Regulation of the Escherichia coli rrnB P2 Promoter

The seven rRNA operons in Escherichia coli each contain twopromoters, rrn P1 and rrn P2. Most previous studies have focusedon the rrn P1 promoters. Here we report a systematic analysisof the activity and regulation of the rrnB P2 promoter in orderto define the intrinsic properties of rrn P2 promoters and tounderstand better their contributions to rRNA synthesis whenthey are in their natural setting downstream of rrn P1 promoters.In contrast to the conclusions reached in some previous studies,we find that rrnB P2 is regulated: it displays clear responsesto amino acid availability (stringent control), rRNA gene dose(feedback control), and changes in growth rate (growth rate-dependentcontrol). Stringent control of rrnB P2 requires the alarmoneppGpp, but growth rate-dependent control of rrnB P2 does notrequire ppGpp. The rrnB P2 core promoter sequence (-37 to +7)is sufficient to serve as the target for growth rate-dependentregulation.

INTRODUCTION

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Introduction

To meet the requirement for protein synthesis as nutritionalconditions change, bacteria modulate ribosome synthesis primarilyby controlling transcription initiation from rRNA promoters.Escherichia coli contains seven rRNA operons (rrnA, -B, -C,-D, -E, -G, and -H), each of which has two promoters, rrn P1and rrn P2, separated by $~$ 120 bp (16). The rrn P1 and rrn P2promoters have many sequence characteristics in common: nearconsensus -10 and -35 hexamers, separated by 16 bp, that bindthe ${sigma}$ subunit of RNA polymerase (RNAP) (16); an A+T-rich regionupstream of the -35 hexamer (UP element) that increases transcriptionby binding the C-terminal domains of the ${alpha}$ subunits of RNAP (27,28); and a G+C-rich region (the discriminator) (38) betweenthe -10 hexamer and the transcription start site that is requiredfor proper regulation (4, 8, 15, 25) (see Fig. 1 for the rrnBP2 sequence).

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FIG. 1. Sequence of the rrnB P2 promoter region from -112 to +21. The most frequent rrnB P2 transcription start site is designated +1. The -10 and -35 hexamers are indicated in bold, start sites are underlined, and the upstream (-112, -68, -53, and -37) or downstream (+7 and +21) endpoints used for construction of lacZ fusions are indicated by ${lceil}$ or ${rciel}$ , respectively. The -15C insertion is indicated by the symbol ^, and substitutions are identified below the arrows.

Regulation of rRNA transcription initiation is often analyzedin terms of the responses of rRNA promoters to three experimentalsituations. First, rrn P1 activity changes in proportion tothe steady-state growth rate (growth rate-dependent control[12, 22; reviewed in reference 13]), coordinating the numberof ribosomes with the need for protein synthesis. Second, rrnP1 activity changes in inverse proportion to changes in rRNAgene dose (feedback regulation [14; reviewed in reference 13]),maintaining ribosome synthesis homeostatically. Third, relA-dependentinhibition of rrn P1 activity is observed following amino acidstarvation (stringent control [reviewed in reference 6]), preventingan overinvestment of energy in ribosome synthesis when the substratesfor protein synthesis are unavailable.

The literature is less clear about the regulation of the rrnP2 promoters. Early studies indicated that rrn P2 promotersare growth rate dependent (but less so than rrn P1 promoters)and not stringently controlled (31-33), but subsequent studiessuggested the opposite, that rrn P2 promoters are not growthrate dependent (12, 40) and are stringently controlled (11,15, 19). Studies employing rrnB P2-lacZ or rrnB P2-cat fusionsas reporters (12, 40) suggested that rrn P2 promoters are moreactive than rrn P1 promoters, in contrast to conclusions basedon direct measurements of RNAs transcribed from rrn P1 and rrnP2 promoters (7, 20, 31, 40). Furthermore, inhibition of rrnP2 promoters by transcription originating upstream from therrn P1 promoters (promoter occlusion [11]) qualifies conclusionsabout the intrinsic activity and regulation of rrn P2 derivedfrom examination of constructs containing both promoters (7,19, 20, 31-33).

Here we systematically reinvestigated the activity and regulationof rrnB P2 by using newly constructed reporters of rrnB P2 promoteractivity, as well as direct measurement of transcripts by primerextension. We found that rrnB P2 is less active during steady-stategrowth than we concluded previously and that it is regulatedin response to growth rate, amino acid starvation, and rRNAgene dose. In addition, we show that stringent control of rrnBP2 requires ppGpp but that growth rate-dependent regulationof rrnB P2 does not require ppGpp. The rrnB P2 core promotersequence is sufficient to serve as the regulatory target forgrowth rate-dependent control.

MATERIALS AND METHODS

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Materials and Methods

Media and growth conditions. Cells were grown at 30°C in Luria-Bertani (LB) medium, brainheart infusion medium (Difco), or either M9 (21) or morpholinepropanesulfonicacid (23) minimal medium containing 0.4% glucose or glycerol,with or without 0.8% Casamino Acids (Difco) plus tryptophan(40 µg/ml). When required, ampicillin (Sigma) was includedat 100 µg/ml. To induce a stringent response, the serineanalog serine hydroxamate (1 mg/ml; Sigma) was added to an exponentiallygrowing culture supplemented with amino acids (37).

Strains. The strains and plasmids used in this study are listed in Table1. Antibiotic resistance cassettes replacing the relA and spoTgenes were transduced into ${lambda}$ lysogens by using phage P1vir (10).All promoter derivatives were generated by PCR from plasmidscontaining a wild-type promoter(s). Primers for the PCR weredesigned to include an EcoRI site at the upstream junction ofthe promoter sequence and a HindIII site at the downstream junction.The PCR products were cleaved with EcoRI and HindIII, ligatedinto plasmid pRLG770 (29), sequenced, and then cloned into bacteriophage ${lambda}$ derivatives containing lacZ to form promoter-lacZ fusions aspreviously described (26). In all lacZ fusion constructs (referredto as system 1 [26]), the DNA sequence downstream from the indicatedpromoter fragment is identical and derives from trp-lac DNAsequences. Promoter constructs are named by the endpoints ofthe DNA fragments used for their construction, where +1 is thetranscription start site. rrnB P2 utilizes multiple transcriptionstart sites; +1 refers to the most frequently utilized site,a C residue 7 bp downstream from the -10 hexamer (Fig. 1; H.D.M.and R.L.G., data not shown).

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TABLE 1. Strains, plasmids, and promoter constructs used in this study

The rrnB P2-lacZ fusion described previously (12) was constructedby cleavage of rrnB DNA with FnuDII, ligation with a HindIIIlinker, and then fusion of this fragment to a ${lambda}$ phage arm containinglacZ. This resulted in an rrnB P2 promoter with the downstreamDNA sequence 5'-CCCGGGGAAGCTT...-3', beginning with position+1. This sequence differs from the wild-type rrnB P2 sequenceby C-to-G transversions at +5 and +7 (Fig. 1).

In vivo ß-galactosidase assays. Cultures of lysogens containing promoter-lacZ fusions were started(A₆₀₀ of $~$ 0.02) from fresh colonies and shaken at 30°C forabout 4 generations to an A₆₀₀ of $~$ 0.35, and ß-galactosidaseactivity was measured following sonication as previously described(12, 21).

In vivo primer extension. Cultures of lysogens containing promoter-lacZ fusions were startedfrom fresh colonies (starting A₆₀₀ of $~$ 0.02) and shaken at 30°Cfor about 4 generations to an A₆₀₀ of $~$ 0.35. RNA was extractedby a boiling lysis procedure, and reverse transcription wasperformed as previously described (15, 35). Note that the absoluteactivities of promoters making different RNAs are not comparable,since the efficiency of extension by reverse transcriptase varieson different templates. To ensure that any differences in RNAlevels observed at different growth rates did not result fromchanges in RNA half-life, half-lives were measured directlyafter addition of rifampin. No differences in RNA half-lifewere detected (data not shown). Furthermore, control promoterswhose activities did not change with growth rate when examinedby using promoter-lacZ fusions but that had the same mRNA sequencesas the growth rate-dependent promoters examined here did notchange with growth rate when examined by primer extension (datanot shown).

Feedback assays. A multicopy plasmid containing an intact rrnB operon codingfor functional 16S, 23S, and 5S rRNAs (pNO1301) or an rrnB operoncoding for nonfunctional 16S and 23S rRNAs (pNO1302) was transformedinto lysogens carrying promoter-lacZ fusions. Cultures wereshaken at 30°C in M9 medium supplemented with glucose andampicillin to an A₆₀₀ of $~$ 0.35, and ß-galactosidaseactivity was measured as described above. As described in thefigure legends, the activity of a promoter-lacZ fusion in astrain containing pNO1301 and that of a promoter-lacZ fusionin a strain containing pNO1302 were compared and the ratio wasthen normalized to that obtained from a control strain containinga lacUV5 promoter-lacZ fusion transformed with the same plasmids(12).

RESULTS

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Results

Sequence determinants for rrnB P2 activity. In order to investigate the behavior of rrnB P2 systematically,we created a series of rrnB P2-lacZ fusions containing promoterfragments with different upstream endpoints and the same downstreamendpoint: -112 to +7, -52 to +7, and -37 to +7 (Fig. 1). Wemeasured the activities of these fusions at a relatively lowgrowth rate (i.e., when most of the rRNA transcription in thecell originates from the rrn P2 promoters [see below and reference31]). rrnB P2 promoters with upstream endpoints of -112 and-52 had similar activities and were fourfold more active thanthe -37 to +7 construct (Fig. 2). The stimulatory effect ofthe -37 to -52 region is consistent with the previous findingthat rrnB P2 contains an UP element; i.e., that the rrnB P2sequence upstream of the -35 hexamer stimulates transcriptionboth in vivo and in vitro (27, 28). These results also suggestthat, unlike the rrn P1 promoters, which are activated by thetranscription factor Fis (29), rrnB P2 does not require activationby a transcription factor that binds upstream of the UP element.

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FIG. 2. Relative activities of rrnB P2 promoter variants. ß-Galactosidase activities from single-copy rrnB P2 promoter-lacZ fusions were measured in M9 minimal medium containing 0.4% glycerol as a carbon source. The rrnB P2(-68 to +7; C+5G,C+7G) fusion is the construct reported in reference 12. The average and standard deviation of at least three independent experiments are shown for each promoter.

The rrnB P2-lacZ fusion used in our previous studies (12, 15)was five- to sixfold more active than the P2(-112 to +7) orP2(-52 to +7) fusion described above (Fig. 2). This construct[now referred to as P2(-68 to +7; C+5G,C+7G)] contains a differentupstream endpoint and C-to-G transversions at positions +5 and+7 (i.e., downstream of the transcription start site [Fig. 1and Materials and Methods]). Since sequences upstream of -52did not appear to affect transcription, we tested whether thedifference in activity between the original and new constructsresulted from the substitutions at +5 and +7 by introducingthese transversions into the P2(-112 to +7)-lacZ fusion. TheP2(-112 to +7; C+5G,C+7G) construct was five- to sixfold moreactive than the wild-type P2(-112 to +7) promoter (Fig. 2).

In theory, the increase in ß-galactosidase activitycould have arisen from either an increase in mRNA half-lifeor a more favorable RNAP-promoter interaction. We found thatthe C+5G,C+7G mutations did not alter mRNA half-life in vivo,but the mutations did alter characteristics of the open complexformed with RNAP in vitro (data not shown; see Discussion).Therefore, we now consider our previously constructed rrnB P2promoter-lacZ fusion, P2(-68 to +7; C+5G,C+7G), to be a mutantthat does not accurately reflect wild-type promoter interactionswith RNAP. As a result, we reevaluated our previous conclusionsconcerning the regulation of the rrnB P2 promoter (12, 15).

The rrnB P2 promoter is subject to growth rate-dependent control. To evaluate growth rate dependence of transcription from therrnB P2 promoter, cultures were grown in various media supportingdifferent steady-state growth rates and promoter activitieswere determined. The activities of promoter-lacZ fusions containingthe rrnB P2 -37 to +7, -52 to +7, or -112 to +7 sequence increasedat least threefold over a fourfold range of growth rates (Fig.3A to C). Promoter-lacZ fusions with an rrnB P2 downstream endpointof +21 (with an upstream endpoint of either -112 [Fig. 3D] or-37 [data not shown]) exhibited regulation similar to that ofrrnB P2 promoters with a downstream endpoint of +7, suggestingthat these junctions represent wild-type promoter behavior.These regulation patterns are quite different from those exhibitedby the control lacUV5 promoter (Fig. 3E) or by the mutant promoterconstruct P2(-112 to +7; C+5G,C+7G) (Fig. 3F and reference 12;see also reference 40). These results suggest that sequencesupstream from -37 or downstream from +7 are not required forgrowth rate-dependent regulation of rrnB P2 and that the substitutionsat +5 and/or +7 affect properties of the rrnB P2 promoter thatare important for its regulation (see Discussion).

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FIG. 3. Promoter activities as a function of growth rate. ß-Galactosidase activities were measured at different growth rates, obtained by growing cells in different media as described in Materials and Methods: M9 medium with 0.4% glycerol, M9 medium with 0.4% glucose, M9 medium with 0.4% glycerol plus 0.8% Casamino Acids plus tryptophan, M9 medium with 0.4% glucose plus 0.8% Casamino Acids plus tryptophan, and LB medium. Linear regressions were drawn by using SigmaPlot 5.0 (Jandel Scientific). The endpoints of the wild-type and mutant rrnB P2 promoter fragments used to construct the fusions are indicated in the panels. The lacUV5 promoter-lacZ fusion shown in panel E has been described previously (12). To enable visual comparison of the slopes, the activity of each promoter was normalized to a value of 1.0 at a growth rate of 0.9 doubling per hour (8). Strain designations and observed promoter activities (in Miller units) at a growth rate of 0.9 doubling per hour are as follows: A, RLG3851, 784 ± 34 U; B, RLG3863, 1,749 ± 72 U; C, RLG5014, 1,958 ± 236 U; D, RLG3914, 4,189 ± 252 U; E, RLG4993, 408 ± 66 U; F, RLG3915, 7,211 ± 300 U; G, RLG3898, 3,167 ± 281 U; H, RLG3897, 2,240 ± 104 U. Data from at least two independent experiments are shown for each construct.

Previous studies of rrnB P1 revealed a number of features inthe core promoter important for regulation, including the lengthof the spacer (16 bp) between the -10 and -35 hexamers and theidentity of the sequence in the discriminator region betweenthe -10 hexamer and the transcription start site (4, 8). Weconstructed mutations in the rrnB P2 spacer or discriminatorregion to determine whether changing these features would alterregulation of this promoter. Either a 1-bp insertion in therrnB P2 spacer, P2(-112 to +7; Cins-15), or a 3-bp substitutionin the discriminator region, P2(-112 to +7; C-5A,A-4T,C-3A),significantly reduced growth rate regulation of the promoter(Fig. 3G and H), suggesting that similar sequence features inrrn P1 and rrn P2 might contribute to their regulation.

Growth rate-dependent regulation of the rrnB P2 promoter in tandem with rrnB P1. In their natural setting in rRNA operons, the rrn P2 promotersare located about 120 bp downstream from the rrn P1 promoters.To measure the activities of rrnB P2 when it is alone or intandem with rrnB P1, we directly measured RNA synthesis by usingquantitative primer extension. Transcription from the isolatedrrnB P2 promoter increased as a function of growth rate (Fig.4A), in agreement with the results obtained with lacZ fusions.However, in the presence of transcription from rrnB P1 (promoteractivity illustrated in Fig. 4B), rrnB P2 promoter activitydid not increase with growth rate and actually decreased slightly(Fig. 4A). These results are consistent with previous indications(11) that when the rrn P2 promoter is in its natural setting,its activity is occluded by transcription originating from rrnP1. Occlusion is most apparent when rrnB P1 activity is greatest,i.e., at high growth rates, and is sufficient to mask the intrinsicgrowth rate dependence of rrnB P2.

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FIG. 4. Transcription from rrnB P2 in the presence and absence of rrnB P1. (A) RNA transcribed from rrnB P2 promoter-lacZ fusions was measured directly by primer extension from lysogens grown in morpholinepropanesulfonic acid medium supplemented with 0.4% glycerol, 0.4% glucose, or 0.4% glucose plus 0.8% Casamino Acids plus tryptophan or in LB medium. Symbols: •, RLG5014, rrnB P2(-112 to +7); ${blacktriangledown}$ , RLG3871, rrnB P1P2(-152 of P1 to +7 of P2). The average and standard deviation of at least three independent experiments are shown. Promoter activities are expressed in arbitrary units. (B) RNA transcribed from rrnB P1, in the context of the rrnB P1P2(-152 of P1 to +7 of P2) promoter-lacZ fusion, was measured directly by primer extension from lysogens grown in the same media used for the experiment whose results are shown in panel A. The average and standard deviation of at least three independent experiments are shown. Promoter activity is expressed as a percentage of maximum promoter activity. Absolute promoter activity should not be compared to that of rrnB P2 (see Materials and Methods). (C) ß-Galactosidase activities from the same promoter-lacZ fusions described in panels A and B and from an rrnB P1-lacZ fusion. Symbols: •, RLG3871, rrnB P1P2(-152 of P1 to +7 of P2); ${blacksquare}$ , RLG4757, rrnB P1(-152 to +50); ${blacktriangledown}$ , RLG5014, rrnB P2(-112 to +7). The average of two independent experiments is shown for each promoter. The dashed line is a plot of the sum of the activities from the isolated rrnB P1-lacZ and rrnB P2-lacZ fusion constructs.

We next measured whether a lacZ fusion to a DNA fragment containingthe intact rrnB P1P2 region would report the sum of the twoisolated promoter activities (Fig. 4C). The isolated rrnB P1and rrnB P2 promoters were each growth rate dependent (althoughto different extents [31]). As expected from the results shownin Fig. 4A, the activity of the tandem rrnB P1P2-lacZ fusionat low growth rates reflected the sum of the activities fromthe isolated promoters (Fig. 4C, dashed line), since occlusionof rrnB P2 is negligible when rrnB P1 is least active. In contrast,at high growth rates, the activity of the rrnB P1P2 fusion wassubstantially lower than that of the sum of the two isolatedpromoters (Fig. 4C, dashed line), consistent with the observedocclusion of rrnB P2 by rrnB P1 under these growth conditions(Fig. 4A). We conclude that the lacZ fusion to the tandem rRNApromoters accurately reports expression from the intact rrnBP1P2 promoter region, describing the sum of the activities fromthe rrnB P1 promoter and the occluded rrnB P2 promoter.

rrnB P2 is stringently controlled but does not require ppGpp for growth rate-dependent control. To determine whether the rrnB P2 promoter is stringently controlled,we used primer extension to measure its expression followingamino acid starvation in a wild-type strain. Figure 5A showsthat transcription from wild-type rrnB P2 was strongly inhibited,as reported previously for rrnB P1 (11, 15, 19). The rrnB P2promoter with an altered discriminator sequence, P2(-112 to+7; C-5A,A-4T,C-3A), was inhibited only slightly (15, 38), andinhibition of wild-type rrnB P2 did not occur in a relA mutant(Fig. 5A). Thus, the results confirm previous conclusions (11,15, 19, 35) that rrnB P2 is stringently controlled by the relAproduct, ppGpp.

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FIG. 5. rrnB P2 is stringently controlled but does not require ppGpp for growth rate-dependent regulation. (A) RNA transcribed from rrnB P2 promoter-lacZ fusions was measured directly by primer extension following amino acid starvation induced by serine hydroxamate addition to a culture growing exponentially in LB medium (A₆₀₀ of $~$ 0.3). The identity of the rrnB P2 promoter and the strain background are indicated for each sample. Symbols: •, RLG5014, wild-type (WT) rrnB P2(-112 to +7); ${blacksquare}$ , RLG3987, mutant rrnB P2(-112 to +7; C-5A,A-4T,C-3A); ${blacktriangledown}$ , RLG6982, wild-type rrnB P2(-112 to +7) in a relA strain. The average and standard deviation of three independent experiments are shown. (B) ß-Galactosidase activity from an rrnB P2 core promoter-lacZ fusion, P2(-37 to +7), as a function of growth rate in wild-type and relA spoT mutant strains. Symbols: ${blacktriangledown}$ , RLG3851, wild-type strain; •, RLG3866, relA spoT mutant strain. Data from two independent experiments are shown for each strain.

ppGpp concentrations change inversely with growth rate (6, 30).To determine whether ppGpp is required for growth rate-dependentcontrol of rrnB P2, we measured the activity of an rrnB P2-lacZfusion at different growth rates in a wild-type strain and ina strain lacking the two genes capable of ppGpp synthesis, relAand spoT. Growth rate regulation of rrnB P2 was very similarin the mutant and wild-type strains (Fig. 5B), indicating thata regulator(s) other than ppGpp is sufficient to maintain normallevels of transcription from rrnB P2 in steady-state growth.However, we have not ruled out the possibility that ppGpp, whenpresent, contributes to this regulation.

Increased rRNA gene dose reduces transcription from rrnB P2. Total rRNA synthesis is gene dose independent, because transcriptionfrom individual rRNA operons is reduced in inverse proportionto the increase in rRNA gene number (14). It was shown previouslythat rrnB P1 promoter-lacZ fusions were inhibited by the presenceof extra rRNA operons on multicopy plasmids, while the mutantrrnB P2 promoter-lacZ fusion P2(-68 to +7; C+5G,C+7G) was unaffected(12). To determine whether the wild-type rrnB P2 promoter isfeedback regulated, we compared the activities of wild-typeP2(-112 to +7) and two mutant promoter-lacZ fusions, P2(-112to +7; C+5G,C+7G) and P2(-112 to +7; C-5A,A-4T,C-3A), in thepresence of a multicopy plasmid harboring an intact rrnB operonor control plasmids (see the legend to Fig. 6 and reference12). The wild-type rrnB P2 and rrnB P1 promoters were inhibitedabout 30 and 50%, respectively, while the mutant rrnB P2 promoterswere inhibited less than 10% by the presence of extra rRNA operons(Fig. 6). Thus, rrnB P2 promoter activity is feedback regulated,although to a lesser extent than rrnB P1.

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FIG. 6. rrnB P2 promoter activity is inhibited by the presence of extra rRNA operons. Lysogens containing promoter-lacZ fusions were transformed with a multicopy plasmid containing either an intact rrnB operon (pNO1301) or an rrnB operon containing a large deletion in the 16S and 23S rRNA genes (pNO1302). Promoter activity is expressed as the ratio of the ß-galactosidase activity from a promoter-lacZ fusion in a strain containing pNO1301 to that in a strain containing pNO1302 and then normalized to the value obtained from a control lacUV5 promoter as previously described (12). RLG4993, lacUV5 (-59 to +36); RLG3848, rrnB P1 (-61 to +50); RLG5014, rrnB P2(-112 to +7); RLG3987, rrnB P2(-112 to +7; C-5A,A-4T,C-3A); RLG3915, rrnB P2(-112 to +7, C+5G,C +7G). The average results and standard deviations of experiments with three independent transformants are shown.

DISCUSSION

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Discussion

rrn P2 promoters are regulated. We have shown that the rrnB P2 promoter (in the absence of rrnBP1) has intrinsic regulatory characteristics similar to thoseof the rrn P1 promoters. rrnB P2 activity increases with growthrate, core promoter sequences are sufficient for this growthrate-dependent regulation, and ppGpp is not required for thiscontrol. Furthermore, rrnB P2 is feedback inhibited by the presenceof extra rRNA operons and is stringently controlled (11, 15).Thus, our studies suggest that both the rrn P1 and rrn P2 promotersare coordinately regulated (although not to the same extent).As a consequence of being regulated to different degrees, therrn P1 and rrn P2 promoters have different functions: the rrnP1 promoters are most responsible for the large amounts of rRNArequired during rapid growth, while the rrn P2 promoters areresponsible for most of the rRNA expression that occurs at lowgrowth rates (Fig. 4B and reference 31).

In some previous studies (18, 41), changes in rrnB P2 promoteractivity with growth rate and amino acid limitation were attributedto changes in the concentration of free RNAP. We suggest thatthis conclusion is not likely to be correct, since (i) UP elementsreduce the concentration of RNAP required for transcription(26), yet growth rate-dependent regulation of rrnB P2 was notaffected by deletion of its UP element (Fig. 3); (ii) rrnB P2activity was unaffected by conditions that should reduce theeffective RNAP concentration within the cell (i.e., by the presenceof multicopy plasmid pNO1302, whose transcription titrates outRNAP [reference 2 and data not shown]); and (iii) rrnB P2 activitywas affected by conditions that do not alter the cell's RNAPconcentration (i.e., by the presence of multicopy plasmid pNO1301[Fig. 6 and references 2, 11, 26, and 30]). Thus, the activityof rrnB P2 is unlikely to be a valid indicator of the free cellularRNAP concentration.

Mechanism of regulation of rrn P2 promoters. The rrnB P2 promoter forms a relatively short-lived open complexwith RNAP (H.D.M. and R.L.G., unpublished data), accountingfor its sensitivity to inhibition by ppGpp (3). However, sincerrnB P2 is growth rate dependent even in relA spoT mutant strains,mechanisms in addition to those involving ppGpp must contributeto control of the rrn P2 promoters. One mechanism contributingto regulation of the rrn P1 promoters involves sensing of changinginitiating nucleoside triphosphate concentrations (4, 5, 9,35). rrnB P2 responds to changing concentrations of its initiatingnucleoside triphosphate, primarily CTP, in vitro and in vivo(H.D.M and R.L.G., unpublished data). We suggest, therefore,that when the rrn P2 promoters are responsible for most rRNAsynthesis, ribosome production is determined, at least in part,by CTP levels.

The small DNA-binding proteins Fis and H-NS affect the activityof the rrn P1 promoters in vitro and in vivo (1, 29, 36). Wetested rrnB P2 promoter activity in strains with fis or hnsdeleted in vivo and observed that rrnB P2 activity was increasedin both mutants (data not shown). However, neither protein hasbeen implicated in direct negative control of rrn P2 promoters(e.g., see reference 36). Since rrnB P2 promoter activity isregulated homeostatically (Fig. 6), it is likely that the increasein rrnB P2 promoter activity in the strain lacking fis resultsindirectly from the loss of activation of rrn P1 (feedback derepression[13]). In contrast, the activities of numerous promoters havebeen shown to increase in hns strains (34). Further work isrequired to determine whether the effect of hns on rrnB P2 promoteractivity is specific and/or direct.

The loss of regulation of the rrnB P2 promoter that resultedfrom the transversions at +5 and +7 was somewhat unexpected,since sequences downstream of +1 that affect regulation of rrnP1 promoters have not been identified (8). The C+5G and C+7Gmutations increase the half-life of the rrnB P2 open complexin vitro (H.D.M. and R.L.G. unpublished), consistent with themodel in which regulation of rrnB P2 results from its intrinsickinetic characteristics. Since RNAP clamps down on the templatein the downstream region of the transcription elongation complex(17) and this region is protected by RNAP in footprints of transcriptioninitiation complexes (24), it is possible that direct contactsof RNAP with DNA are affected by the mutations at +5 and/or+7. We will address the molecular mechanism of regulation ofrrnB P2 in a subsequent report.

ACKNOWLEDGMENTS

We thank Wilma Ross and other members of our laboratory forhelpful comments and strain construction.

H.D.M. was supported by a National Institutes of Health GeneticsPredoctoral Training Grant. Research in our laboratory is supportedby grant RO1 GM37048 from the National Institutes of Healthto R.L.G.

FOOTNOTES

* Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin, 1550 Linden Dr., Madison, WI 53706. Phone: (608) 262-9813. Fax: (608) 262-9865. E-mail: rgourse{at}bact.wisc.edu.

REFERENCES

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