Vol. 174, No. 23

JOURNAL OF BACrERIOLOGY, Dec. 1992, p. 7648-7655

0021-9193/92/237648-08$02.00/0

In Vitro Transcription from the Escherichia coli ilvIH Promoter DEBRA AKER WILLINS AND JOSEPH M. CALVO* Section of Biochemistry, Molecular and Cell Biology, Comnell University, Ithaca, New York 14853 Received 24 June 1992/Accepted 21 September 1992

Lrp (leucine-responsive regulatory protein) activates the expression of the Escherichia coli ilvIH operon in vivo and mediates the repression of the operon by exogenous leucine. In previous studies, operon expression in vivo was measured with transcriptional fusions of lacZ to the ilvIH promoter. Here, ilvIH mRNA was measured directly by primer extension. The steady-state level of ilvIH mRNA was 11-fold higher in a wild-type parent strain than in a derivative lacking Lrp. A two-step procedure was developed for measuring ilvIH mRNA synthesized in vitro. RNA was synthesized with plasmid templates and purified RNA polymerase, and then ilvIH mRNA was measured by primer extension. In vitro, mRNA synthesis was initiated at two sites, one corresponding to the in vivo site (promoter P1) and the other corresponding to a site about 60 bp further upstream (promoter P2). Purified Lrp stimulated transcription two- to fivefold from promoter Pl, whereas it decreased transcription more than fivefold from promoter P2. Transcription from promoter P1 was stimulated by Lrp with templates containing the wild-type ilvIH promoter but not with templates containing mutations in an Lrp binding site. Furthermore, under at least some conditions, leucine reversed the stimulatory effect of Lrp. Taken together with the results of mutational analyses, these results establish that Lrp acts directly to stimulate transcription from the ilvIH promoter. Furthermore, they suggest that the ilvIH promoter is recognized by a sigma 70 RNA polymerase.

Lrp (leucine-responsive regulatory protein), a newly recognized global regulatory protein in Escherichia coli, regulates the expression of at least 12 operons (3, 11). Here we address Lrp-mediated regulation of ilvIH, an E. coli operon that encodes an enzyme catalyzing the first step in branchedchain amino acid biosynthesis (2). This operon is unusual in that sequences required for optimal expression extend at least 250 bp upstream of the presumed start point of transcription (6). Lrp (formerly called IHB) binds ilvIH DNA in this region and activates transcription of the ilvIH operon (13, 15). Like many of the operons regulated by Lrp, ilvIH is also regulated by leucine (3, 11). Exogenous leucine reduces transcription of the ilvIH operon 5- to 10-fold in vivo, an effect that is mediated by Lrp (6, 13, 15, 19). From an analysis of 5' endpoints of ilvIH mRNAs formed in vivo and in vitro, Haughn et al. identified four ilvIH promoters, P4 (most upstream), P3, P2, and P1 (6). In vivo, most ilvIH transcripts apparently arose from promoter P1, whereas in vitro, most transcripts arose from promoter P2, with lesser amounts arising from P3 and P4 and only insignificant amounts arising from P1 (6). These latter in vitro experiments, which used purified RNA polymerase, were performed before the discovery of Lrp. The work described here was undertaken to explore some questions regarding the regulation of ilvIH transcription that could be analyzed with an in vitro system. (i) The major start point for transcription in vitro in the absence of Lrp (P2) was different from that for transcription in vivo (P1). Does Lrp stimulate transcription in vitro from P1, and does it repress transcription from other start points? (ii) Lrp was known from in vivo studies to be necessary for the activation of ilvIH transcription (13 and this study). Is Lrp sufficient for the activation of transcription in vitro, or are additional *

factors necessary? (iii) If Lrp affects ilvIH transcription in vitro, does it do so at the same concentration at which binding to specific sites is known to occur? (iv) Does leucine prevent the effects of Lrp on transcription in vitro? We investigated these questions by measuring the levels of ilvIH transcription by primer extension.

MATERIALS AND METHODS Definition of the base-numbering convention. The major start points for ilvIH transcription in vivo spanned 10 nucleotides: 5' TTACACATJTT 3' (6). For the purposes of this study, the middle A was assigned a position of +1. All positions described here are assigned relative to that position. This system differs from the numbering convention that we used previously (6). Strains and growth conditions. The following strains were used: CV1058 F- ara thi A(lac-pro) ilvIH::Mu dI1734(pCV7) and CV1060 F- ara thi A(lac-pro) ilvIH::Mu d11734 lrp-35::

TnlO(pCV7). The strains were grown at 37°C in a minimal medium containing M9 salts (9), 0.2% glucose, 5 ,ug of thiamine per ml, 50 ,ug of proline per ml, and micronutrients (16). When present, leucine was added to 100 ,g/ml and ampicillin was added to 100 ,g/ml. Plasmid pCV7 contains a PvuII-HindIII fragment of ilvIH DNA (nucleotides -766 to +2147) cloned into the ampcontaining PvuII-HindIII fragment of pBR322 (19). It carries the ilvIH promoter-regulatory region, ilvI, and most of ilvH

(6, 19).

Plasmid pCV156 and its derivatives pCV157, pCV158, and pCV159 (20a) contain the region from -333 to +29 of ilvIH cloned upstream of the promoterless lacZ gene in plasmid pRS415 (18). Plasmid pCV157 carries five clustered substitution mutations between -76 and -71, which create a XhoI site; plasmid pCV158 carries a deletion of 8 bp between -76

Corresponding author. 7648

VOL. 174, 1992

and -69; and plasmid pCV159 carries a deletion of 10 bp between -75 and -66 and an additional change from T to C at -76 (20a). Isolation of cellular RNA. RNA was isolated from strains CV1058, CV1060, and CV1059 grown in minimal medium supplemented with proline and ampicillin in the presence or absence of leucine. Strain CV1060 (1rp-35::Tn1O) grew very slowly in the medium containing leucine. The protocol used was the same as that of Gilman (4) with the addition that RNA was extracted twice with phenol and then twice with ether before precipitation with ethanol. Isolation of plasmid DNA for in vitro transcription. Cells were grown at 37°C in 1 liter of LB or TB medium (17) containing 100 pg of ampicillin per ml. When the A550 reached 1.0 or higher, 200 mg of chloramphenicol was added, and incubation was continued at 37°C overnight. Cells were harvested by centrifugation at 10,000 x g for 15 min, and a crude preparation of nucleic acid was isolated by an alkaline lysis procedure. The crude preparation of nucleic acid was purified by the following procedures: precipitation with isopropanol, treatment with RNase and then with proteinase K, extraction with phenol, and precipitation with ethanol. The resulting plasmid DNA was purified further by polyethylene glycol precipitation (with final concentrations of 1 M NaCl and 10% polyethylene glycol), extraction with phenol, and two ethanol precipitations. The final pellet was resuspended in 0.5 ml of water, and its concentration was measured by monitoring the A26. Primers. Chemically synthesized oligonucleotides were suspended in water to a concentration of 152 ,uM and used directly as primers without purification. The names of the primers indicate both the RNA and the position to which the primer hybridizes. For example, primer ilv +40/+59 hybridizes to ilvIH RNA at positions +40 to +59. The following primers were used: ilv +40/+59, 5' CCATCTCGGCTCCA GACAAC 3'; amp +77/+96, 5' CAGGAAGGCAAAATGC CGCA 3' (the amp promoter position was determined as described by Hawley and McClure [7]); and ilv-lac +45/+64, 5' GCGCGTCGCCGCT'T'CATCG 3' (hybridizes to ilvIH RNA from the ilvIH-lacZ fusion in plasmid pCV156 and its derivatives). Primers were labelled with 32p in a 26-,u reaction mixture containing 60 ,uCi of [(-32P]ATP (3,000 Ci/mmol; Amersham), 0.7 puM primer, 13 U of polynucleotide kinase (New England BioLabs), 70 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, and 5 mM dithiothreitol. After incubation for 30 min at 37°C, the reaction was stopped by 5 min of incubation at 65°C. In vitro transcription. A standard reaction mixture contained the following in a final volume of 50 pul: 1 nM pCV7 DNA template (circular or linearized with PstI), 20 U of RNasin (an RNase inhibitor; Promega), 25 to 75 nM Lrp, 42 mM Tris-HCl (pH 8), 8.5% glycerol, 0.12 mM EDTA, 1 mM dithiothreitol, 10 mM magnesium chloride, 50 mM potassium glutamate, 17 mM NaCl, and 50 ,g of acetylated bovine serum albumin per ml. The samples were incubated at 37°C for 10 min to allow Lrp to bind to DNA. Two U of E. coli RNA polymerase (RNAP) (Promega) was added, and the reaction mixture was incubated again at 37°C for 10 min to allow RNAP to bind to the DNA template. Transcription was initiated by the addition of nucleoside triphosphates (Pharmacia) to a concentration of 0.2 mM. After 10 min at 37°C, the reaction was stopped by the addition of ice-cold water containing 50 ,ug of E. coli tRNA (Sigma R-4251, purified by phenol extraction) and 1 p.l of each 32P-labelled primer; enough water was added to bring the reaction

ilvIH TRANSCRIPTION

7649

volume to 100 p.l. This step was followed immediately by phenol extraction and ether extraction. The RNA was precipitated with 1/4 volume of 10 M ammonium acetate and 3 volumes of cold ethanol. Deviations from these standard conditions are detailed in the relevant figure legends. Primer extension. Primer extension was performed with either cellular RNA or RNA isolated after in vitro transcription. Cellular RNA was hybridized with primers as follows. Fifty micrograms of RNA was brought to a volume of 100 ,ul with water containing 50 ,ug of tRNA, and then 1 p.l of each 32P-labelled primer was added. The mixture was extracted with phenol, extracted with ether, and then precipitated with 1/4 volume of 10 M ammonium acetate and 3 volumes of cold ethanol. RNA made in vitro was hybridized with primers at the last step in the in vitro transcription protocol (see above). Both kinds of RNA were processed identically after hybridization and ethanol precipitation. Primer extension was performed as follows. Pellets containing primer hybridized to RNA were resuspended in 15 p.l of water, and then the remaining reaction components were added to bring the volume to 25 p.l. The reaction mixture contained 50 mM Tris-HCl (pH 8), 10 mM magnesium chloride, 9 mM dithiothreitol, 50 mM KCl, 0.5 mM each deoxynucleoside triphosphate, 25 U of RNasin, and 7.5 U of reverse transcriptase (avian myeloblastosis virus super reverse transcriptase; Molecular Genetic Resources). Samples were incubated at 45°C for 20 min, and the reaction was stopped by the addition of EDTA to 20 mM. Cold water containing 10 p.g of sonicated calf thymus DNA (Sigma D-1501) was added, bringing the volume to 100 p.l. Samples were extracted once with phenol and once with ether before nucleic acid was precipitated with 1/10 volume of 3 M sodium acetate and 3 volumes of cold ethanol. Pellets were resuspended in 5 ,ul of formamide containing 0.04% bromphenol blue and 0.02% xylene cyanol. 32P-labelled DNA fragments (MspI-cut pBR322 plasmid DNA) used as size standards were also resuspended in formamide containing those dyes. Samples were denatured by being heated at 100°C for 2 min. Primer extension products were fractionated on an 8% acrylamide-50% urea sequencing-type gel (29:1 ratio of acrylamide to bisacrylamide) with 2/3x Tris-borate-EDTA buffer (lx buffer is 90 mM Tris-HCl [pH 8], 90 mM borate, and 3 mM EDTA) at 2,000 V for about 1 h until the bromphenol blue dye was near the bottom of the gel. The gel was removed and exposed to X-ray film overnight. Appropriate segments of the gel were excised and mixed with 5 ml of biodegradable counting scintillant (Amersham). The 32p_ label was quantitated in a scintillation counter. For some experiments, the gel was dried under vacuum before bands were excised. For experiments done with in vitro-made RNA, background determinations were made from slices taken from several locations that did not have an appreciable signal, and an average value was used as a background value for the whole gel. For experiments done with cellular RNA, it was important to have individual background determinations for every lane. Samples for such background determinations were taken at the same vertical position. For the products analyzed, the numbers of counts (after subtraction of the background signal) were linear with the amount of RNA used in the primer extension reaction (data not shown). RESULTS In vivo steady-state levels of ilvIH transcripts. To provide a basis for comparison with in vitro studies, we first measured

:1~ '. ~

WILLINS AND CALVO

7650

A.

RNA from strain:

lrp

CV1060

allele : Irp::Tn]O Leu

ilv P22

+

J. BACTERIOL.

B.

CV1058 lrp+

in in vivo vitro

+

>

i /v P 1>

.........i

p. ... . .

P1/amp: 1.7 0.4 12.3 3.0 FIG. 1. (A) Effect of Lrp on transcription in vivo. Cellular RNA was isolated from strains CV1060 (Irp-35::TnlO) and CV1058 (Irp+) grown in the absence or in the presence of 100 ,ug of leucine per ml. The amounts of ilvIH and amp transcripts were determined by hybridization to 32P-labelled primers ilv +40/+59 and amp +77/+96 and extension from these primers with reverse transcriptase. The sizes of the primer extension products (amp, 96 bases; ilv P1, 59 and 56 bases) were determined by comparison with size standards not shown. P1/amp is the ratio of counts per minute for these transcripts, determined by scintillation counting. In a repetition of this experiment, the P1/amp ratios were, respectively, 2.0, 0.7, 28.4, and 4.0. (B) Comparison of 5' endpoints of ilvIH mRNA synthesized in vivo and in vitro. For the in vivo lane, cellular RNA was isolated from strain CV1058 grown in the absence of leucine. For the in vitro lane, RNA was synthesized in vitro from a plasmid pCV7 DNA template with purified RNAP. The amounts of ilvIH and amp transcripts were determined as described in panel A. Conditions for the in vitro transcription were as described in Materials and Methods (standard reaction conditions), with 1 nM uncut pCV7 DNA as the template but no Lrp. the steady-state levels of transcripts made in vivo in both a wild-type strain (lrp+; strain CV1058) and a strain carrying a TnlO insertion inactivating the lrp gene (strain CV1060) (13). Both strains contained a chromosomal ilvIH-lacZ transcriptional fusion and multicopy plasmid pCV7, carrying most of the ilvIH operon and an amp gene. RNA was isolated from strains grown in the absence or presence of leucine, and ilvIH transcripts were measured quantitatively by primer extension with a 32P-labelled primer, ilv +40/+59. At the same time, amp transcripts were measured by primer extension with a 3P-labelled primer, amp +77/+96. The latter primer provided an internal control for the amount of RNA analyzed. All data were normalized to the amp +77/+96 signal. RNA from strain CV1058 (lrp') yielded major primer extension products of approximately 59 and 56 bases and a minor product of 51 bases, corresponding to 5' endpoints mapping to positions +1, +4, and +9 (Fig. 1A). Assuming that these 5' endpoints represent start points of transcription, these endpoints define promoter P1. Primer extension from the amp primer yielded an expected product of 96 bases. When the same strain was grown in the presence of

leucine, the levels of the major transcripts (quantitated together) were reduced fourfold in one experiment (Fig. 1A; see P1/amp ratios at the bottom of the figure) and sevenfold in another (data not shown). The extent of leucine repression of steady-state ilvIH mRNA levels observed here was similar to the 10-fold leucine repression observed by Squires et al. (19). Similar experiments were performed with strain CV1060 (1rp-35::TnlO), which is isogenic with strain CV1058 except that it carries a transposon insertion in the lrp gene (13). No Lrp was detected in crude cell extracts of this strain in a gel retardation assay (13) or in a Western blot (immunoblot) probed with anti-Lrp antibody (16a). In one experiment, strain CV1060 had 7-fold less transcription from promoter P1 than did the wild-type strain (Fig. 1A; both strains were grown in the absence of leucine), and in a second experiment, it had 14-fold less transcription (data not shown). The same transcription start points were observed for CV1060 (1rp-35::TnlO) as for CV1058 (wild type). Leucine added to cultures of CV1060 caused an apparent fourfold decrease in ilvIH mRNA levels. In previous studies, in which promoter expression was measured with a transcriptional fusion to lacZ, there was almost no effect of leucine on ilvIH promoter expression in an lrp-35::TnlO strain (13). For this comparison of two weak signals, ,-galactosidase data may be more reliable than primer extension data because the enzyme assay is more sensitive and has a relatively high signal/ background ratio. These results, which indicate that Lrp is necessary for high-level expression from the ilvIH promoter, confirmed the results of earlier studies in which expression was measured by means of an ilvIH-lacZ transcriptional fusion (13). In vitro transcription from the ilvIH promoters. In vitro transcription experiments were performed with plasmid pCV7 DNA as a template and purified RNAP. Transcription products were purified and analyzed by primer extension with the ilv +40/+59 and amp +77/+96 primers as described for the experiments with in vivo RNA. Three major primer extension products from the ilv +40/+59 primer were observed. Two of them, 56 and 59 bases in length, were identical to two of the primer extension products observed with in vivo RNA and corresponded to transcripts mapping to positions +1 and +4 (Fig. 1B). Since these in vitro experiments were done with highly purified components, the 5' endpoints can be equated with transcription start points for promoter P1 rather than with endpoints generated by processing. Thus, transcription in vitro is initiated faithfully at promoter P1, the primary promoter that is used in vivo. The third major primer extension product from the ilv +40/+59 primer was 120 bases in length and corresponded to transcription initiated at -60 from promoter P2. Promoter P2, although not an active promoter in vivo, was about as active as promoter P1 in vitro. Stimulation of ilvIH transcription in vitro by Lrp. The Lrp used here, purified to >98% purity (22), did not contain any RNase activity when used at concentrations as high as 300 nM. Furthermore, 300 nM Lrp did not increase or decrease expression from the lambda PR promoter in in vitro transcription experiments, suggesting that it did not have nonspecific effects on transcription in vitro. In preliminary experiments, we found that Lrp stimulated transcription from promoter P1 and reduced transcription from promoter P2. Focusing attention on promoter P1, we found conditions that resulted in maximal stimulation of transcription by Lrp. These conditions, defined in Materials

ilvIH TRANSCRIPTION

VOL. 174, 1992 15

10 PS

5

o

l

0

*

*

*

*

50

*

100

Lrp (nM) FIG. 2. Lrp titration. In vitro experiments of the type shown in Fig. 1B were performed in the presence of increasing amounts of Lrp. P1/amp is the ratio of counts per minute for these transcripts, determined by scintillation counting. Open circles and closed circles represent the results of two independent experiments. These experiments were performed under standard conditions with 1 nM PstIcut pCV7 DNA as the template.

and Methods, are hereafter referred to as standard conditions. Lrp binds to at least six sites upstream of the ilvIH promoter, two "upstream" sites located between -253 and -211 and four "downstream" promoter-proximal sites located between -141 and -46 (20a). In a gel retardation assay with the usual binding buffer replaced by in vitro transcription buffer, about 8 nM Lrp was required for 50% saturation of the upstream binding region and about 45 nM Lrp was required for 50% saturation of the downstream binding region (data not shown). To compare the concentration of Lrp needed for the stimulation of in vitro transcription with that needed for binding, we performed in vitro transcription under standard conditions with different concentrations of Lrp. Stimulation of transcription from promoter P1, which followed a smooth curve with no indication of a biphasic transition, was half-maximal at about 15 nM Lrp and near maximal at between 50 and 100 nM Lrp (Fig. 2). With even higher concentrations of Lrp, transcription from promoter P1 decreased from its maximal level (data not shown). Maximal stimulation in this experiment was fivefold, and in numerous other experiments it varied between two- and fourfold. In general, stimulation of transcription was observed over the same Lrp concentration range as that required for Lrp binding to ilvIH DNA. However, it was difficult to determine from this experiment which Lrp binding regions had to be occupied for stimulation to occur. The concentration of Lrp needed for a half-maximal effect in stimulating transcription did not correspond exactly to the concentration needed for half-maximal binding to either the upstream or the downstream region. This result may be due to differences in Lrp binding constants under in vitro transcription conditions versus gel retardation assay conditions. In vitro transcription with DNA templates with defective Lrp binding sites. In vitro transcription was performed with DNA from plasmid pCV156 as a template (wild type) and with DNAs from three plasmids that were derived from pCV156 and that carry mutations in the -72 Lrp binding site. Figure 3 depicts the position of the -72 site relative to the ilvIH promoters (asterisks represent the -72 site mutations). Plasmid pCV157 contains five base-pair substitutions creating an XhoI site between -76 and -71, and plasmids

7651

pCV158 and pCV159 contain 8- and 10-bp deletions, respectively, at the XhoI site (Fig. 3) (20a). In vitro, the binding of Lrp to the downstream binding region was reduced more than 10-fold by each of the three mutations, and in vivo, expression from the ilvIH promoters was reduced 5- to 8-fold (20a). Plasmid pCV156 and its derivatives were constructed by cloning the promoter and regulatory region of ilvIH into plasmid pRS415 (18) and therefore do not contain ilvIH DNA in the region of the ilv +40/+59 primer. A new primer was made: ilv-lac +45/+64. RNA transcribed from pCV156 and its derivatives yielded weak signals with this primer compared with those observed in experiments with primer ilv +40/+59. To obtain adequate signals, we performed in vitro transcription with fivefold more DNA, Lrp, and RNAP (versus standard conditions). The reactions were performed with a circular DNA template. With RNA transcribed from pCV156 and its derivatives, primer ilv-lac +45/+64 generated extension products of 64 and 61 bases for P1 transcripts and 124 bases for P2 transcripts. Primer amp +77/+96, used in previous experiments as an internal control, was also used here to detect amp transcripts, since the amp gene is present on these plasmids as well. The basal levels of in vitro transcription obtained with wild-type and mutant DNAs used as templates were about the same (Fig. 4). However, whereas Lrp stimulated transcription from the wild-type template by 2.7-fold, it had no effect or a lesser effect on transcription from the mutant templates (0.8-, 1.2-, and 1.6-fold stimulation). These results indicate that Lrp binding to the -72 site is necessary for the activation of ilvIH transcription from promoter P1. It is important to note that in the absence of Lrp, these mutant DNA templates yielded little or no transcription from promoter P2 (Fig. 4). This result was expected because the mutations are within the -10 region of promoter P2 (Fig. 3). Effect of leucine on Lrp-stimulated in vitro transcription. Leucine was added to in vitro transcription reaction mixtures at a concentration (37 mM) that was found by Ricca et al. (15) to reduce Lrp binding to ilvIH DNA by 2.5-fold. Under standard conditions, transcription in vitro from promoter P1 was not appreciably affected by leucine (Fig. 5).

The leucine effect was also examined under conditions that differed from the standard conditions in containing 110 mM NaCl (rather than 50 mM potassium glutamate) and 12-foldhigher levels of Lrp. Under these nonstandard conditions, ilvIH transcription in the presence of Lrp was reduced twofold by leucine (Fig. 5). Effect of a crude cell extract on in vitro transcription. A small amount of crude cell extract from an Lrp-deficient strain (1rp-35::TnlO) was added to in vitro transcription reaction mixtures to determine its effect on ilvIH transcription (Fig. 6). This experiment was done with a circular pCV7 DNA template to minimize problems with nucleases in the crude extract. With the addition of the crude extract, the basal level of ilvIH transcription from P1 decreased, whereas the level of amp transcription increased. When P1 transcription was normalized to amp, P1 basal-level transcription decreased by at least a factor of 5, but the Lrp stimulation factor increased from 1.5 to 3.8 (Fig. 6). This result indicated that additional factors in the crude extract can influence Lrp stimulation of ilvIH transcription. DISCUSSION Lrp has recently attracted attention as an E. coli master regulatory protein that influences the expression of more

7652

WILLINS AND CALVO

J. BACTERIOL.

Upstream

Downstream

region

region

P2 -200

-300 I

I

+1

-100

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I

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-60

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mutations: CCTGCTCGAGCTTATTAC

Xho

A8 AlO FIG. 3. ilvIH promoter-regulatory region. The upper part of the diagram depicts the relative positions of RNAP (grey ovals) and Lrp (black boxes) binding sites. Arrows represent ilvIH in vitro transcripts, and the position of hybridization of the ilv +40/+59 primer is also shown. The open box represents the ilvI coding sequence. The lower part of the diagram shows the DNA sequence near the -72 Lrp binding site and promoter P2 (-35 and -10 regions are indicated). Mutations in the -72 Lrp binding site are depicted at the bottom of the figure. Asterisks represent changes from the wild-type sequence, and brackets represent nucleotides deleted in the A8 and A10 mutations. The A10 mutation includes a T-to-C change at -76.

than 12 different operons (3, 11). Some of these operons found by chance, others were found by two-dimensional electrophoretic analysis, and yet others were found by analysis of mutants created by placMu transposition (3, 8, 11 and references cited therein). One of the most striking aspects of the Lrp regulon is the number of different patterns of regulation that are caused by the interaction of Lrp and leucine. Like some other important global regulators, for example, Crp (14), Lrp activates the expression of some operons and represses the expression of others. For some of the operons activated by Lrp, that activation is negated by leucine (as is the case for the ilvIH operon), requires leucine, or is independent of leucine (3, 11). Similarly, for operons whose expression is repressed by Lrp, the same three subcategories have been recognized; leucine negates the effect, leucine is required for the effect, or leucine has no effect (3, 11). Thus, in total, six different regulatory patterns involving Lrp and leucine have been observed. For operons controlled by Lrp, does Lrp act directly to activate or repress the transcription of target operons, or does it act indirectly by affecting the expression of some other regulatory gene which, in turn, affects the expression were

of a target operon? Several lines of evidence, including the evidence presented here, indicate that Lrp acts directly to activate the transcription of the ilvIH operon. Lrp binds to specific sites upstream of the ilvIH promoter (15), and mutations in each of five distinct binding sites reduce the extent of Lrp binding in vitro and the expression of the operon in vivo (20a). Furthermore, the characteristics of two classes of lrp mutations are consistent with a direct role of Lrp in activation. lrp mutations that prevent Lrp from binding ilvIH DNA in vitro prevent ilvIH operon expression in vivo (12a). Other mutations, such as lrp-i, alter the proportions of Lrp-DNA complexes formed in vitro and reduce the effect that leucine has on binding (15). These mutations also prevent the repression of the ilvIH operon by leucine in vivo (13, 20). The results presented here offer strong support for the idea that Lrp directly stimulates ilvIH expression. We demonstrated that purified Lrp stimulates transcription from the ilvIH promoter in vitro at concentrations similar to those required for Lrp binding to ilvIH DNA. The in vitro transcription system displays four important characteristics of the in vivo system: transcription is initiated at or near +1,

ilvIH TRANSCRIPTION

VOL. 174, 1992

Plasmid: pCV156 pCV157 pCVI]58 pCV159

Mutation: Lrp:

None -

+

Xho -

A8 +

-

Lrp:

Al0 +

-

Leu:

+

ilv P2

>

4b

anmp

>

4____*-a am 4mm

il/ P1

>

P1/amp:

Conditions:

7653

Standard Non-standard -

+

+ +

-

-+

+

-

+

ilv P2 > amp

>

ilv PI

>

.b

a; I.u.

2.7 1.7 1.5 1.3 1.5 1.1 1.8 FIG. 4. In vitro transcription with wild-type and mutant templates defective for Lrp binding. RNA was made in vitro from plasmid pCV156-derived DNA templates in the absence or presence of Lrp. Plasmid pCV156 carries a wild-type copy of the ilvIH regulatory region. Plasmids pCV157, pCV158, and pCV159 are identical to pCV156 except that they carry mutations in the -72 Lrp binding site (X7ho A8, and 10 mutations, respectively; sequences are shown in Fig. 3). RNA transcribed from ilvIH and amp was detected by primer extension from 32P-labelled ilv-lac +45/+64 and amp +77/+96 primers, respectively, and quantitated as described in the legend to Fig. 1A. The conditions in this experiment differed from the standard conditions in containing 0.19 U of RNAP per pA; 5 nM uncut plasmid and 98 nM Lrp were used. 1.0

Lrp stimulates transcription, mutations in a critical Lrp binding site reduce transcription, and leucine reduces transcription (albeit only under certain conditions in vitro). The stimulatory effects of Lrp in vitro (2- to 4-fold) were not as high as those in vivo (about 11-fold), suggesting either that we have not established optimal in vitro conditions or that some other factors modulate Lrp action. In this regard, we found that the addition of a crude extract to in vitro transcription reactions increased the degree of Lrp-mediated stimulation up to twofold. Genetic experiments established that Lrp is necessary for ilvIH expression (13). The work reported here strongly suggests that Lrp by itself can activate transcription from the ilvIH promoter. The Lrp that was used in these experiments was >98% pure, but we cannot exclude the possibility that a second stimulatory factor is present in the impurities. When Lrp acts as an activator, does it act from a distance, perhaps through some sort of looping mechanism, or from sites adjacent to or overlapping the promoter? In a recent analysis, Collado-Vides et al. (1) and Gralla (5) suggested that E. coli and Salmonella typhimurium promoters that were positively controlled could be divided into two groups. One of them was a large collection of sigma 70 promoters in which activation can be understood in terms of a direct interaction between a neighboring activator and RNAP. The other group was made up of a much smaller number of sigma 54 promoters. For this latter group, binding sites for activator proteins were located far upstream of the promoter and could be moved even further away without destroying activator function. The activation of these sigma 54 promoters is imagined to occur through a looping mechanism

P1/amp: 3.6 11.3 9.9 0.4 0.9 0.5 FIG. 5. Effect of leucine on in vitro transcription. RNA was made in vitro with plasmid pCV7 DNA as a template in the absence of Lrp, in the presence of Lrp, or in the presence of Lrp and 37 mM leucine. The transcription reaction was carried out under standard conditions or under nonstandard conditions as described below. RNA transcribed from ilvIH and amp was detected by primer extension from 32P-labelled primers and quantitated as described in the legend to Fig. 1A. The experiment was repeated, with essentially the same results. Reactions performed under standard conditions contained 1 nM PstI-cut pCV7, 25 nM Lrp, 0.04 U of RNAP per ,ul, and 50 mM potassium glutamate in the transcription buffer (other details are described in Materials and Methods). Nonstandard reaction conditions differed from standard reaction conditions in containing 2 nM PstI-cut pCV7, 295 nM Lrp, 0.08 U of RNAP per pl, and 110 mM NaCl instead of potassium glutamate in the transcription buffer; also, preincubation of Lrp with the DNA template was omitted. bringing the activator in contact with RNAP, and there is experimental evidence for some systems supporting this view (21). For Lrp activation of ilvIH transcription, choosing between these two models is complicated by the fact that there are at least six binding sites for Lrp, extending from -253 to -46. Two of those sites, centered at -72 and -52, are at positions that might allow direct neighbor interactions between Lrp and RNAP (1). However, a recent mutational analysis indicated that the -52 site is not important for ilvIH expression (20a). The fact that purified sigma 70 RNAP correctly initiated transcription in vitro in our studies suggests that the ilvIH promoter is a sigma 70 promoter. Furthermore, experiments performed by E. Ricca and M. Sacco and their colleagues (1Sa) and confirmed by us (20a) have shown that moving Lrp binding sites further upstream destroys the ability of Lrp to activate ilvIH expression in vivo. Considering all of these results leads us to the following view. The activation of ilvIH transcription probably requires the binding of Lrp to the site centered at position -72, at which Lrp can presumably contact RNAP bound at promoter P1. The role of the remaining sites most likely is to increase occupancy of the site centered at position -72 through cooperative binding of Lrp. Evidence for such cooperativity is the subject of another paper (20b). In the absence of Lrp, transcription was initiated in vitro at both promoters P1 and P2. The addition of Lrp increased transcription from P1 but decreased transcription from P2. This result was expected because the Lrp binding site

7654

WILLINS AND CALVO

Additions: Lrp:

J. BAcTERIOL.

None Crude ext. -

+ -

ilv P2

>

IWO

anmp

>

4

+

_

il/ P1 > P1/amp: 6.9 12.1 1.4 4.1 FIG. 6. Effect of a crude cell extract (Crude ext.) on in vitro transcription. Transcription from plasmid pCV7 in the presence of purified RNAP was examined in the presence or absence of Lrp (298% purity) and the presence or absence of a crude cell extract from Lrp-deficient strain CV1008 (lrp-35::TnlO). RNA transcribed from ilvIH and amp was detected by primer extension from 32p_ labelled primers and quantitated as described in the legend to Fig. 1A. In a repctition of this experiment, the Pl/amp ratios were, respectively, 5.6, 8.4, 0.4, and 1.4. These experiments were performed under standard conditions with 1 nM uncut pCV7 and 25 nM Lrp. The crude extract was added to 0.2 ,ug of protein per pl.

centered at -72 overlaps promoter P2. Indeed, the mutations that destroyed this site also destroyed promoter P2. This situation, of two closely spaced promoters, only one of which is active in vivo, is reminiscent of the situation for the lac and ilvG operons (10, 12). For the lac operon, an upstream P2 promoter (not used in vivo) overlaps the downstream P1 promoter (used in vivo); the start points of P1 and P2 are 22 bases apart. Crp-cyclic AMP (cAMP), which stimulated lac expression in vivo, reduced RNAP occupation of the P2 promoter and increased occupation of the P1 promoter in vitro (10). The two tandem promoters of the ilvG operon (one productive in vivo and the other unproductive) were regulated in a similar way by integration host factor (12). For regulation of the lac operon, Meiklejohn and Gralla (10) argued that the exclusion of RNAP from promoter P2 did not totally account for the function of Crp-cAMP in stimulating transcription from promoter P1, because a lac promoter mutation inactivating P2 did not increase lac expression in the absence of Crp-cAMP (23). They concluded that Crp-cAMP had two functions that were both necessary for the stimulation of lac transcription: removing RNAP from the unproductive P2 promoter and directly stimulating transcription from promoter P1 (10). Lrp seems to act in a similar fashion: in vitro, it stimulated transcription from the downstream P1 promoter and reduced transcription from the apparently unproductive P2 promoter. Here also, the removal of RNAP from the P2 promoter was not sufficient to stimulate transcription from promoter P1. Mutations that destroy the P2 promoter (-72 Lrp binding site mutations) did not increase transcription from promoter P1 in vitro in the absence of Lrp. This result suggests that Lrp, like Crp-cAMP, may have a dual function in activating transcription from promoter P1 and clearing RNAP from the unproductive P2 promoter.

ACKNOWLEDGMENTS We thank Stanley Zahler, Jeffrey Roberts, Bonnie Tyler, and Jerry Grandoni for helpful comments. We also acknowledge the use of strains donated by Jill Platko and Qing Wang and thank them for helpful discussions. This work was supported by grant GM39496 from the National Institutes of Health. D.A.W. was supported by Public Health Service training grant 5T32GM07273-12 from the National Institutes of Health.

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VOL. 174, 1992 nichia coli K-12. J. Bacteriol. 147:797-804. 20. Ursini, M. V., P. Arcari, and M. DeFelice. 1981. Acetohydroxyacid synthase isoenzymes of Escherichia coli K-12: a transacting regulatory locus for ilvIH gene expression. Mol. Gen. Genet. 181:491-496. 20a.Wang, Q., and J. M. Calvo. Unpublished data. 20b.Wang, Q., and J. M. Calvo. Submitted for publication. 21. Wedel, A., D. S. Weiss, D. Popham, P. Droge, and S. Kustu. 1990. A bacterial enhancer functions to tether a transcriptional

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activator near a promoter. Science 248:486-490. 22. Willins, D. A., C. W. Ryan, J. V. Platko, and J. M. Calvo. 1991. Characterization of Lrp, an Escherichia coli regulatory protein that mediates a global response to leucine. J. Biol. Chem. 266:10768-10774. 23. Yu, X.-M., and W. S. Reznikoff. 1985. Deletion analysis of the Escherichia coli lactose promoter P2. Nucleic Acids Res. 13: 2457-2468.