ10 region and the initially reported transcription start site (7) underlined.…”

“…The characterization of pyrimidine-mediated regulation of upp expression was essentially as described for the codBA operon (157). Initially, reiterative transcription at the upp promoter was examined in vitro, using reaction mixtures containing various physiological UTP concentrations.…”