INTRODUCTION

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The carAB operon of Escherichia coli encodes the two subunits of carbamoyl phosphate synthetase. This enzyme catalyzes the formation of carbamoyl phosphate, which is an intermediate in the pyrimidine nucleotide and arginine biosynthetic pathways. Expression of the carAB operon is subject to cumulative repression by the end products of each pathway (10). Transcription of the operon is initiated at two tandem promoters designated P1 and P2 (Fig. 1A). Initiation at P1, the more upstream promoter, is negatively regulated by the availability of pyrimidine nucleotides (4, 24). This regulation is poorly understood but requires, at least to some extent, the trans-acting factors CarP and integration host factor (IHF) (9). These proteins bind upstream of promoter P1 and may induce structural modifications and/or the assembly of a nucleoprotein complex required for regulation (7, 8). Initiation at promoter P2 is regulated by arginine-dependent binding of the arginine repressor, ArgR, to two operator sequences that flank the transcriptional start site(s) (4, 24). ArgR binding at promoter P2 does not inhibit transcription initiated at promoter P1.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Organization of the carAB regulatory region and sequences of relevant promoter regions. (A) Locations of carAB promoters P1 and P2 and the binding sites for IHF (hatched box), CarP (open boxes), and ArgR (filled boxes). (B) Sequences of the carAB P1 and pyrBI promoter regions. The 10 regions (underlined) and the start sites (boldface) are indicated.

The initially transcribed region of the pyrimidine-sensitive carAB promoter P1 contains the sequence GTTTGC (nontemplate strand), with initiation occurring at the first G residue (Fig. 1B) (4, 24). This region contains a run of three T residues at which reiterative transcription is reported to occur (16). Reiterative transcription is the repetitive addition of a nucleotide (UMP in the case of carAB) caused by slippage between a homopolymeric stretch of nascent transcript and a stretch of 3 complementary nucleotides in the DNA template (15). Recently, it has been shown that reiterative transcription involving the repetitive addition of U residues plays a central role in pyrimidine-mediated regulation of pyrBI, codBA, and upp expression in E. coli, although the control mechanisms may differ significantly (19, 27, 31).

In the case of the pyrBI operon, which encodes the two subunits of the pyrimidine nucleotide biosynthetic enzyme aspartate transcarbamylase, a high intracellular level of UTP induces reiterative transcription after synthesis of the nascent transcript AAUUU (Fig. 1B). This reaction produces transcripts with the sequence AAUUUU_n (where n = 1 to >30), which cannot be extended downstream to include additional leader and structural gene sequences and which are eventually released from the initiation complex. Consequently, the synthesis of AAUUUU_n transcripts precludes the synthesis of short transcripts with the sequence AAUUUG, which are made by normal elongation and can be extended to produce full-length pyrBI transcripts. In this way, pyrBI expression is regulated over a sevenfold range by pyrimidine availability and intracellular UTP levels (19).

In this study, we examined the role of reiterative transcription in pyrimidine-mediated regulation of carAB expression. Our results show that regulation does occur in part through a mechanism involving UTP-sensitive reiterative transcription much like that described for the pyrBI operon. This regulation appears to be independent of regulation mediated by CarP and IHF.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. E. coli K-12 strain MC4100 [F araD139 (argF-lac)U169 rpsL150 thiA1 relA1 deoC1 ptsF25 flbB5301 rbsR] (6) served as a source of chromosomal DNA. Strain CLT42 [MC4100 araD⁺ car-94] (29) was used as the parent in the construction of  lysogens. Plasmid pDLC126 (33) was used to construct carAp₁::lacZ operon fusions. This plasmid contains a galK::lacZ gene fusion without a promoter. The galK::lacZ gene fusion includes the galK ribosome binding site, the first 54 codons of galK, two spacer codons, and all of lacZ except the first eight codons. Plasmid pDLC126 also contains a unique BamHI site just upstream of the galK::lacZ gene fusion. Operon fusions were made by digesting plasmid pDLC126 with BamHI and ligating the linear plasmid to a BamHI restriction fragment containing either the wild-type or a mutant carAB P1 promoter region. Fusion constructions were screened by restriction mapping and confirmed by DNA sequence analysis with a Sequenase kit (U.S. Biochemicals).

Restriction digests, ligations, and transformations. Conditions for restriction digests, ligations, and transformations were as previously described (29).

DNA preparations and site-directed mutagenesis. Plasmid DNA was isolated by use of Qiagen plasmid kits. Chromosomal DNA was prepared as previously described (22). Two types of carAB P1 promoter region fragments were prepared: a short version that does not contain the binding sites for CarP and IHF and a long version that contains these sites. The short fragment, which contains nucleotides 58 to +40 of the promoter region (counting from the transcriptional start site) and flanking DNA containing BamHI restriction sites, was amplified by PCR. The template in the PCR was chromosomal DNA from strain MC4100. The forward and reverse DNA primers were 5'-CGCGGATCCAGCTGATATAAAAAATCCCGCC and 5'-CGCGGATCCAGCTGAATCAATGCAAATCTGC, respectively, with nonpromoter sequences including restriction sites shown in italics. PCR conditions were as follows: 94°C for 4 min; then 94°C for 1 min, 37°C for 1 min, and 72°C for 30 s for 30 cycles; and finally 72°C for 5 min. The long carAB P1 promoter region fragment contains nucleotides 355 to +40 of the promoter region and flanking DNA containing BamHI restriction sites. This fragment was amplified by PCR as described above except the forward primer was 5'-CGCGGATCCAGCTGCCACAAAATATTTGTTATGGTGCA. The resulting PCR products (both short and long versions) were digested with BamHI to trim the fragment ends. The fragments were then inserted separately into plasmid pDLC126 to create carAp₁::lacZ operon fusions and also into plasmid pALTER-1 (Promega) for use in site-directed mutagenesis.

In vitro transcription. Purified RNA polymerase holoenzyme containing ⁷⁰ was prepared as previously described (5, 11). The DNA templates were 207-bp EcoRV restriction fragments containing the short carAB P1 promoter region (with either a wild-type or a mutant promoter) and a downstream segment that included an efficient (~98%) intrinsic transcriptional terminator, the pyrBI attenuator (32). The presence of the pyrBI attenuator facilitates RNA polymerase release from the DNA template in our multiple-round transcription assay, thereby increasing the synthesis of long (referred to as full-length) transcripts initiated at the carAB P1 promoter and terminated at the attenuator. A general diagram of the DNA templates is shown in Fig. 2. As a control in one experiment, we also used a previously described template containing the pyrBI promoter-leader region (19). All templates were prepared and their concentrations and purity were determined as previously described (26).

View larger version (7K): [in this window] [in a new window]   FIG. 2.   DNA template for in vitro transcription. bp 1 to 27 are from the multiple cloning site of plasmid pBluescript II SK() (Stratagene), bp 28 to 135 are from the BamHI fragment containing the short carAB P1 promoter region (see "DNA preparations and site-directed mutagenesis" in Materials and Methods), and bp 136 to 207 are from a PCR-generated DNA that contains the pyrBI attenuator (att). A transcript initiated at promoter P1 and terminated at the end of the pyrBI attenuator contains 96 nucleotides.

Transfer of carAp₁::lacZ operon fusions from plasmids to the E. coli chromosome. Wild-type and mutant carAp₁::lacZ operon fusions carried on derivatives of plasmid pDLC126 were individually transferred to the chromosome of strain CLT42 by using phage RZ5 (28). The presence of a single prophage at the  attachment site was confirmed by PCR analysis (25). In this procedure, the concentration of each primer was 500 nM.

Media and culture methods. Cells used for enzyme assays and RNA isolations were grown at 37°C with shaking in NC medium (1) supplemented with 10 mM NH₄Cl, 0.4% (wt/vol) glucose, 0.015 mM thiamine hydrochloride, 1 mM arginine, and either 1 mM uracil or 0.25 mM UMP. Cell growth was monitored and culture samples were harvested during the exponential phase of growth at the same cell density as previously described (18).

Enzyme assays. Cell extracts were prepared by sonic oscillation (28). -Galactosidase activity (22) and protein concentrations (21) were determined as previously described.

Isolation of cellular RNA and primer extension mapping. Cellular RNA was isolated quantitatively as described by Wilson et al. (33). Primer extension mapping of the 5' ends of carAp₁::lacZ transcripts was performed as previously described (20) except that 45 µg of RNA from uracil-grown cells and 30 µg of RNA from UMP-grown cells were used for analysis. The different amounts of RNA, which were isolated from the same mass of cells, reflect the different levels of stable RNA in cells growing at different rates. The primer used in these experiments was 5'-CGCGGATCCAGCTGAATCAATGCAAATCTGC (the reverse primer for PCR synthesis described above), which was labeled with ³²P at the 5' end. The 29 nucleotides at the 3' end of this primer hybridize to nucleotides 24 to 52 in the carAp₁::lacZ transcript. Although this primer would also be expected to hybridize to carAB transcripts, these transcripts are eliminated by mutation in the strains used here (see below).

	RESULTS

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Demonstration of UTP-sensitive reiterative transcription at the carAB P1 promoter. To confirm that reiterative transcription occurs at the carAB P1 promoter and determine whether this reaction is sensitive to the concentration of UTP, we transcribed a DNA template containing this promoter in a reaction mixture containing 200 µM (each) ATP, CTP, and [-³²P]GTP and either 1,000 or 50 µM UTP. The UTP concentrations selected are similar to those found in E. coli grown under conditions of pyrimidine excess or severe pyrimidine limitation, respectively (2, 23). As a control, we also transcribed a template containing the pyrBI promoter under the same conditions as described above, except that [-³²P]ATP was used instead of [-³²P]GTP to 5' end label the transcripts. Transcripts synthesized in the reactions were separated by gel electrophoresis and visualized by autoradiography.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Analysis of transcripts initiated at the carAB P1 promoter. DNA templates containing either the pyrBI (control) or the carAB P1 promoter were transcribed in vitro in reaction mixtures containing either 1,000 or 50 µM UTP and analyzed as described in the text. An autoradiograph of the 25% polyacrylamide sequencing gel used to separate the transcripts is shown. The radiolabel included in the reactions is indicated below the gel. The lengths (in nucleotides) of AAUn transcripts initiated at the pyrBI promoter and the pyrBI aborted transcript with the sequence AAUUUG (open arrow) are shown on the left. The lengths of GUn transcripts initiated at the carAB P1 promoter and carAB aborted transcripts containing five or more residues (i.e., GUUUG, GUUUGC, GUUUGCCAG, GUUUGCCAGA, and GUUUGCCAGAA) (filled arrows) are shown on the right. Note that the identities of the very minor bands below GUU and GUUU and above GUUUGC are unknown, although the latter band may be GUUUUG (see below). Full-length transcripts are also indicated (bracket). Heterogeneity in the lengths of these transcripts is due to termination at multiple sites within the intrinsic terminator (data not shown).

Additional evidence for the sequence assignments of short carAB transcripts. To provide additional evidence in support of our sequence assignments for the carAB transcripts produced by reiterative transcription and simple abortive initiation, we transcribed the carAB P1 promoter template in reaction mixtures containing selected nucleoside triphosphates and different radiolabels. With a reaction mixture containing only 200 µM [-³²P]GTP, no transcripts were synthesized (Fig. 4, lane 3). With a reaction mixture containing 200 µM [-³²P]GTP plus 1,000 µM UTP, large amounts of transcripts that appeared identical to the putative GU_n and GUUUG transcripts generated with all four nucleoside triphosphates including 1,000 µM UTP were produced (Fig. 4, compare lanes 1 and 4). These results indicate that the putative GU_n and GUUUG transcripts indeed contain only G and U residues, consistent with their assigned sequence. For the latter reaction, we did not detect transcripts that migrated with the 9-, 10-, and 11-mer transcripts presumably produced by simple abortive initiation. This result indicates that these transcripts require CTP and/or ATP for synthesis, consistent with their assigned sequences of GUUUGCCAG, GUUUGCCAGA, and GUUUGCCAGAA, respectively.

View larger version (39K): [in this window] [in a new window]   FIG. 4.   Nucleotide requirements for the synthesis of short transcripts initiated at the carAB P1 promoter. The nucleotides included in each reaction mixture and their concentrations are shown below the gel, with the following abbreviations: A, 200 µM ATP; C, 200 µM CTP; G, 200 µM GTP; U, 1,000 µM UTP; and u, 50 µM UTP (-, no nucleotide). The 32P-labeled nucleotide (i.e., -GTP or -ATP) included in the reaction mixture is also indicated. Mock reactions without RNA polymerase were also analyzed to show bands that were introduced by contaminants in the radiolabel preparations used in this experiment. An autoradiograph of the 25% polyacrylamide gel used to separate the transcripts is shown. The lengths (in nucleotides) of GUn transcripts, aborted transcripts containing five or more residues (i.e., GUUUG, GUUUGC, GUUUGCCAG, GUUUGCCAGA, and GUUUGCCAGAA) (arrows), and full-length transcripts (bracket) are indicated on the left.

Effects of UTP concentration on transcription from the carAB P1 promoter. To examine in more detail the effects of UTP concentration on transcription from the carAB P1 promoter, we transcribed the promoter region in reaction mixtures containing 200 µM (each) ATP, CTP, and [-³²P]GTP and various concentrations of UTP ranging from 20 to 1,000 µM. The results show that the level of each GUUUU_n transcript increased linearly over a wide range when the UTP concentration was increased from 20 to 500 µM (Fig. 5A). This increase was 10-fold in the case of GUUUU and approximately 60-fold for longer GUUUU_n transcripts. Above 500 µM, transcript levels were constant or increased slightly, indicating that the UTP concentration was at or near the saturating level for U addition to the transcript. The pattern of increase for one of these transcripts, GU₉, is shown in Fig. 5B. Thus, reiterative transcription at the carAB P1 promoter appears to be induced by increasing UTP concentrations similarly to the situation described for the pyrBI promoter (19).

View larger version (27K): [in this window] [in a new window]   FIG. 5.   Effects of UTP concentration on transcription from the carAB P1 promoter. Standard in vitro transcription reactions in which the UTP concentration was varied as indicated were performed. End-labeled transcripts were separated on a 25% polyacrylamide sequencing gel, visualized by autoradiography, and quantitated. (A) Autoradiograph of the gel. Full-length transcripts (bracket) and nucleotide lengths of transcripts produced by simple abortive initiation are indicated on the left. Nucleotide lengths of GUUUUn transcripts produced by reiterative transcription are shown on the right. (B) The levels of the full-length, GU9, and GU3GC2AG transcripts plotted as a function of UTP concentration. The units for transcript levels are arbitrary.

Construction and in vitro characterization of carAB P1 promoter mutations that eliminate reiterative transcription. As the first step in assessing the role of reiterative transcription in pyrimidine-mediated regulation of carAB expression, we constructed two site-directed mutations that introduce either a T-to-G or a T-to-C change at position 3 of the initially transcribed region of the carAB P1 promoter (Fig. 1B). These mutations interrupt the run of three T residues in the initially transcribed region, which is expected to abolish reiterative transcription. To show that the mutations had the anticipated effect, wild-type and mutant DNA templates were transcribed in reaction mixtures containing 200 µM (each) ATP, CTP, and [-³²P]GTP and 1,000 µM UTP. Analysis of the transcript products indicated that reiterative transcription was in fact eliminated by both mutations, as judged by the absence of a long, regularly spaced ladder of transcripts (Fig. 6, compare lane 1 with lanes 3 and 5).

View larger version (25K): [in this window] [in a new window]   FIG. 6.   Effects on transcription of the T-to-G and T-to-C mutations at position +3 of the carAB P1 promoter region. Standard in vitro transcription reactions were performed at high (1,000 µM) and low (50 µM) UTP concentrations using either the wild-type (wt) or a mutant DNA template. End-labeled transcripts were separated on a 25% polyacrylamide sequencing gel and visualized by autoradiography. Full-length transcripts (bracket) and nucleotide lengths of transcripts produced with the T-to-C mutant template are indicated on the right. Note that minor (unmarked) bands in the lower part of the autoradiograph are either contaminants in the [-32P]GTP preparation or bands which have arisen from an unknown source.

Regulatory effects of carAB P1 promoter mutations that eliminate reiterative transcription. To directly measure the contribution of reiterative transcription to regulation, we constructed a set of carAp₁::lacZ operon fusion strains that contain either the short or the long version of the wild-type or a mutant promoter region. (Note that only the long promoter fragment contains the binding sites for the regulatory proteins CarP and IHF.) The lacZ operon fusions were created by individually inserting a promoter region into plasmid pDLC126 and then transferring the fusion onto phage RZ5 by recombination. The recombinant phages were used to infect strain CLT42 (car-94 lacZYA), and lysogens carrying a single prophage at the chromosomal  attachment site were isolated. These strains were designated CLT5174 (short wild-type fusion), CLT5175 (short T-to-G mutant fusion), CLT5177 (short T-to-C mutant fusion), CLT5192 (long wild-type fusion), CLT5202 (long T-to-G mutant fusion), and CLT5203 (long T-to-C mutant fusion). These strains are pyrimidine (and arginine) auxotrophs because the car-94 mutation inactivates carbamoyl phosphate synthetase. The six strains were grown in glucose-minimal salts (plus arginine) medium containing either uracil or UMP as the pyrimidine source, which provides a condition of pyrimidine excess or limitation, respectively. The levels of -galactosidase in these strains were assayed as an indicator of expression from the carAB P1 promoter (Table 1).

Quantitative primer extension mapping of carAp₁::lacZ transcripts initiated at the wild-type and mutant promoters of short operon fusions. Quantitative primer extension mapping was used to confirm the start sites and measure the levels of carAp₁::lacZ transcripts synthesized in the three short operon fusion strains grown on uracil or UMP. With respect to start site(s), the results show that essentially all wild-type and T-to-C mutant transcripts were initiated at the G residue located 7 bases downstream from the promoter 10 region (i.e., the previously identified wild-type start site) (Fig. 7). For the T-to-G mutant, the wild-type start site was used predominantly, but two minor start sites were also employed. The minor start sites are a C residue and the mutant G residue located 6 and 9 bases downstream from the 10 region, respectively (Fig. 1B). Presumably, these minor start sites do not affect regulation, and levels of T-to-G mutant transcripts described below are the sum of the predominant and two minor transcript bands. With respect to transcript levels, the data showed that the wild-type transcript level was 2.5-fold higher in UMP-grown cells than in cells grown on uracil (Fig. 7). In contrast, the pyrimidine source had much smaller effects on the level of transcript produced with the mutant fusions. Overall, the carAp₁::lacZ transcript levels roughly paralleled the -galactosidase activities shown in Table 1. Note that transcripts from the resident carAB operon of the parent strain CLT42 were not detected by this primer extension assay (Fig. 7). Apparently, these transcripts are effectively eliminated by the car-94 mutation.

View larger version (41K): [in this window] [in a new window]   FIG. 7.   Levels of carAp1::lacZ transcripts synthesized in the short operon fusion strains CLT5174 (wild type [wt]), CLT5175 (T-to-G mutant), and CLT5177 (T-to-C mutant). Cellular RNA was quantitatively isolated from cells grown on either uracil (R) or UMP (M), and transcript levels were measured by primer extension mapping as described in Materials and Methods. An autoradiograph of the 10% polyacrylamide sequencing gel used to analyze the primer extension products is shown. The dideoxy sequencing ladder (G, A, T, C) of the wild-type carAB P1 promoter region, which was used to identify transcripts, was generated with the same primer that was used for primer extension. Note that the sequence is of the template strand. Identical results were obtained by using a sequencing ladder produced with either mutant promoter region. Primer extension product levels, which correspond to transcript levels, were measured with a PhosphorImager. Relative levels are indicated below the appropriate lanes of the autoradiograph. Essentially the same results were obtained in a second, completely independent experiment. As a control, the autoradiograph also shows that transcripts initiated at the carAB P1 promoter were not detected in parent strain CLT42 grown on either uracil or UMP.

	DISCUSSION

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The in vitro studies described here demonstrate that reiterative transcription involving the repetitive addition of U residues occurs during initiation at the carAB P1 promoter. This reaction produces transcripts with the sequence GUUUU_n (where n = 1 to >30), which are apparently released from the transcription initiation complex. The extent of reiterative transcription is directly proportional to the UTP concentration up to approximately 500 µM. Above this concentration, reiterative transcription is stimulated to a much lesser extent. These experiments also indicate an inverse relationship between the levels of reiterative transcription and the synthesis of simple aborted and full-length (i.e., normally elongated) transcripts initiated at the carAB P1 promoter. Furthermore, we showed in vitro that substitution mutations within the run of three T residues in the initially transcribed region of the carAB P1 promoter completely eliminate reiterative transcription.

Our in vivo studies with carAp₁::lacZ operon fusion strains indicate that pyrimidine-mediated regulation of operon expression occurs over an 18-fold range, when measured with the long operon fusion that includes the binding sites for CarP and IHF. When the binding sites for these regulatory proteins are deleted, as in the short operon fusion, the range of regulation is reduced to between two- and threefold. Introduction of a promoter mutation that abolishes reiterative transcription into carAp₁::lacZ operon fusions showed that nearly all regulation of the short operon fusion was eliminated and that the range of regulation with the long operon fusion was reduced by a factor of 2 to 3. Thus, UTP-sensitive reiterative transcription appears to account for about two- to threefold of the total pyrimidine-mediated regulation, while the remainder of this regulation (i.e., six- to ninefold) presumably occurs by an independent mechanism involving CarP and IHF. The estimated range of regulation by reiterative transcription is consistent with the observed steady-state levels of carAp₁::lacZ transcripts produced with short operon fusions containing wild-type and mutant promoters. Primer extension mapping of full-length wild-type transcripts reveals an essentially unique 5' end without extra U residues, indicating that only transcripts that avoid reiterative transcription can be extended to include downstream sequence.

Taken together, our results indicate that regulation of carAp₁::lacZ (and carAB) operon expression by UTP-sensitive reiterative transcription occurs by a mechanism analogous to that described for the pyrBI operon and suggest the following regulatory model. Transcription is initiated at the G start site in a UTP concentration-independent manner. After the nascent transcript is extended normally to include four bases and has the sequence GUUU, weak base pairing between the sequences UUU in the transcript and AAA in the DNA template facilitates rapid and reversible slippage between the two strands (Fig. 8). This slippage presumably occurs as a 1-base, upstream shift of the transcript (or at least of the 3' end of the transcript) (12). When the UTP level is high and the nascent transcript is shifted upstream, the last A of the template AAA sequence directs the efficient addition of the first "extra" U residue to the 3' end of the transcript. This transcript can be released from the initiation complex, or another round of slippage and U addition can occur. Repetition of this cycle generates transcripts with long runs of U residues at their 3' ends. Transcripts containing one or more extra U residues are effectively excluded from the normal mode of transcript elongation (i.e., nonrepetitive nucleotide addition). When the UTP level is low, slippage followed by correct repositioning of the GUUU transcript without the addition of an extra U residue are the most likely reactions and permit normal template-directed addition of a G residue to the 3' end of the transcript. This addition results in more-stable base pairing between the transcript and template and the apparent loss of alternative alignments for the 3' end of the transcript, which can preclude further slippage. The GUUUG transcript can then be released as a simple aborted transcript or extended further downstream with a certain probability that this transcript will become a full-length carAB transcript.

View larger version (22K): [in this window] [in a new window]   FIG. 8.   Model showing the alternative fates of the nascent GUUU transcript initiated at the carAB P1 promoter. The GUUU [or GUUU(G/U)] transcript is shown hybridized to the template strand of the initially transcribed region. The curved lines in the complexes indicate the melted strands of DNA. The precise positioning of the 5' end of the transcript in "slipped" complexes is unknown.

Although described in terms of high and low UTP concentrations, it is important to note that the model does not propose an on-off switch for carAB expression, except perhaps at extreme UTP concentrations. Instead, the model suggests that there is a gradient of operon expression that balances the level of synthesis of carbamoyl phosphate synthetase with the cell's need for UTP. The UTP concentration determines the frequency of selecting one of two mutually exclusive transcription pathways. One pathway, which is enhanced by an increase in the UTP concentration, leads to the synthesis of nonproductive transcripts. The other pathway, favored by a lowering of the UTP concentration, permits the synthesis of transcripts capable of being translated. Although not included in the model, it is also possible that GTP levels affect carAB expression by influencing the addition of a U or G residue at position 5 of the nascent transcript (17). Physiological conditions that allow GTP levels to modulate the levels of reiterative and productive transcription at the carAB P1 promoter remain to be established.

The full range of pyrimidine-mediated regulation of the carAB P1 promoter apparently requires two independent control mechanisms. Similar situations have been described for a number of E. coli promoters. One function of multiple control mechanisms is regulation of gene expression in response to a wide range of concentrations of a particular effector molecule, with each control mechanism sensitive to a different range of effector concentrations (19, 35). This type of regulation may exist for the carAB P1 promoter. The experiments described in this paper, using pyrimidine auxotrophs grown under conditions of pyrimidine excess or limitation, indicate that the range of CarP-IHF-mediated regulation is essentially the same as that previously measured for pyrimidine prototrophs grown in minimal media with or without a uracil supplement (7, 9). This result indicates that virtually all CarP-IHF-mediated regulation occurs within a range of UTP concentrations between 900 µM and approximately 1.4 mM (23, 30). On the other hand, our results suggest that the bulk of regulation by reiterative transcription occurs at UTP concentrations found in pyrimidine-limited cells (i.e., from 900 to approximately 50 µM). These low UTP levels may be transiently experienced by pyrimidine prototrophs following a shift from a pyrimidine-rich to a pyrimidine-poor environment. Furthermore, we have observed that little (1.4-fold) pyrimidine-mediated regulation of short carAp₁::lacZ fusion expression occurs with pyrimidine prototrophs grown in the presence or absence of uracil (13). Thus, regulation of the carAB P1 promoter appears to occur predominantly via a CarP-IHF-mediated mechanism at high UTP concentrations and via UTP-sensitive reiterative transcription at lower UTP concentrations. Note that the regulatory effector of CarP-IHF-mediated regulation does not have to be UTP, but only a molecule whose concentration reflects changes in the UTP level. The identity of this effector awaits further characterization of CarP-IHF-mediated regulation.

An interesting difference revealed by the present study is that the range of regulation provided by UTP-sensitive reiterative transcription at the carAB P1 promoter is much smaller (by roughly a factor of 3) than that observed with the pyrBI promoter (19). Presumably, this effect is due to differences in promoter sequence. There are two obvious differences that could contribute to or account for the effect. First, the sequences at the 5' end of the carAB and pyrBI transcripts (preceding the U run) are different: G for carAB and AA for pyrBI. Conceivably, the G residue at the end of the carAB transcript could provide stronger base pairing with the template than that provided by the two A residues at the end of the pyrBI transcript. Stronger base pairing would be expected to suppress reiterative transcription and the range of regulation. Second, the locations of the transcription start sites are different for the two promoters: position 7 (counting from the 10 region) for carAB P1 and position 8 for pyrBI (Fig. 1). We recently discovered, using variants of the carAB P1 promoter, that moving the start site from position 7 to position 8 enhances regulation by reiterative transcription by more than twofold (13). The reason for this enhancement is not known. However, this result suggests for the first time that the architecture of the transcription initiation complex can significantly affect reiterative transcription. On the basis of these observations, it appears that the cell could control the expression of numerous operons by employing a similar UTP-sensitive reiterative transcription reaction and achieve different ranges of regulation through minor variations in promoter sequence and/or organization.