JouRNAL OF BACTERIOLOGY, Aug. 1992, p. 5156-5160 0021-9193/92/155156-05$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 174, No. 15

Activation Defects Caused by Mutations in Escherichia coli rpoA Are Promoter Specific GARY N. GUSSIN,1* CAROL OLSON,1 KAZUHIKO IGARASHI,2 AND AKIRA ISHIHAMA2 Biology Department, University of Iowa, Iowa City, Iowa 52242,1 and Department oI Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan Received 3 March 1992/Accepted 1 June 1992

Escherchia coli RNA polymerases containing mutated a subunits were tested for their ability to respond to three different positive regulators (activators) in vitro. The two a (rpoA) mutants, a-256 and a-235, have deletions of the C-terminal 73 and 94 amino acids, respectively. In runoff transcription assays catalyzed by reconstituted holoenzyme, the effects of the mutations on each of three promoters tested were different: activation of the XpRm promoter by cI protein (repressor) was nearly normal, activation of the XpRE promoter by cI protein was reduced approximately fivefold, and direct activation of the trpPB promoter of Pseudomonas aeruginosa was completely inhibited. We also found that the reconstituted mutant enzyme was defective in recognition of trpPI in the absence of activator. The differential responses of the three promoters to their activators in the presence of the mutant enzymes indicate that the location of an activator-binding site does not by itself determine the region of RNA polymerase with which the activator interacts.

Recently, several groups have reported evidence implicating the a subunit of Escherichia coli RNA polymerase (RNAP) in positive control of transcription (13, 20, 21, 23, 26). Since at least some activators appear to interact directly with RNAP (18, 27), it is possible that activation is mediated by direct contact between particular activators and a. The simplest explanation of the available data would be that the contact point is in the C-terminal third of a, since most rpoA mutations that cause defects in activation are located in this region. However, since RNAP is a multisubunit enzyme (a%p'o%), indirect effects of the mutations on RNAP function cannot be excluded. The effects of two particular mutant subunits, a-235 and a-256, which have deletions of the C-terminal 94 and 73 amino acids, respectively, have been tested extensively in vitro, by using RNAP holoenzyme reconstituted with purified mutant a protein (20, 21). Transcription catalyzed by the mutant enzymes was activated normally when cyclic AMPcatabolite gene activator protein (CAP) was bound near the -35 region of the galPi promoter at a site whose midpoint is at -41.5 (41.5 nucleotides preceding the transcription start site) but not when the activator was bound upstream from the lacPl promoter at a site with a midpoint at -61.5. Extension of these studies to several other activators led to the hypothesis that activators bound close to the -35 regions of their target promoters would, in general, contact RNAP in a region not affected by the a deletions, while activators bound farther upstream would contact the region removed by the deletions (20, 21). To test this hypothesis, we examined activation of three promoters: (i) X pRm, which is activated by binding of cI protein (repressor) to OR2 (24); (ii) XpRE, which is activated by binding of cII protein to two TTGC sequences that flank the -35 region (29); and (iii) the Pseudomonas aeruginosa promoter, trpPB, which is activated by TrpI protein bound to recognition site II, the weaker of two TrpI recognition sites, in the presence of indoleglycerol phosphate (InGP) (5, 6, 9). With respect to the corresponding transcription start sites, *

Corresponding author. 5156

the three activator-binding sites are centered at -42, -32.5, and -42, respectively. We found that the three promoters responded differently to the deletion mutant RNAPs in the presence of their activators: activation of PRM was nearly normal, activation of PRE was greatly diminished, and activation of trpPB was abolished. Since the binding sites for TrpB and cI protein (repressor) are identically located with respect to their target promoters, the position of the activator-binding site cannot by itself be used to predict the region of RNAP that will be contacted by the activator. Transcription in vitro. Runoff transcription assays have, been described previously (8). Activator proteins, when present, were allowed to incubate with the DNA template (1.5 to 2 nM, as indicated) for 10 min, following which RNAP holoenzyme (final concentration of active enzyme, 15 nM) was added for an additional 5 to 30 min to allow open complexes to form. Nucleoside triphosphates and heparin (50 ,ug/ml) were then added, and transcription was allowed to proceed for 15 min. Reactions were carried out at 37°C. Samples were analyzed by electrophoresis in 7 M urea-6% polyacrylamide gels; autoradiograms were scanned on a Bio-Rad video densitometer. Pure repressor was a gift from R. T. Sauer; CII and TrpI were purified in the laboratory of G. N. Gussin by J. Ferm and J. Gao, respectively (5, 25). Final concentrations of activators (TrpI, 4.0 ,ug/ml; CII, 3.1 ,ug/ml; repressor, 7.2 ,ug/ml) were the minimal concentrations that yielded maximal transcription with the native holoenzyme. RNAP. The native RNAP holoenzyme was purified according to the procedure of Burgess and Jendrisak (3), with minor modifications. Wild-type and mutant holoenzymes were also reconstituted from dissociated subunits to produce core enzymes, which were then incubated with a fourfold excess of purified sigma subunit to produce holoenzyme (21). Effects of mutant holoenzymes on activation. Since transcription from each promoter was assayed in the presence of heparin, initiation was limited to one round from each template molecule. Thus, in each case, the synthesis of radioactive RNA is a measure of the number of open complexes formed during a specified time (usually 10 min) in

NOTES

VOL. 174, 1992

native wt

reconstituted wt

5157

TABLE 1. Activity of mutant enzymes in response to specific activators of PRM and PRE'

a-256 a-235

Holoenzyme activity

Reconstituted

Promoter

Native Wild type

rpoA256

rpoA235

7.4 1.1

7.2 0.8

5.8 0.6

6.9 0.7

2.9

2.3

2.9 1.0

2.3 0.8

1.4

2.3

11.6 9.9 9.0 0.91 0.70

8.6 10.0 13.0 1.3 1.0

7.4 8.0 2.2 0.28 0.21

7.6 9.1 1.9 0.21 0.16

P+ RM

PR

PR

PRM PRM + CI Activation ratiob Relative activation

FIG. 1. Activation of pRM in the presence of mutant RNAP. Activation of transcription from PRM in vitro was assayed in the presence (+) or absence (-) of repressor, as outlined in the text. A 194-bp XAluI fragment containingpRm andpR was inserted between two strong transcription terminators in plasmid pSW305, constructed by S. Woody in our laboratory. The resulting recombinant, pWF-6, was cleaved at flanking PvuII and SmaI sites to yield a 1,065-bp template for synthesis of terminated transcripts which were 265 and 363 nucleotides long from PR and PRM, respectively. The final template concentration was 2 nM.

which RNAP and DNA were incubated prior to the addition of substrate nucleoside triphosphates and heparin. To standardize conditions, we titrated each RNAP preparation on a DNA fragment containing only one strong promoter, the late promoterPR' of bacteriophage X. The estimated fractions of active enzyme were 40% for purified wild-type holoenzyme (not reconstituted), 15% for reconstituted wild-type holoenzyme, and 5% for the reconstituted mutant enzymes. In each experiment, the concentrations of active enzyme were the same for all enzyme preparations. Activation of pRM. Assays of X PRM activity were performed at 0.1 M KCI, which preferentially decreases basal activity in the absence of cI protein. The DNA fragment used also contains the major lambda lytic promoterPR. The results of a typical experiment are presented in Fig. 1. In the absence of repressor (lanes labeled -), PR forms open complexes efficiently, but PRM is only weakly expressed. In the presence of repressor, PR is repressed and

PRM

is

activated (lanes labeled +). Figure 1 demonstrates that both the basal and activated levels of activity Of PRM are approximately the same for reconstituted wild-type and a-235 mutant RNAP, while the activity of the reconstituted a-256 holoenzyme is somewhat lower than that of the other enzyme preparations. Table 1 lists the results of densitometric quantification of the data for one experiment. The ratios of activities in the presence and absence of cI protein (activation ratios) were approximately 2.6, 2.9, 2.3, and 3.3 for the purified RNAP holoenzyme, reconstituted wild-type enzyme, reconstituted a-256, and reconstituted a-235 mutant enzyme, respectively. Thus, in spite of the somewhat lower basal activity of the a-256 enzyme, it responded nearly as well to the activator as did wild-type RNAP. To test whether the ability of the mutant enzymes to be activated was dependent on the presence of excess RNAP, one experiment was performed with 3 to 4 nM instead of 15 nM (active) enzyme. However, even at this low RNAP concentration, the degree of activation by the mutant enzymes was approximately the same as that for reconstituted wild-type RNAP (data not shown). Activation of pRE. Similar experiments were performed with X PRE (Fig. 2). In this case, we used 0.05 M KC1 to increase the rate of open complex formation (25). Neverthe-

ratio" POOP POOP + CII pRE + CIld Normalized activity' Relative normalized activityf

2.6 0.9

3.3 1.1

a Activity was measured densitometrically in arbitrary units. b Values for PRM + CI divided by those for PRM in the absence of CI. c Activation ratio normalized with respect to the ratio for reconstituted wild-type RNAP. d Material in the band designated by the upper arrow in Fig. 2 has been ignored. Therefore, the value for the native enzyme, for which there was a higher proportion of material in the upper band, is underestimated. If this band represents a transcript that is initiated atpRE and proceeds to the end of a dimeric template, the transcript would be 950 nucleotides long. On a molar basis, the fractions of RNA molecules in thepRE band would be 0.81 and 0.95 for the native and reconstituted wild-type enzymes, respectively. I Normalized activity: values forpRE + CII divided by those forp"p + CII. f Normalized activity divided by the activity for reconstituted wild-type RNAP.

less, because of the relatively low RNAP concentration (15 nM), there was only a barely detectable signal from PRE in the absence of cII protein (lanes labeled -). When cII protein was added, two new major bands appeared. One

(PRE) corresponds in position to that expected for the truncated transcript produced by synthesis to the end of the linear template; the other (upper arrow in Fig. 2) is at the position of a longer cIT-dependent transcript that may result from dimerization of the template (11). For the native wild-type enzyme, there were roughly equal amounts of the two transcripts; however, for the reconstituted wild-type enzyme, approximately 80% of the cII-dependent material migrated as authenticPRE transcript. Since the ratios of the two transcripts were the same for all three reconstituted enzymes, we ignored the upper band in determining the amount of cII-dependent transcript. Addition of cII protein significantly increasedpRE activity for both wild-type enzymes, but the mutant enzymes were stimulated to a much lesser extent (Table 1). Since the DNA template also contains the Xp., promoter, which should be unaffected by cII protein, densitometer readings were normalized to poop activity. The degree of activation (relative normalized activity), defined as 100% for the reconstituted wild-type enzyme, was about 21% for the ax-256-containing RNAP and 16% for the a-235 enzyme (Table 1). (As mentioned previously, we observed a weak signal [not visible in Fig. 2] from PRE in the absence of cIT protein for all four enzyme preparations. The bands could not be quantified accurately, but visual inspection indicates that the activities of the reconstituted wild-type and ot-256 enzymes were approximately equal and that the activity of the a-235 enzyme was only slightly lower. Therefore, the mutant enzymes appear to be defective primarily in their response to the cII protein rather than in their ability to recognize PRE.)

5158

NOTES

J. BACTE;RIOL.

native wt

-

reconstituted wt

ax-256 ax-235

+

+- -

-4-

'PRE

P4-op FIG. 2. Activation of pRE in the presence of mutant RNAP. A 651-bp BglII fragment containingpRE was subcloned into the BamHI site of pKlOON (12) to yield plasmid pSB200; cleavage at flanking EcoRI and HindlIl sites yielded a 683-bp fragment that was used as template (final concentration, 1.5 nM). The template produced a 77-nucleotide transcript fromp.p and a runoff transcript which was 267 nucleotides long fromPRE. The upper arrow indicates a longer cII-dependent transcript that may arise through dimerization of the template.

Activation of trpP.. The effects of the a subunit deletions on tipPB activation by TrpI and InGP were much more dramatic (Fig. 3a). As for PRE, these experiments were performed at 0.05 M KCl. For both native and reconstituted wild-type enzyme, there was a weak signal from the closely linked and divergent tipP, promoter in the absence of TrpI

(a) native wt

reconstituted wt a-256 a-235

protein. Addition of TrpI repressed tbpP1 and activated trpPB, as is expected since the TrpI recognition sites I and II overlap t-pP, (4). However, following a 10-min preincubation, the mutant enzymes yielded virtually no signal either from trpP, in the absence of activator or from trpPB whether or not activator was present. Previous results indicated that the reconstituted mutant enzymes form open complexes less efficiently than the wild-type enzyme at weak promoters (20, 21). In fact, the two tip promoters are very poor matches to the E. coli consensus promoter (see reference 8), and therefore, it is not surprising that they are poorly recognized by the mutant RNAP preparations. To enhance the signal from these promoters, we repeated the experiment and exposed X-ray film to the polyacrylamide gel for 3 weeks (instead of 6 to 34 h). Under these conditions (Fig. 3b), trpPB (in contrast to trpP,) is recognized equally well by all three reconstituted enzymes in the absence of TrpI and InGP, but it is very poorly activated by the mutant enzymes in the presence of TrpI and InGP. The key point is that in the absence of TrpI and InGP, the intensity of the trpPB-specific band is approximately the same for all three reconstituted enzyme preparations, but the intensity of the corresponding band in the presence of activator is much greater for the wild-type enzyme than for the mutant enzymes. The small increase in intensity of the tipPB band on addition of TrpI and InGP in experiments with the mutant enzymes is an indirect effect due to the overlap between the two promoters (6). That is, TrpI can indirectly activate tipPB by eliminating interference from open complexes formed at tipP1 (9, 10). Thus, at trpPB there is no direct activation of the mutant enzymes by TrpI and InGP. Because of the long exposure time, we did not quantify these data. We attempted to increase the level of transcription by changing the time allowed for open complex formation from 10 to 30 min. However, no increase in transcription was observed, and the results of a single experiment were essentially the same as those obtained previously. Role of the a subunit C terminus in activation. These results indicate that the ability of the mutant RNAPs to initiate transcription in response to specific activators is not simply a function of the position of the activator-binding site.

(b) reconstituted wt a-256 a-235

trpPB -

4- trp PB

trpP_

*-

trpP1

FIG. 3. Activation of trpPB in the presence of mutant RNAP. Experiments were performed in the presence (+) or absence (-) of TrpI protein as outlined in the text. The template (final concentration, 1.5 nM) was a 528-bp EcoRI-HindIII fragment derived from cleavage of the M13 derivative M2100 (10) at sites in the polylinker. Runoff transcription products were 169 and 256 nucleotides long from tipP1 and trpPB, respectively. The data shown were from two separate experiments in which the X-ray film was developed after exposure for 12 h (a) or 3 weeks (b).

VOL. 174, 1992

The positions of the binding sites for activators of PRM and

trpPB are identical with respect to the two promoters (and

nearly identical to the position of the CAP site relative to galPI). YetPRM (like galPl) is activated virtually normally, while the ability of TrpI and InGP to activate trpPB by direct interaction with the mutant RNAPs is completely abolished. The simplest interpretation of these results is that RNAP possesses two (or more) contact sites for activators, one of which is defined by the rpoA deletions. The ability to contact one or the other activation site on RNAP would depend both on the location of the DNA recognition site for a particular activator and on the structure of the activator. An additional factor may be the sequence of the -35 region of the promoter, which could influence the conformation of RNAP (19). The intermediate response of mutant enzymes to cII protein can be interpreted in either of two ways: (i) the deletions may not remove the entire cII contact site on the ao subunit or (ii) the cII protein may interact with two distinct sites on RNAP. Since the cII-binding site consists of two TTGC sequences that flank the -35 region (29), its contacts with RNAP may be very different from those of the repressor (cI protein) or TrpI. One problem in interpreting these results is that activation may not be mediated in all cases by a direct interaction between an activator protein and RNAP. DNA bending or some other DNA structural change may be induced by the activator (28), in which case bending rather than proteinprotein contacts could be necessary for activation (2). However, there is substantial evidence for RNAP-activator contact in several cases. The main direct evidence is the electron-microscopic visualization of DNA looping mediated by an interaction between RNAP bound at the glnA promoter and NtrC bound to upstream binding sites (27). In addition, there are cI mutations that cause a defect in activation of PRM but do not affect DNA binding (18). The mutations affect a surface of the cI protein that could be expected to interact with RNAP. Similar mutations have now been isolated in CAP (1, 7). In addition, a single amino acid change in a region analogous to that in which the cI mutations are located has been shown to prevent cII-mediated activation of PRE without affecting the ability of cII protein to bind DNA (16). Finally, the binding of several activators (including cI and cII proteins) to their binding sites and of RNAP to the corresponding promoter is synergistic (17, 19, 22). Evidence that the a subunit itself contacts activator proteins is indirect. The best evidence is that rpoA mutations can suppress or be suppressed by mutations in specific activators (12, 24, 27), although a sufficient number of mutually suppressing pairs have not been isolated yet to test the allele specificity of suppression. Additional evidence comes from footprinting studies of synergistic binding of CAP and RNAP at both the lac and gal promoters. When RNAP containing a deleted a subunit is used, binding of CAP and RNAP is synergistic only at galP, not at lacP (22). The correlation between CAP's ability to activate and its ability to interact synergistically with RNAP in the footprinting assay suggests that there is a direct interaction between CAP and the C-terminal region of the a subunit only when CAP is bound upstream from lacP. In the gal case, CAP could interact with some other region of the a subunit or with some other subunit of RNAP. Because our assays depend on the rate of open complex formation at each promoter, the relative activities of the mutant and wild-type enzymes could be expected to vary with the time allowed for open complex formation prior to the addition of substrates.

NOTES

5159

Since the response Of PRE to cII protein was intermediate with the mutant enzymes, experiments with PRE were performed once with preincubation times of 5, 10, and 20 min (data not shown). Unexpectedly, the relative activities of the mutant enzyme in the presence of cII protein were approximately the same at all three preincubation times. These data suggest that the response of the mutant RNAP to activator is heterogeneous: a few promoters appear to be fully activated and able to form open complexes within 5 min, but a large fraction (80 to 85%) are not activated at all. Conceivably, only a fraction of the mutant enzyme molecules are in a conformation that enables them to interact synergistically with cII protein. Finally, it would have been surprising if these rpoA deletions had had no other effect than to prevent certain activators from stimulating RNAP. An effect of the mutations on enzyme stability has been described previously (15). In addition, the mutant reconstituted enzymes are defective in initiation from very weak promoters. Promoters that have been tested with the mutant enzymes include the E. coli trpP, lacUV5, lacP, rpU, tac, uxuAB, galP,pstS, and ompC promoters and the pBR322 P4 and RNA-I promoters (20, 21). With the exception of trpP. (Fig. 3b), the -35 and -10 regions of promoters that are transcribed nearly normally (in the absence of activator) by the mutant enzymes agree much more closely with the consensus sequence (14) than do the corresponding regions of promoters that are transcribed poorly (galPI, lacP2, trpPI,pRE,pstS, and pBR322 P4). It is possible that the mutant enzymes are defective to some extent in transcription initiation at all the promoters tested (including strong promoters likepR and p.p), but the defect may be masked because the preincubation times in our experiments are much longer than the times required for open complex formation at strong promoters. A more detailed kinetic analysis would be required to test this possibility or to reveal more subtle differences in the response of the mutant and wild-type enzymes to each of the activators tested. We thank Raymond Fong for purified wild-type RNAP and DNA fragments. This work was supported by grants-in-aid from the Japanese Ministry of Education, Science and Culture to A.I. and by USPHS grant AI17508 to G.N.G. REFERENCES 1. Bell, A., K. Gaston, R. Williams, K. Chapman, A. Kolb, H. Buc, S. Minchin, J. Williams, and S. Busby. 1990. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription. Nucleic Acids Res. 18:7243-7250. 2. Bracco, L., D. Kotlarz, A. Kolb, S. Diekman, and H. Buc. 1989. Synthetic curved DNA sequences can act as transcriptional activators in Escherichia coli. EMBO J. 8:4289-4296. 3. Burgess, R. R., and J. J. Jendrisak. 1975. A procedure for the rapid large-scale purification of Eschenchia coli RNA polymerase involving polymin P precipitation and DNA cellulose chromatography. Biochemistry 14:4634-4638. 4. Chang, M., and I. P. Crawford. 1990. The roles of indoleglycerol phosphate and the TrpI protein in the expression of trpBA from Pseudomonas aeruginosa. Nucleic Acids Res. 18:979-988. 5. Chang, M., and I. P. Crawford. 1991. In vitro determination of the effect of indoleglycerol phosphate on the interaction of purified TrpI protein with its DNA binding sites. J. Bacteriol. 173:1590-1597. 6. Chang, M., A. Hadero, and I. P. Crawford. 1989. Sequence of the Pseudomonas aeruginosa trpI activator gene and relatedness of trpI to other procaryotic regulatory genes. J. Bacteriol. 171:172-183. 7. Eschenlauer, A. C., and W. S. Reznikoff. 1991. Escherichia coli

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