Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro1

doi:10.1006/jmbi.2000.4327 available online at http://www.idealibrary.com on

J. Mol. Biol. (2001) 305, 673±688

Mechanism of Regulation of Transcription Initiation by ppGpp. I. Effects of ppGpp on Transcription Initiation in Vivo and in Vitro Melanie M. Barker, Tamas Gaal, Cathleen A. Josaitis and Richard L. Gourse* Department of Bacteriology University of WisconsinMadison, 1550 Linden Drive Madison, WI 53706, USA

To determine the role of ppGpp in both negative and positive regulation of transcription initiation during exponential growth in Escherichia coli, we examined transcription in vivo and in vitro from the growth-ratedependent rRNA promoter rrnB P1 and from the inversely growth-ratedependent amino acid biosynthesis/transport promoters PargI, PhisG, PlysC, PpheA, PthrABC, and PlivJ. rrnB P1 promoter activity was slightly higher at all growth-rates in strains unable to synthesize ppGpp (
relA
spoT) than in wild-type strains. Consistent with this observation and with the large decrease in rRNA transcription during the stringent response (when ppGpp levels are much higher), ppGpp inhibited transcription from rrnB P1 in vitro. In contrast, amino acid promoter activity was considerably lower in
relA
spoT strains than in wild-type strains, but ppGpp had no effect on amino acid promoter activity in vitro. Detailed kinetic analysis in vitro indicated that open complexes at amino acid promoters formed much more slowly and were much longer-lived than rrnB P1 open complexes. ppGpp did not increase the rates of association with, or escape from, amino acid promoters in vitro, consistent with its failure to stimulate transcription directly. In contrast, ppGpp decreased the half-lives of open complexes at all promoters, whether the half-life was seconds (rrnB P1) or hours (amino acid promoters). The results described here and in the accompanying paper indicate that ppGpp directly inhibits transcription, but only from promoters like rrnB P1 that make short-lived open complexes. The results indicate that stimulation of amino acid promoters occurs indirectly. The accompanying paper evaluates potential models for positive control of amino acid promoters by ppGpp that might explain the requirement of ppGpp for amino acid prototrophy. # 2001 Academic Press

*Corresponding author

Keywords: ppGpp; RNA polymerase; stringent response; rRNA regulation; amino acid biosynthesis

Introduction Upon amino acid starvation of Escherichia coli, the unusual nucleotides guanosine 50 -diphosphate Present address: C. A. Josaitis, Allergan, Inc., 575 Anton Boulevard, Suite 900, Costa Mesa, CA 92626, USA. Abbreviations used: RNAP, RNA polymerase; ppGpp, guanosine 50 -diphosphate 30 -diphosphate and guanosine 50 -triphosphate 30 -diphosphate; rrn operons, ribosomal RNA operons; BSA, bovine serum albumin. E-mail address of the corresponding author: [email protected] 0022-2836/01/040673±16 $35.00/0

30 -diphosphate and guanosine 50 -triphosphate 30 -diphosphate (referred to here collectively as ppGpp) directly or indirectly alter the expression of a large number of gene products in order to adjust cellular metabolism to starvation conditions.1 This complex physiological phenomenon is known as the stringent response and is characterized by an abrupt cessation of stable RNA (rRNA and tRNA) transcription.1 The high levels of ppGpp induced by starvation directly or indirectly reduce transcription from rrnB P1, a representative stable RNA promoter, about tenfold, and the target sequences required for inhibition # 2001 Academic Press

674 reside in the core promoter ( 41 to ‡1 relative to the transcription start site).2 Speci®c mutations in the core promoter render rrnB P1 insensitive to ppGpp in vivo, further supporting the idea that inhibition occurs at the level of transcription initiation.2 ppGpp also slows transcription elongation,3,4 but this inhibitory effect is alleviated in rRNA operons by the rRNA-speci®c antitermination machinery.5,6 During exponential growth, the ribosome synthesis rate and rrn P1 promoter activity increase in proportion to the growth-rate, a relationship called growth-rate-dependent control.7 RelA, a ribosomeassociated ppGpp synthetase, and SpoT, most likely a bifunctional ppGpp synthetase/degradase, synthesize ``basal'' levels of ppGpp that vary inversely with the growth-rate, but are tenfold to 100fold lower than those induced in a stringent response.8,9 rRNA transcription increases almost normally with growth-rate in strains devoid of ppGpp (
relA
spoT), but is slightly higher at all growth-rates than in wild-type strains.10,11 This suggests that basal levels of ppGpp directly or indirectly inhibit rRNA synthesis slightly, even though ppGpp is not required for growthrate-dependent regulation. We have proposed that variation in the concentrations of NTPs and in the concentration of the rrn P1 transcription factor FIS are responsible, at least in part, for growth-ratedependent regulation of rRNA promoters,12 ± 14 although this model remains controversial.15,16 Bacteria adapt to amino acid starvation by decreasing production of the translational machinery, and by increasing production of enzymes needed for synthesis and transport of amino acids. This increase is relA-dependent, indicating that ppGpp is directly or indirectly required for induction of enzyme synthesis.17 ± 26 ppGpp apparently increases expression at the level of transcription initiation, since the relA-dependent increase in histidine biosynthetic promoter (PhisG) activity following starvation is disrupted by speci®c promoter mutations.27,28 Since the stimulation by ppGpp was observed in S-30 extracts (i.e. in a coupled transcription-translation system), it has been suggested that factors in addition to ppGpp might be required.29 ± 31 Genetic and biochemical evidence suggest that ppGpp interacts directly with RNA polymerase (RNAP).
relA
spoT strains are unable to mount a stringent response, and are incapable of growth in medium containing 19 amino acids and missing any one of ten or 11 speci®c amino acids.9 Selection for growth of
relA
spoT strains on media lacking amino acids identi®ed mutations in the genes coding for the b, b0 , and s subunits of RNAP.1,32 ± 35 These and other results suggest that the mutant RNAPs might restore prototrophy by mimicking the conformation of RNAP bound to ppGpp. Furthermore, a mutation in the gene encoding the b subunit reduced RNAP's sensitivity to ppGpp in vivo,36 and a ppGpp analog crosslinked to the b

Effects of ppGpp on Transcription Initiation

subunit of RNAP in vitro,37 suggesting that b forms at least part of the ppGpp-binding site. The DNA sequences that account for the sensitivity of a promoter to the effects of ppGpp are unclear. However, promoters whose activities increase during a stringent response have A ‡ Trich discriminators (promoter DNA sequences between the 10 hexamer and transcription start site), and promoters whose activities decrease have G ‡ C-rich discriminators, suggesting that the discriminator sequence is one of the determinants for regulation by ppGpp.2,38 Typically, regulators of transcription initiation are proteins that bind to speci®c sequences overlapping or near the RNAP binding site.39 ppGpp functions in a manner different from regulatory proteins that bind to DNA, and it functions as both a negative and positive regulator. In order to understand the mechanism of regulation by ppGpp in vivo, we address here and in the accompanying paper whether regulation by ppGpp is direct or indirect. We show that the presence of basal levels of ppGpp leads to slightly reduced rRNA promoter activity and strongly increased amino acid promoter activity in vivo, and that ppGpp inhibits rrnB P1 activity in vitro but has little or no effect on amino acid promoter activity in vitro. We then explore the kinetic mechanism of promoters affected by ppGpp, we determine the step(s) affected by ppGpp, and we propose a model for how ppGpp localizes its inhibitory effects to some promoters and its stimulatory effects to others. The results strongly support a model for direct negative control and indirect positive control of transcription by ppGpp.

Results Choice of representative promoters To study positive control of transcription by ppGpp, we chose a series of promoters whose gene products increased during a stringent response, and/or that synthesized amino acids required for growth of
relA
spoT strains. These included promoters for arginine (PargI), histidine (PhisG), phenylalanine (PpheA), and lysine, methionine, and threonine (PlysC and PthrABC) biosynthesis, and the branched-chain amino acid transport promoter (PlivJ). The rRNA promoter rrnB P1 was chosen as a representative negatively regulated promoter, and lacUV5 was chosen as a control promoter. Single copy promoter-lacZ fusions were constructed on the bacterial chromosome to examine the behavior of the promoters in vivo. The promoter fragments chosen for these constructs minimized the potential for interactions with regulators other than RNAP by containing only sequences required for transcription initiation (ca 60 to ‡15; Figures 1 and 2). Since PargI contains ArgR repressor binding sites overlapping the core promoter,47 PargI promoter activity was measured in an argR mutant strain.48 We are not aware of activator or

675

Effects of ppGpp on Transcription Initiation

Figure 1. DNA sequences from positions 40 to ‡5 with respect to the transcription start site in the promoters used in this study. Reported major start sites and potential 10 and 35 hexamers are underlined and in bold.2,40 ± 46 The underlined bases in the rrnB P1(dis) and thrABC(dis) sequences indicate 3 bp substitutions in the discriminator region (see the text). The Es70 consensus sequences are shown for comparison.

slightly higher in the
relA
spoT strain than in the wild-type strain at all growth-rates.10 We conclude that basal levels of ppGpp slightly inhibit rRNA transcription initiation directly or indirectly during exponential growth. We tested the effects of promoter mutations on regulation by ppGpp in vivo. PthrABC and rrnB P1 promoter variants with altered discriminator sequences, PthrABC (dis) and rrnB P1(dis), were much less affected by the presence of ppGpp than the wild-type promoters (compare Figure 2(k) and (l) with (g) and (i)), supporting the conclusion that ppGpp directly or indirectly affects transcription initiation, and that the discriminator sequence is important for this regulation.2,27,38 ppGpp inhibits rrnB P1 but does not stimulate amino acid promoters in vitro

repressor binding sites within any of the other promoter fragments used in our constructs. Strains devoid of ppGpp have lower amino acid promoter activities and slightly higher rRNA promoter activities than wild-type strains We measured promoter activities in wild-type and
relA
spoT backgrounds in media supporting different growth-rates in order to assess the effects of basal levels of ppGpp on transcription in vivo (Figure 2). Amino acid promoter activities varied inversely with growth-rate in wild-type cells, correlating with basal ppGpp levels, but were substantially lower in
relA
spoT strains (Figure 2(a)-(g)). At the slowest growth-rates, transcription was as much as eightfold lower in the
relA
spoT strain than in the wild-type strain (Figure 2(d) and (f)). Transcription from a control promoter (lacUV5) varied little with growth-rate and decreased only slightly (1.4-fold) in the
relA
spoT strain (Figure 2(h)). Since the promoter fragments used for making the lacZ fusions contained little (Figure 2(a)-(e) and (g)) or no (Figure 2(f)) sequences downstream of the transcription start site, we conclude that ppGpp directly or indirectly stimulates transcription initiation from amino acid promoters. These results also suggest that the amino acid auxotrophy of
relA
spoT strains derives, at least in part, from a requirement for ppGpp for transcription initiation from amino acid biosynthetic promoters (see Discussion). rrnB P1 promoter activity increased with growth-rate in both the wild-type and
relA
spoT strain, and was slightly higher at all growth-rates (1.1 to 1.4-fold) in the
relA
spoT strain (Figure 2(i)), as observed previously.10,11 Since rRNA and tRNA constitute a large fraction of total RNA and are long-lived while mRNA is not, total RNA levels can be used as a measure of stable RNA transcription. Like the activity of the rrnB P1 promoter-lacZ fusion, the RNA/protein ratio (Figure 2(j)) increased with growth-rate and was

We next asked whether ppGpp acts directly by measuring its effects in vitro on transcription with puri®ed RNAP (Es70). ppGpp decreased transcription from the negatively-regulated rrnB P1 promoter about threefold (Figure 3(a); Table 1), and 60 mM ppGpp was suf®cient for maximal inhibition (Figure 3(b)). As reported previously for rrnB P149 and for some other promoters inhibited during the stringent response,50 ± 52 the degree of inhibition by ppGpp in vitro increased with salt concentration (Figure 3(c)). Inhibition was also apparent in single round transcription assays and

Table 1. Correlation between RNAP-promoter complex half-lives and inhibition by ppGpp in vitro RPo t1/2 (min)a ppGpp

Transcription in vitro ‡/ ppGppb

A. High salt rrnB P1 rrnD P1 rrnB P1(dis) lPL lacUV5 PargI PhisG PlivJ PlysC PpheA PthrABC

<0.25 <0.25 7 60 >600 900 >800 400 >800 750 400

0.32 0.28 0.81 0.90 0.94 1.12 0.93 1.31 1.07 1.22 1.27

B. Low salt rrnB P1 rrnB P1(dis)

0.7 20

0.99 1.04

Promoter

a Half-life measurements were performed as for Figure 4. High and low salt lifetimes were measured at 150 mM NaCl and 30 mM NaCl, respectively. Half-lives shown represent the average of two or more experiments. Half-lives varied by up to 30 % between experiments but the relative differences in halflives between promoters were always observed. b Transcription in the presence of ppGpp divided by transcription in the absence of ppGpp was performed as for Figure 3. High and low salt transcription was performed at 200 mM NaCl and 30 mM NaCl, respectively. The data shown represent the average of two or more experiments. The error was generally 10 % or less.

676

Effects of ppGpp on Transcription Initiation

Figure 2. Transcription activities in the presence and absence of ppGpp in vivo. (a) PargI ( 45 to ‡32); (b) PhisG ( 60 to ‡16); (c) PlivJ ( 60 to ‡13); (d) PlysC ( 59 to ‡18); (e) PpheA ( 73 to ‡10); (f) PthrABC ( 72 to ‡1); (g) PthrABC ( 72 to ‡16); (h) lacUV5 ( 59 to ‡36); (i) rrnB P1 ( 66 to ‡9); (j) stable RNA to protein ratio; (k) PthrABC(dis) ( 72 to ‡16); (l) rrnB P1(dis) ( 66 to ‡9). The numbers in parentheses are the endpoints of the promoter fragments used for constructing promoter-lacZ fusions. See Table 3 for strain numbers. In each panel except (j), the transcription activities are the b-galactosidase activities (Miller units  10 2) from promoter-lacZ fusions in wildtype (®lled circles) or
relA
spoT strains (open circles). (j) Stable RNA to protein ratios (mg/mg). b-Galactosidase activities and RNA/protein ratios are from cultures grown in media supporting different growth-rates (see Materials and Methods). The lower growth-rates are not present in the
relA
spoT graphs, since
relA
spoT strains do not grow in medium lacking amino acids. A PthrABC-lacZ fusion extending further upstream ( 167 to ‡16) was also tested. Its activity and regulation were virtually identical with those of the ( 72 to ‡16) construct (E. Espelie, M.M.B. & R.L.G.; data not shown). The mechanism responsible for the difference in PthrABC promoter activity when the downstream endpoint was ‡16 rather than ‡1 was not investigated further. The PhisG, rrnB P1, and rrnB P1(dis) promoter-lacZ fusions were constructed using a fusion system that results in about tenfold lower activity than the system used for the other fusions, so absolute activities should not be compared directly with the activities determined for the other fusions (see Materials and Methods). Each panel includes data points from two or three independent experiments.

was observed even at lower NaCl concentrations on a linear template (data not shown). The degree of inhibition by ppGpp was the same at RNAP concentrations over a 100-fold range, even when the rrnB P1 promoter was fully occupied (Figure 3(d)). Together, these results suggest that ppGpp might inhibit a step after initial binding of RNAP to the promoter (see below). In contrast, ppGpp had little or no effect on the unregulated promoters rrnB P1(dis) and lacUV5, on the vector-derived RNA-1 promoter, or on the positively regulated promoters PargI, PthrABC, PhisG, PlivJ, PlysC, and PpheA (Figure 3(a); Table 1). No stimulation of amino acid promoter activity was observed under a wide variety of conditions (data not shown). We conclude that inhibition of transcription initiation by ppGpp is direct and promoter-speci®c, and that stimulation of transcription initiation by ppGpp might be indirect. Kinetic analysis of transcription in vitro We next identi®ed the step in the kinetic mechanism of rrnB P1 affected by ppGpp in vitro and

potential steps in the mechanism of amino acid promoters that might be targeted for regulation in vivo. The multistep mechanism of promoter recognition can be abbreviated as:53 R ‡ P „ RPc „ I „ RPo !! RPe In this simpli®ed scheme, the RNAP holoenzyme (R) associates with the promoter (P) in rapid equilibrium to form a competitor-sensitive closed complex (RPc), and then this complex undergoes reversible isomerizations through at least one intermediate (I) to form a competitor-resistant open complex (RPo) in which the promoter DNA near the start site is locally unwound. Upon addition of NTPs, RNAP may temporarily remain at the promoter, forming and releasing small abortive RNAs (typically 2-12 nucleotides long) before escaping the promoter, releasing s, and forming a stable elongation complex (RPe). Each promoter has a unique set of kinetic parameters, determined by its DNA sequence and in¯uenced by the solution conditions, and the overall rate of initiation may be limited by one or

Effects of ppGpp on Transcription Initiation

677

Figure 3. Transcription in vitro in the presence and absence of ppGpp. Multiple-round transcription reactions were performed on supercoiled plasmid templates as described in Materials and Methods. (a) Transcription in the presence of 200 mM ppGpp (evennumbered lanes) or in absence of ppGpp (odd-numbered lanes). The transcription buffer contained 200 mM NaCl. Lanes 1 and 2, rrnB P1 (pRLG862); lanes 3 and 4, rrnB P1(dis) (pRLG2989); lanes 5 and 6, lacUV5 (pRLG2222); lanes 7 and 8, PargI (pRLG5069); lanes 9 and 10, PthrABC (pRLG5073). The RNA-1 transcript from the plasmid origin of replication region is also indicated. Twofold less transcription reaction was loaded onto the gel for rrnB P1(dis) to allow easier visual comparison. (b) Fraction of transcription (‡ ppGpp/ ppGpp) as a function of ppGpp concentration. Filled circles, rrnB P1 promoter; open circles, RNA-1 promoter. The buffer contained 170 mM NaCl. (c) Fraction of transcription (‡200 mM ppGpp/ ppGpp) as a function of NaCl concentration. Filled circles, rrnB P1 promoter; open circles, RNA-1 promoter. (d) Fraction of transcription (‡200 mM ppGpp/ ppGpp) as a function of RNAP concentration. Filled circles, rrnB P1 promoter; open circles, RNA-1 promoter. The buffer contained 170 mM NaCl, 50 nM RNAP was suf®cient for maximal transcription both in the presence and absence of ppGpp.

more of the steps described. A regulator such as ppGpp could act at any step to increase or decrease transcription as long as that step contributes to the overall rate of transcription initiation.53 ppGpp decreases the half-life of the open complex rrnB P1 forms an open complex with a half-life at least one to two orders of magnitude shorter than most promoters, suggesting that the observed dissociation rate of the open complex (a composite of multiple elementary steps; see Appendix) might be rate-limiting for transcription. In addition, some mutant RNAPs that partially mimic the effects of ppGpp on transcription from rrn P1 promoters form even shorter-lived open complexes than wildtype RNAP.12,33,54,55 Since this short-lived open complex is a likely target for an inhibitor of rrn P1 transcription,56,57 we previously proposed that ppGpp might function by decreasing the lifetime of the open complex.12 To test this hypothesis directly, RNAP-promoter complexes were pre-formed on supercoiled templates in the absence or presence of ppGpp. After addition of the competitor heparin to prevent reassociation of RNAP, the fraction of complexes remaining at subsequent times was quanti®ed by transcription (see Materials and Methods). ppGpp decreased the half-life of the rrnB P1 open complex two- to threefold (Figure 4(a)). However, ppGpp also decreased the half-lives of the rrnB P1(dis), PargI, and PthrABC open complexes two- to threefold, even though transcription from these promoters was not inhibited by ppGpp in vitro or in vivo (Figure 4(b)-(d)). Figure 4 shows that the open complexes formed by the rrnB P1(dis), PargI, and PthrABC promoters were much longer-lived than the rrnB P1 open complex. This suggests that promoter-speci®c effects of ppGpp on transcription

might result from the relative contribution of the observed dissociation rate of the open complex to the overall rate of transcription in different promoters rather than from promoter-speci®c effects of ppGpp on the half-life of the open complex. We examined more promoters to evaluate further whether the half-life of a promoter's open complex correlates with inhibition of transcription by ppGpp in vitro. Table 1A shows that, like rrnB P1, rrnD P1 formed a short-lived open complex, and transcription from this promoter was inhibited by ppGpp in vitro. In contrast, the lacUV5, lPL, Phis, PlivJ, PlysC, and PpheA promoters all made much longer-lived open complexes, and none of these promoters was inhibited by ppGpp in vitro. The lifetime of the open complex is a function of the ionic conditions.58 The salt-dependence of the effect of ppGpp on transcription from rrnB P1 (Figure 3(c)) is consistent with the idea that inhibition occurs by decreasing the open complex halflife. ppGpp inhibited transcription from rrnB P1 in vitro at 150 mM NaCl when the half-life was a few seconds or less, but it did not inhibit transcription of rrnB P1 at 30 mM NaCl when the half-life was almost one minute (Table 1). Apparently, the lifetime of the open complex is not rate-limiting for transcription from rrnB P1 at the lower salt concentration. The rrnB P1 open complex is also longerlived on supercoiled than on linear templates, explaining why ppGpp can inhibit transcription on linear templates at lower salt concentrations than on supercoiled templates (data not shown). We propose that ppGpp inhibits transcription initiation only when the open complex dissociates rapidly compared to the competing rates of NTP addition and promoter escape, and that the half-life of the open complex in high salt is an indicator of sensitivity of a promoter to inhibition by ppGpp both in vitro and in vivo.

678

Effects of ppGpp on Transcription Initiation

in vivo, and from lacUV5, an unregulated promoter. The N25antiDSR promoter, which was previously shown to be escape-limited,62 was included as an additional control to con®rm that our assay could detect differences in clearance. After complexes were formed on promoter fragments, heparin and NTPs were added, and run-off RNA products (<60 nt) were measured as a function of time (Figure 5(a)). As reported previously,62 RNAP synthesized many abortive products and escaped the N25antiDSR promoter only very slowly. In contrast, RNAP cleared the amino acid and control promoters rapidly with minimal abortive product production (Figure 5(a) and (b); data not shown). These results suggest that the amino acid promo-

Figure 4. ppGpp decreases the half-lives of open complexes in vitro. Half-lives were measured as described in Materials and Methods with supercoiled templates in the presence (®lled circles) and absence (open circles) of 1 mM ppGpp. (a) rrnB P1 (pRLG589), (b) rrnB P1(dis) (pRLG2989), (c) PargI (pRLG5069), (d) PthrABC (pRLG5073). (a) and (b) were measured in buffer containing 30 mM NaCl; (c) and (d) were measured in buffer containing 150 mM NaCl. The half-lives were not measured at the same salt concentration, because the rrnB P1 open complex decayed too quickly for precise measurement at the high salt concentrations used for measuring the half-lives of the open complexes of other promoters. Therefore the absolute lifetimes of the open complexes at rrnB P1 and the amino acid promoters should not be compared directly (see Table 1).

Effect of ppGpp on promoter escape RNAP must disrupt several contacts with the promoter in the process of promoter escape. There are several examples in the literature of promoters whose activities in vivo are limited by slow promoter escape, despite being able to bind RNAP quickly and/or to form long-lived open complexes.59 ± 61 Since the results described in the previous section indicated that amino acid promoters formed very long-lived complexes with RNAP (t1/2 ˆ several hours), we asked whether these promoters might be limited for promoter escape, and whether ppGpp might increase transcription initiation in vivo by decreasing the lifetime of the open complex suf®ciently to facilitate promoter clearance. We compared the rates of promoter escape from PargI and PlivJ, two promoters positively regulated

Figure 5. Promoter escape. The rate of escape was measured for four promoters in the presence and absence of 200 mM ppGpp. RNAP was added to supercoiled templates, samples were withdrawn at 0.33, 0.67, 1, 2, 4, 8, 16, and 32 minutes after NTP addition for PargI, lacUV5, and PlivJ, and at 0.5, 1, 2, 4, 8, 16, 32, 47.5, 64, and 90 minutes for N25antiDSR, and transcripts were resolved by PAGE. (a) A representative gel; arrows indicate increasing time after NTP addition. The asterisk (*) indicates the position of the full-length transcript. (b) The rate of escape of RNAP from N25antiDSR, lacUV5, PargI, and PlivJ. The amounts of full-length transcript for each promoter at each time-point are expressed as a fraction of that promoter's plateau value. (c) Escape in the presence and absence of ppGpp. Full-length transcripts (from PargI in this case) were plotted as a function of time. The paused transcripts near full-length in size were included for purposes of quanti®cation. ppGpp did not increase the rate of escape (and in fact decreased elongation slightly; see the text). Furthermore, the maximum number of full-length transcripts (plateau) was the same in the presence and in the absence of ppGpp.

679

Effects of ppGpp on Transcription Initiation

ters are not clearance-limited even though they form very long-lived complexes with RNAP. We next measured the effect of ppGpp on promoter escape. ppGpp did not decrease abortive initiation or increase the rate of full-length transcript formation from the PargI, PlivJ, lacUV5, or N25antiDSR promoters (Figure 5(a) and (c); data not shown). In fact, ppGpp slightly decreased the rate of full-length transcript formation because of the inhibitory effect of ppGpp on transcription elongation reported previously.3 Inhibitory effects of ppGpp on transcription from the amino acid promoters were not observed (Figures 2 and 3), presumably because NTP concentrations are higher under those conditions and not limiting for transcription elongation (see Materials and Methods). We conclude, contrary to our earlier speculation,33 that ppGpp does not increase transcription from amino acid promoters by stimulating the rate of promoter escape.

Effect of ppGpp on the rate of association of RNAP with promoters We tested the effect of ppGpp on the rate of open complex formation for rrnB P1, lacUV5, and PargI by performing t plots63 using nitrocellulose ®lter-binding assays.64 In these kinetic assays, tobs, the inverse of the rate of open complex formation (kobs), is plotted versus the inverse of the RNAP concentration (Figure 6; see Materials and Methods). As shown in Figure 6(a) and Table 2, ppGpp decreased the composite isomerization rate constant ki for rrnB P1 open complex formation two- to threefold (see Appendix for de®nitions of composite rate constants), and it decreased the half-life of the open complex two- to threefold (Figure 4(a)). We propose that because of the unusually short-lived open complex at rrnB P1, the effects on both ki and on the half-life likely derive from an effect of ppGpp on only one elementary

rate constant that is present in both kinetic parameters (see Discussion and Appendix). ppGpp had only a very slight or no effect on the overall association constant ka or on the isomerization constant ki of the lacUV5 and PargI promoters (Figure 6(b) and (c); Table 2). However, RNAP associated about sevenfold more slowly with PargI than with lacUV5. In combination with the fact that the amino acid promoters were not limited for promoter escape and formed long-lived open complexes, our data suggest that this slow association rate with RNAP might be rate-limiting for PargI transcription initiation in vivo. The data in the accompanying paper suggest that amino acid promoters' slow association rates are in fact central to their indirect regulation by ppGpp in vivo.

Discussion ppGpp inhibits rRNA transcription directly and specifically ppGpp inhibited transcription initiation from the rrnB P1 promoter by puri®ed RNAP in vitro (Figure 3), accurately re¯ecting the speci®city observed in vivo. We conclude that ppGpp inhibits rRNA transcription directly. The maximal inhibitory effect by ppGpp observed on transcription from rrnB P1 in vitro was somewhat smaller than the tenfold decrease observed at high ppGpp concentrations following amino acid starvation in vivo.2 This quantitative difference might have resulted from: (a) suboptimal solution conditions; (b) the absence of a hypothetical cellular component that enhances the negative effect of ppGpp in vivo; or (c) indirect effects of ppGpp production that magnify the negative effect of ppGpp on rrn P1 promoter activity in vivo. For example, rrn P1 promoters require high concentrations of the initiating nucleotide (ATP or GTP) for maximal transcription in vivo,12 and the reduction in ATP and GTP pools that occurs during a stringent response65,66 might exacerbate

Figure 6. Effect of ppGpp on open complex formation. Assays were performed as described (Materials and Methods) in the presence (®lled circles) and in the absence (open circles) of 200 mM ppGpp and the results are displayed as t-plots.63 Note that the X and Y axis values differ in each plot, and the absolute values of the derived kinetic constants (Table 2) should not be compared between rrnB P1 and the other promoters, because they were measured under different solution conditions. (a) rrnB P1; (b) lacUV5; (c) PargI.

680

Effects of ppGpp on Transcription Initiation

Table 2. Summary of kinetic constants Promoter

ppGpp

ka  10

a

rrnB P1 lacUV5 PargI

‡ ‡ ‡

5

(M

1

s 1)

1900 (500) 900 (380) 7.4 (0.9) 8.3 (1.2) 1.0 (0.1) 1.3 (0.1)

ki  103 (s 1)

t1/2 (min)

42 (3.0) 16 (1.5) 523 529 4.2 (1.3) 5.1 (0.9)

>180 >180 49 17 158 62

a

Since the rrnB P1 open complex is very short-lived on a linear DNA fragment, rrnB P1 measurements were performed in buffer containing a low salt concentration, ATP and CTP (see the text and Materials and Methods). Therefore, rrnB P1's kinetic constants cannot be compared to the kinetic constants for lacUV5 and PargI. Furthermore, t1/2 for rrnB P1 refers to the lifetime of the NTPstabilized complex (RPAC), while t1/2 for lacUV5 and PargI refer to the lifetime of the open complex (RPo).

rRNA transcription inhibition. Furthermore, negative DNA supercoiling decreases following a stringent response, although this decrease occurs more slowly than the initial drop in stable RNA transcription.49,67 Since rrn P1 promoters are more active on supercoiled DNA than on relaxed templates,49,57 reduced supercoiling could amplify ppGpp's initial inhibitory effects. ppGpp inhibits rRNA transcription by decreasing the half-life of the short-lived rrn P1 open complex ppGpp decreased the half-life of the rrnB P1 open complex, consistent with previous observations that ppGpp inhibits transcription or lowers rrn P1 occupancy even when ppGpp is added after open complex formation,68 ± 70 that ppGpp reduces rrnB P1 open complex occupancy, but not closed complex occupancy,71 and with studies on other stringently regulated promoters.72,73 However, previous studies have not determined why ppGpp inhibits only certain promoters. It has been suggested that ppGpp acts by altering kinetic constants only of stringently regulated promoters, for example by changing the conformation of RNAP, making it unable to interact with a speci®c set of promoters, or by binding to RNAP only at speci®c promoters.74 In contrast, we observed that ppGpp decreased the open complex half-lives of all promoters, but that promoters whose transcription was unaffected by ppGpp in vitro formed open complexes orders of magnitude longer-lived than rrnB P1. In these long-lived open complexes, RNAP encounters the substrates for transcription and clears the promoter long before dissociation occurs. Since promoters inhibited by ppGpp all form relatively short-lived open complexes (rrnB P1,56 PpyrBI,75 rrnD P1 and P2,76 rrnB P2,72 PhisR,67 rpoD P1 and P2,54 PleuV77,78), we suggest that ppGpp's inhibitory speci®city results not from differences in the way it interacts with RNAP in different complexes, but from the intrinsic kinetic parameters of the inhibited promoters, i.e. from the short half-lives of their open complexes. Our results indicate that ppGpp does not affect initial binding of RNAP to the promoter, consistent with the observation that ppGpp inhibited tran-

scription from rrnB P1 even when the promoter was saturated with RNAP. However, ppGpp decreased rrnB P1's composite isomerization rate constant (ki) two- to threefold and decreased the half-life of its open complex two- to threefold (Figure 5, Table 2; see also Appendix). Both the half-life of the open complex and ki are functions of several elementary rate constants, and for most promoters these kinetic parameters do not contain elementary rate constants in common. However, we suggest in the Appendix that because rrnB P1 makes such a short-lived open complex, effects of ppGpp on only one elementary rate constant (an increase in the reverse isomerization rate k 2 in the general three-step mechanism53) could account for the effects observed on both open complex half-life and ki. ppGpp acts as a positive regulator of amino acid promoter activity during balanced growth We examined the effects of basal levels of ppGpp on amino acid promoters by comparing transcription at different growth-rates in wild-type and
relA
spoT strains. Transcription initiation from amino acid promoters was as much as eightfold lower in strains lacking ppGpp (Figure 2). These results indicate that basal levels of ppGpp are needed, either directly or indirectly, for ef®cient transcription initiation from amino acid promoters during balanced growth (see also 9,28). Additional operon-speci®c regulatory mechanisms, responding to limitations for individual amino acids, are of course superimposed on the more general ppGppdependent system affecting transcription initiation. The conclusion that ppGpp increases the initiation step in transcription from amino acid promoters is consistent with previous reports that attenuation of the his operon is not affected by ppGpp,18,28 that addition of ppGpp to cell-free transcription-translation assays does not affect PhisG or Plac expression if added post-transcriptionally,17,79 and that ppGpp does not affect maturation and activity of the ArgI and ArgF enzymes in vitro.47 Although one report concluded that ppGpp stimulates translation of the argCBH operon,80 on balance it seems unlikely that ppGpp is required for steps in amino acid biosynthesis

681

Effects of ppGpp on Transcription Initiation

after transcription initiation, although we have not rigorously excluded this possibility. The reduction in transcription initiation from certain amino acid promoters in
relA
spoT strains and the resulting underproduction of those amino acids could explain the observed amino acid polyauxotrophy. Alternatively, particular metabolic imbalances resulting from the underexpression of particular operons could be responsible for the polyauxotrophy. For example, by underproducing only some enzymes in a biosynthetic pathway,
relA
spoT strains could theoretically overproduce an amino acid precursor that is toxic in high abundance. Supplementation of the amino acid would shut off that pathway, relieving toxicity. Our experiments do not address why strains lacking ppGpp are auxotrophic for only certain amino acids and not for others. Some amino acid promoters simply may not require ppGpp for expression.
relA strains are not amino acid auxotrophs, but they have unusual growth sensitivities in media containing certain amino acid combinations, and they are hypersensitive to a variety of amino acid analogs.17,81 ± 83 The small reduction in ppGpp levels resulting from loss of relA alone leads to only slightly reduced amino acid promoter activity compared to the wild-type strain (data not shown). Nevertheless, this reduction in promoter activity could be suf®cient to explain the observed sensitivities of
relA strains. ppGpp-dependent stimulation of amino acid promoters may occur indirectly Since we did not observe stimulation of amino acid promoter activity by ppGpp in vitro with puri®ed RNAP, we propose that ppGpp stimulates amino acid promoters indirectly. Stimulation could result from effects of ppGpp on expression of one or more regulators, from effects of ppGpp in conjunction with a factor not present in the in vitro transcription reaction, or from indirect effects of ppGpp that do not involve other factors (see the accompanying paper). Since relA and spoT affect the abundance of a very large number of proteins in the cell,84 ± 87 we examined the potential effects on amino acid promoters of two such regulators, sS (the stationary phase sigma factor) and Lrp (leucine-responsive protein), both of whose expression was reported previously to be stimulated by ppGpp in vivo.88,89 Strains lacking rpoS or lrp did not exhibit either reduced amino acid promoter activity or reduced regulation (data not shown). We conclude that a requirement for ppGpp for expression of sS or Lrp cannot account for ppGpp's effects on transcription from amino acid promoters. Our kinetic analysis indicates that amino acid promoters have two characteristics that correlate with positive control by ppGpp. First, they form very long-lived complexes with RNAP (Table 1); as a result, the reduction in open complex half-life

caused by ppGpp does not reduce transcripton from these promoters (Table 1). Second, amino acid promoters associate with RNAP very slowly (Figure 6 and data not shown) and they require relatively high concentrations of RNAP for maximal transcription in vitro and in vivo.55 Although the data presented here do not de®ne the mechanism responsible for the positive effect of ppGpp on transcription initiation from amino acid promoters, it likely involves increasing the RNAP association rate. Models for stimulation of amino acid promoters are evaluated further in the accompanying paper.

Materials and Methods Strain constructions Strains and plasmids are listed in Tables 3 and 4, respectively. The PargI, PlysC, PpheA, PthrABC, and rrnB P1 promoters were generated by PCR from the Escherichia coli chromosome. PhisG and PlivJ promoters were constructed by extending overlapping synthetic oligonucleotides with Sequenase (US Biochemicals). Promoter fragments were generated with EcoRI sites upstream and HindIII sites downstream of the promoter sequences, ligated into pRLG770, and then cloned into bacteriophage l to form promoter-lacZ fusions as described.90 RLG4418, RLG3739, and RLG5651 are monolysogens containing ``system I'' promoter-lacZ fusions and all other monolysogens contain ``system II'' promoter-lacZ fusions.90 For comparison, the same promoter produces approximately tenfold less b-galactosidase activity in system I than in system II. Two fusion systems were used because system II fusions containing very strong promoters are lethal or reduce cell growth and because the background activity in system I fusions is too high for accurate analysis of weak promoters. Transductions of relA, spoT, rpoS, lrp, and argR mutations were performed with phage P1vir91 using the donor strains listed in Table 3.
relA
spoT strains were veri®ed as amino acid auxotrophs. argR transductants were selected in minimal medium for resistance to trimethoprim (2 mg/ml) and screened in minimal medium for resistance to canavanine (100 mg/ml). PargI activity increased 400-fold in an argR strain, but PargI activity was dependent on the presence of ppGpp and increased inversely with growth-rate in both argR ‡ and argR backgrounds. b -Galactosidase activity assays Cells were grown at 30  C in Brain-Heart Infusion (Difco), LB, or M9 minimal medium91 containing 0.4 % (w/v) glucose, glycerol, or succinate, with 20 amino acids (80 mg/ml each) or 0.8 % (v/v) Casamino acids (Difco) plus tryptophan (40 mg/ml). Liquid cultures were inoculated to an A600 of about 0.02 from colonies on agar containing the exact same medium (to avoid upshifts or downshifts). Cultures were grown for about 4 generations to an A600 of approximately 0.35, harvested, lysed,33 and b-galactosidase activity was measured as described.91 Similar results were obtained for both wildtype and
relA
spoT strains when b-galactosidase activities were normalized to protein concentrations (as in the RNA/protein determinations described below) rather than to the A600. Since suppressors accumulate fre-

682

Effects of ppGpp on Transcription Initiation

Table 3. Bacterial strains Name

Genotype

Source

‡

RLG3499ˆMG1655 pyrE lacI lacZ RLG4975, argR::fol RLG857ˆ relA251::kan spoT207::cam RLG3237ˆrpoS::Tn10 RLG6008ˆW3110 lrp-201::Tn10 VH1000 lRLG4814 (contains argI ( 45‡32)-lacZ) RLG4814 argR::fol RLG4978 relA251 spoT207 VH1000 lRLG4418 (contains his ( 60‡16)-lacZ) RLG4418 relA251 spoT207 VH1000 lRLG4422 (contains livJ ( 60‡13)-lacZ) RLG4422 relA251 spoT207 RLG4422 rpoS::Tn10 RLG4422, lrp-201::Tn10 VH1000 lRLG4816 (contains lysC ( 59‡18)-lacZ) RLG4816 relA251 spoT207 VH1000 lRLG4818 (contains pheA ( 73‡10)-lacZ) RLG4818 relA251 spoT207 VH1000 lRLG5080 (contains thrABC ( 72‡1)-lacZ) RLG5080 relA251 spoT207 VH1000 lRLG4819 (contains thrABC ( 72‡16)-lacZ) RLG4819 relA251 spoT207 RLG4819 rpoS::Tn10 RLG4819, lrp-201::Tn10 VH1000 lRLG1368 (contains lacUV5 ( 59‡36)-lacZ) RLG4998 relA251 spoT207 VH1000 lRLG5493 (contains thrABC ( 72‡16) ( 4-6GCC)-lacZ) RLG5493 relA251 spoT207 VH1000 lRLG3739 (contains rrnB P1 ( 66‡9)-lacZ) RLG3739 relA251 spoT207 VH1000 lRLG5651 (contains rrnB P1 ( 66‡9) ( 5-7ATA)-lacZ) RLG5651 relA251 spoT207

VH1000 DS956 CF1693 UM122 BE1 RLG4814 RLG4978 RLG4984 RLG4418 RLG4432 RLG4422 RLG4434 RLG6048 RLG6043 RLG4816 RLG4828 RLG4818 RLG4830 RLG5080 RLG5090 RLG4819 RLG4831 RLG6101 RLG6046 RLG4998 RLG5066 RLG5493 RLG5661 RLG3739 RLG6104 RLG5651 RLG5663

quently,
relA
spoT cultures were never inoculated from liquid cultures, only from freshly grown colonies streaked from frozen stocks, and colony morphology and culture growth-rate were monitored closely. RNA/protein determinations Cells were grown as above and cooled on ice. Cells (14 ml) were pelleted by centrifugation, washed with 7 ml of 15 mM Tris (pH 8.0), 4 mM EDTA, 1 mM DTT, resuspended in 7 ml of the same buffer, and the A600

V.J. Hernandez

48 9

R. Johnson R. Matthews This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work

was measured. After sonication, the RNA/protein ratio and b-galactosidase activity were measured at least in duplicate as described.10 ppGpp purification ppGpp was prepared as a lithium salt from GDP, ATP, and a crude ribosome preparation as described92 and analyzed by UV absorption and thin-layer chromatography.93 Since GDP rather than GTP was used as the substrate, the predominant product was ppGpp

Table 4. Plasmids Name

Description

Source

pRLG770 pRLG589 pRLG862 pRLG2989 pRLG2222 pRLG5069 pRLG5073 pRLG3266 pRLG2983 pRLG4414 pRLG4416 pRLG5071 pRLG5072 pRLG5161 pRLG1507 pRLG1512 pRLG4264 pRLG5196

General transcription vector pRLG770 containing rrnB P1 ( 88‡50) pRLG770 containing rrnB P1 ( 88‡1) PRLG770 containing rrnB P1 (CGC-5-7ATA) ( 88‡1) pRLG770 containing lacUV5 ( 48‡40) pRLG770 containing argI ( 45‡32) pRLG770 containing thrABC ( 72‡16) pRLG770 containing rrnD P1 ( 60‡1) pRLG770 containing lPL ( 1313‡2) pRLG770 containing his ( 60‡16) pRLG770 containing livJ ( 60‡13) pRLG770 containing lysC ( 59‡18) pRLG770 containing pheA ( 73‡10) pDS3 contains N25antiDSR ( 280‡95) pRLG770 with multiple cloning sites pRLG1507 containing rrnB P1 ( 61‡50) pRLG1507 containing lacUV5 ( 48‡40) pRLG1507 containing argI ( 45‡32)

100 100 101 2 100

This work This work

102 2

This This This This

62

work work work work

57 90

S. Aiyar & R.L.G. This work

683

Effects of ppGpp on Transcription Initiation rather than pppGpp. The puri®ed ppGpp was distinguishable from other purine nucleotides (GDP, GTP, AMP, ATP), and indistinguishable from a sample of ppGpp prepared elsewhere (a gift from Mitchell Singer, UC-Davis). ppGpp aliquots were stored dessicated at 80  C and resuspended in water to 10 mM as needed. We estimated 4 Li atoms per ppGpp molecule by conductivity. Therefore, all control reactions were performed with fourfold more LiCl than ppGpp (although no effect of LiCl compared to water was detected on any transcription step). Multiple round in vitro transcription RNAP (Es70) was a generous gift from R. Landick. The enzyme was puri®ed according to Burgess & Jendrisak94 and was 60(10) % active in binding to the lPR promoter.64 All RNAP concentrations reported are active concentrations. Plasmid DNA was isolated on Qiagen-100 columns, extracted twice with phenol/chloroform, ethanol-precipitated, and resuspended in 10 mM Tris-Cl (pH 8.0). Multiple-round transcription was started by addition of 5-25 nM RNAP to supercoiled plasmid templates (0.8-1 nM) in 40 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA, 30220 mM NaCl as indicated, 1 mM ATP, 200 mM CTP and GTP, 10 mM UTP, and [a-32P]UTP (2 mCi) in 10 ml reactions at 30  C. The indicated amount of ppGpp was added to reactions. Transcription was stopped 15 minutes after RNAP addition with an equal volume of formamide loading buffer. Samples were electrophoresed on 7 M urea/5 % polyacrylamide gels, and the dried gels were visualized and quanti®ed by phosphorimaging (ImageQuant Software, Molecular Dynamics). Lifetime of open complexes Open complex lifetime was measured on supercoiled templates using a transcription assay.57 RNAP and plasmid DNA were incubated 30 minutes at 30  C in transcription buffer with the indicated concentration of NaCl, and with 1 mM ppGpp. Aliquots (10 ml) were removed to a tube containing 1.5 ml of NTPs (®nal concentration 1 mM ATP, 200 mM CTP and GTP, 10 mM UTP, and [a-32P]UTP (5 mCi)) at different times after heparin (50 mg/ml ®nal) addition. Transcription reactions were stopped after ten minutes and analyzed as described above. Control experiments indicated that RNAP remained active throughout the time-course. The fraction of complexes remaining was plotted versus time using unweighted linear regression (Sigmaplot, Jandel Scienti®c). Plots resulted in straight lines that intersected the y-axis at or near 1.0, indicating that the observed half-life was for a single species for each promoter. Open complex lifetime on linear templates was measured by ®lter binding.57 For PargI and lacUV5, 10 nM RNAP was incubated with 0.1 nM 32P endlabeled template for 30-90 minutes in transcription buffer, 100 mM NaCl, and 200 mM ppGpp at 25  C. Since rrnB P1 does not form long-lived complexes on linear DNAs, RNAP was bound in the presence of 500 mM ATP and 50 mM CTP for 30 minutes in transcription buffer containing 30 mM KCl. In the presence of ATP and CTP, rrnB P1 forms a long-lived complex,56,95 and the reported half-life for rrnB P1 on a linear fragment (Table 2) is for this long-lived complex. Aliquots (50 ml) were removed at the indicated times after heparin (50 mg/ml ®nal) addition, ®ltered, and washed (1 ml

10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 100 mM NaCl for PargI and lacUV5 or 30 mM KCl for rrnB P1). The [32P]DNA retained on the ®lter (BA85, Schleicher & Ï erenkov counting, corrected Schuell) was quanti®ed by C for background retained in the absence of RNAP (<3 % of input), and the fraction of complexes remaining was plotted versus time as above. Promoter escape The rate of promoter escape was measured according to Hsu.96 Linear templates containing promoter and ¯anking vector sequences were generated by PCR from plasmid templates, puri®ed on Qiaquick PCR Puri®cation columns, eluted in 10 mM Tris (pH 8.0), and quanitifed spectrophotometrically. RNAP (50 nM) was prebound to 20 nM linear template at 30  C for one hour in transcription buffer containing 50 mM NaCl and 200 mM ppGpp. NTPs and heparin were added to (®nal concentrations) 50 mg/ml heparin, 100mM ATP and GTP, 10 mM CTP and UTP, [a-32P]CTP (5 mCi) and/or 5 mCi of [a-32P]UTP), and 5 ml aliquots were removed to tubes containing 5 ml of formamide loading buffer at the indicated times. Reactions were heated for four minutes at 90  C, cooled on ice, and electrophoresed on pre-run 7 M urea/17 % (acrylamide to bisacrylamide, 19:1, w/w) gels with 0.5 TBE top buffer and 0.67 TBE 1 M sodium acetate bottom buffer. Gels were visualized and quanti®ed by phosphorimaging. Association kinetics Association kinetics were measured by performing tplots63 using the nitrocellulose ®lter-binding assay.64 lacUV5 and PargI were measured under identical conditions. In order to measure the effect of ppGpp on association of RNAP with the rrnB P1 promoter, which forms only short-lived complexes, we used a lower salt concentration and added ATP and CTP.56 It is valid to compare rate constants obtained in the presence or absence of ppGpp on a single promoter or between PargI and lacUV5, but because of the differences in solution conditions, it is not valid to compare the absolute values of the rate constants obtained for PargI and lacUV5 with rrnB P1. Restriction fragments containing promoter and ¯anking vector sequences were excised from pSL6 derivatives (see Table 4) with XhoI, extracted twice with phenol/ chloroform, isolated from 3 % Nusieve agarose gels (FMC) using a Qiaquick Gel Extraction Kit (Qiagen), precipitated with ethanol, and resuspended in 10 mM Tris (pH 8.0). Fragments were end-labeled with Sequenase (US Biochemicals) and [a-32P]TTP, ®lled-in with dNTPs, extracted with phenol/chloroform, puri®ed using a G25 Sephadex Quickspin column (Boehringer Mannheim), and [32P]DNA concentrations were estimated on agarose gels by comparison to standards. Association rates of RNAP with promoter DNA fragments were measured in transcription buffer containing 100 mM NaCl at 30  C (or in 30 mM KCl with 500 mM ATP and 50 mM CTP at 25  C for rrnB P1), and 200 mM ppGpp. Aliquots (50 ml) were removed, added to 5 ml heparin (50 mg/ml ®nal), mixed, ®ltered after ten seconds in the presence of heparin, washed, and quantiÏ erenkov counting as described above. The ®ed by C RNAP concentration was always at least ®vefold in excess of the DNA fragment concentration. Comparisons

684 in the presence or absence of ppGpp were always performed concurrently using the same RNAP dilutions. After background subtraction (<3 % of input), the fraction of DNA retained on the ®lter was plotted versus time. A single association rate kobs and multiple plateau levels (fraction of DNA bound at equilibrium) were simultaneously ®t from two to ®ve independent measurements for each RNAP concentration using a weighted non-linear least-squares analysis, NONLIN.97 Traditional t-plot analysis requires that open complex formation goes to completion (i.e. near 100 % promoter occupancy). Plateau values for maximum occupancy were generally 0.80  0.05. kobs was corrected for fractional occupancy.98 We note that this correction was crucial for accurate estimation of ka and ki and was not used in previous analyses of the effects of ppGpp. The associated error was determined by weighted non-linear least-squares analysis99 using Sigmaplot (Jandel Scienti®c). The RNAP complex with rrnB P1 contains ATP and CTP56,95, and therefore NTP binding steps make a slight contribution to ki.

Acknowledgments We thank Wilma Ross, Bob Landick, Oleg Tsodikov, Tom Record, Mike Bartlett, Dick D'Ari, and Irina Artsimovitch for insightful discussions and/or comments on the manuscript, Oleg Tsodikov for advice on t-plot analysis, Reid Johnson, David Sherratt, Rowena Matthews, and Lillian Hsu for strains or plasmids, Erin Espelie for construction and analysis of one of the promoters, John Keener for preparation of the ribosomes used in the ppGpp isolation, and Mitchell Singer and Dan Gentry for advice on ppGpp puri®cation. This work was supported by N.I.H. grant GM37048 to R.L.G., an N.I.H. Molecular Biosciences Predoctoral Traineeship to M.M.B., and a fellowship from P®zer Biotechnology to M.M.B.

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Effects of ppGpp on Transcription Initiation

73.

74. 75.

76. 77.

78. 79.

80.

81. 82. 83.

84. 85.

86.

87. 88.

89. 90.

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687

Effects of ppGpp on Transcription Initiation that dramatically increases promoter strength. J. Mol. Biol. 235, 1421-1435. 91. Miller, J. H. (1972). Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 92. Cashel, M. (1974). Preparation of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) from Escherichia coli ribosomes. Anal. Biochem. 57, 100-107. 93. Cashel, M. & Kalbacher, B. (1970). The control of ribonucleic acid synthesis in Escherichia coli. V. Characterization of a nucleotide associated with the stringent response. J. Biol. Chem. 245, 2309-2318. 94. Burgess, R. R. & Jendrisak, J. J. (1975). A procedure for the rapid, large-scale puri®cation of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry, 14, 4634-4638. 95. Borukhov, S., Sagitov, V., Josaitis, C. A., Gourse, R. L. & Goldfarb, A. (1993). Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli. J. Biol. Chem. 268, 23477-23482. 96. Hsu, L. M. (1996). Quantitative parameters for promoter clearance. Methods Enzymol. 273, 59-71. 97. Johnson, M. L. & Frasier, S. G. (1985). Nonlinear least-squares analysis. Methods Enzymol. 117, 301342. 98. Tsodikov, O. V. & Record, M. T., Jr (1999). General method of analysis of kinetic equations for multistep reversible mechanisms in the single-exponential regime: application to kinetics of open complex formation between Es70 RNA polymerase and lambda PR promoter DNA. Biophys. J. 76, 1320-1329. 99. Record, M. T. J., Ha, J.-H. & Fisher, M. A. (1991). Use of equilibrium and kinetic measurements to determine the thermodynamic origins of stability and speci®city and the mechanism of formation of site speci®c complexes between proteins and helical DNA. Methods Enzymol. 208, 291-343. 100. Ross, W., Thompson, J. F., Newlands, J. T. & Gourse, R. L. (1990). E. coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 9, 3733-3742. 101. Ross, W., Gosink, K. K., Salomon, J., Igarashi, K., Zou, C., Ishihama, A., Severinov, K. & Gourse, R. L. (1993). A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science, 262, 1407-1413. 102. Ross, W., Aiyar, S. E., Salomon, J. & Gourse, R. L. (1998). Escherichia coli promoters with UP elements of different strengths: modular structure of bacterial promoters. J. Bacteriol. 180, 5375-5383.

Appendix Interpretation of Kinetic Parameters In addition to decreasing the half-life of open complexes formed at all promoters tested two- to threefold, ppGpp decreased the composite isomerization rate constant ki of rrnB P1 two- to threefold. ppGpp did not decrease ki of the other promoters examined. It is possible that ppGpp affects two different elementary rate constants in rrnB P1 by the same amount, one contributing to the open complex half-life and another to ki, but it is more likely that ppGpp affects only one rate constant. Here, we propose that ppGpp decreases an

elementary rate constant, de®ned below as k 2, that contributes to the half-life of the open complex, and we propose that this elementary rate constant also contributes to ki in the unusual initiation mechanism of rrnB P1. We suggest that ppGpp affects k 2 in the same way at all promoters, but its overall effect on transcription varies with the kinetic parameters of the particular promoter. In the three-step mechanism,A1,A2 RNAP forms two kinetically signi®cant intermediates I1 and I2 before forming the open complex RPo: k1

k2

k3

kNTP

k

k

k

k

R ‡ P „ I1 „ I2 „ RPo „ RPNTP 1

2

3

NTP

The rate of dissociation from the open complex can be described by an observed composite dissociation rate constant kd. In the multistep transcription initiation mechanism, kd is not equivalent to the off-rate from the closed complex, k 1. The following descriptions of (A1) kd and (A2) ki are derived for this three-step general mechanism without any assumptions about which steps are rate-limiting:A3 1=kd ˆ 1=k

3

‡ …1 ‡ K3 †=k

2

‡ K2 …1 ‡ K3 †=k

1

‡ 1=k …A1†

1=ki ˆ 1=k2 ‡ 1=…k

3

‡ k3 † ‡ 1=‰K2 …k

3

‡ k3 †Š …A2†

where K2 ˆ k2/k 2 and K3 ˆ k3/k 3 are the equilibrium constants of the isomerization steps. Equations (A1) and (A2) can be applied to any promoter, including rrnB P1, as long as the observed kinetics are single-exponential. These expressions for kd and ki can be simpli®ed for lPRA3 and lacUV5A2 under standard conditions based on the nature of the rate-limiting steps at these promoters: 1=kd ˆ …1 ‡ K3 †=k 1=ki ˆ 1=k2

2

…A3† …A4†

Formulae (A3) and (A4) cannot be used to describe the mechanism of rrnB P1, since rrnB P1 does not form a long-lived RPo and the mechanism of initiation at rrnB P1 may not have the same ratelimiting steps as lPR and lacUV5. Although kd and ki both depend on some of the same elementary rate constants (k2, k 2, k3, and k 3) in the more general equations (A1) and (A2), the simpli®ed versions of kd and ki (equations (A3) and (A4)) contain only one term each from equations (A1) and (A2) and do not include the same elementary rate constants. Effect of ppGpp on lacUV5 For lacUV5, under standard conditions the simple expressions of kd (equation (A3)) and ki

1

688

Effects of ppGpp on Transcription Initiation

(equation (A4)) apply.A2 Since ppGpp increases kd for lacUV5, ppGpp must affect one or more of the three contributing rate constants, k 2, k3, or k 3. In order for ppGpp to increase the rate of kd, ppGpp must: (a) increase k 3; (b) decrease k3; or (c) increase k 2. ppGpp does not affect ki for lacUV5, so ppGpp does not affect k2. Effect of ppGpp on rrnB P1 ppGpp increases kd and decreases ki for rrnB P1 (Table 2). (a) According to equation (A2), if ppGpp increased the elementary rate constant k 3, there would be either an increase or no change in the rate of ki (depending on how large k 3 is), but this would be inconsistent with the observation that ppGpp decreases ki. (b) According to equation (A2), if ppGpp decreased k3, there would be either a decrease or no change in the rate of ki, consistent with the observed effect. (c) According to equation (A2), if ppGpp increased k 2, there would be either a decrease or no change in ki, also consistent with the observed result. Thus, effects of ppGpp on k3 and k 2 are both consistent with a decrease in ki for rrnB P1. ppGpp most likely increases k

2

We suggest that ppGpp most likely affects k 2, although our theoretical analysis cannot rigorously eliminate the possibility that ppGpp affects k3. ppGpp increased kd about threefold for all promoters at different monovalent salt concentrations and on supercoiled and linear templates. Although k 2 likely varies from promoter to promoter, an increase in k 2 in expression (A3) would in almost all cases result in a corresponding increase in kd. k3

also likely varies with promoter sequence and with ionic conditions, but in contrast to the situation with k 2, a decrease in k3 in expression (A3) would not always result in a corresponding increase in kd, since sometimes K3 would be insigni®cant relative to 1. It seems unlikely that ppGpp would affect all promoters similarly and to the same extent under all solution conditions were it to act by decreasing k3, and therefore we suggest that ppGpp acts by increasing k 2. If indeed ppGpp affects k 2 and as a result changes ki and kd by the same magnitude, then we can simplify the general expressions of kd and ki for rrnB P1 to: 1=kd ˆ ‰…1 ‡ K3 †…1 ‡ k2 =k 1 †Šk 1=ki ˆ 1=‰…K2 …k3 ‡ k 3 †Š

2

…A5† …A6†

References A1. Roe, J. H., Burgess, R. R. & Record, M. T., Jr (1984). Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the lambda PR promoter. J. Mol. Biol. 176, 495-522. A2. Buc, H. & McClure, W. R. (1985). Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps. Biochemistry, 24, 2712-2723. A3. Tsodikov, O. V. & Record, M. T., Jr (1999). General method of analysis of kinetic equations for multistep reversible mechanisms in the single-exponential regime: application to kinetics of open complex formation between Es70 RNA polymerase and lambda PR promoter DNA. Biophys. J. 76, 1320-1329.

Edited by R. Ebright (Received 16 August 2000; received in revised form 8 November 2000; accepted 8 November 2000)