lac repressor acts by modifying the initial transcribing complex so that it cannot leave the promoter

Cell, Vol. 66, 793-796,

August

23, 1991, Copyright

0 1991 by Cell Press

kc Repressor Acts by Modifying the Initial Transcribing Complex So That It Cannot Leave the Promot6r Jookyung Lee and Alex Goldfarb Department of Microbiology College of Physicians and Surgeons Columbia University New York, New York 10032

Summary RNA polymerase engaged in the joint complex with the lac repressor at the lac UV5 promoter cannot escape into elongation but generates abortive RNA oligomers. The joint complex actively transcrfbes a few initial base pairs in a reaction unusually sensitive to a decrease in the substrate concentration. The joint complex, however, fails to traverse a point in the initial transcribed sequence that normally requires a high concentration of the elongating substrate. Thus, the repressor acts by augmenting a natural high “kNTpnsite (pause site) embedded in the promoter. A lethal RNA polymerase mutation that mimics the effect of the repressor leads to an analogous block of promoter clearance and shortened abortive product pattern on several promoters, reflecting the widespread occurrence of high kNTPsites in promoters. Introduction As the central enzyme of gene expression, DNA-dependent RNA polymerase @NAP) is the ultimate target for a myriad of regulatory mechanisms, all of which modulate the few basic reactions that RNAP performs. Evolutionary conservation of RNAP subunits (recently reviewed in lwabe et al., 1991) indicates that its basic functions and, implicitly, regulatory responses have the same structurefunctional basis in all living organisms. In this work we demonstrate that RNAP catalytic function can serve as a target for negative regulation and describe an RNAP mutation that mimics the regulatory effect of the external factor. The initial steps in the functional cycle of the Escherichia coli RNAP include promoter search and recognition by the holoenzyme (subunit composition a$p’o), formation of the reversible closed complex, and the subsequent isomerization of RNAP into the stable, irreversible open complex, which involves local melting of the DNA double helix (reviewed in Chamberlin, 1974; von Hippel et al., 1984; McClure, 1985). In the presence of the four transcription substrates, the open complex is converted to the initial transcribing complex (ITC) (Krummel and Chamberlin, 1989). At this step, the holoenzyme is stably anchored at the promoter, catalytically generating nested oligomers, up to nine nucleotides in length, in repeated reiterative acts of abortive initiation (Johnston and McClure, 1978; Carpousis and Gralla, 1980, 1985; Krummel and Chamberlin, 1989). The promoter-proximal sequence involved in abortive initiation is known as the initial transcribed se-

quence (ITS). The cyclic abortive reaction WWItUallY ceases as the result of the promoter clearance event, which involves the release of the o factor, relinquishing of the promoter-anchoring contacts, cessation of abortive initiation, and commencement of processive elOngatiOn by the core enzyme (a&Y) (Hansen and McClure, 1980; Carpousis and Gralla, 1985; Straney and Crothers, 1987a; Krummel and Chamberlin, 1989; Metzger et al., 1989). The productive interaction of RNA polymerase with the lac promoter is inhibited by the lac repressor (LacR), which binds to the operator sequence overlapping the promoter site (Jacob and Monod, 1961; Gilbert and Muller-Hill, 1968; Schmitz and Galas, 1979; Miller and Reznikov, 1980). The early in vitro experiments by Pastan and his colleagues (de Crombrugghe et al., 1971; Chen et al., 1971) demonstrated that the LacR-mediated repression could be lifted by adding IPTG, the lac operon inducer, to preformed promoter complexes resistant to rifampicin challenge. This suggested that LacR acts after RNAP has bound to DNA. However, the alternative view that LacR acts by occluding the promoter from RNAP binding (Majors, 1975) has prevailed for nearly two decades. This view was challenged by the work of Straney and Crothers (1987b), who showed that LacR and RNAP coexist on the promoter in a joint nonproductive complex that can be triggered to make RNA by the addition of IPTG. The authors suggested that LacR blocks the closed-to-open complex transition. However, as was noted by Krummel and Chamberlin (1989) the data of Straney and Crothers (1987b) were consistent with an alternative mechanism whereby LacR acted at a later stage, preventing the RNAP escape from the ITC into elongation. To resolve this issue, we examined the properties of promoter complexes formed at the lac UV5 promoter carried on an isolated restriction fragment. Results Heparin-Resistant RNAP-LacR Promoter Responsive to IPTG Induction The initial sequence of the lac transcription

Complex unit is

12345678910 GAAUUGUGAGCGGPPPA + W -, ApA + In the in vitro reaction, transcripts can be initiated either with ATP (pppA) or with dinucleotide primers GpA and ApA. If the reaction is carried out in the absence of CTP, which is first used at position +lO, the transcription is limited to the 9 bp of the ITS, generating abortive products containing only A, U, and G residues. Such conditions are referred to as the “-c” reaction. To determine whether LacR and RNAP can indeed form a dormant promoter complex that would “fire” upon the addition of IPTG, the complexes between RNAP and the

Cell 794

LacR complex, detected heparin sensitive.

Figure

1. Single-Round

Transcription

-

-I-

+

-

-

+

from the lac UV5 Promoter

The 123 bp promoter DNA fragment was preincubated at 37OC for 10 min without (lanes 1) or with (lanes 2 and 3) La&, then RNAP was added followed (after 20 min) by the addition of heparin (to 0.15 mgl ml) and the -C reaction components, which included 1.0 mM primer (ATP in [A] and ApA in [B]) and 50 uM each of unlabeled ATP, GTP, and UTP. After 5 min, [a-“P]CTP (2 Ci/mmol) was added (to 10 PM) either alone (lanes 1 and 2) or together with IPTG (to 1 .O mfvl) and incubation continued for another 10 min followed by electrophoresis. The autoradiograms shown represent sections of the same gel slab rearranged for the convenience of presentation. The indicated position of the runoff 64 nucleotide transcript was established in a multipleround assay. The single-round protocol led to extensive pausing represented by transcripts shorter than the runoff. A control experiment (not shown) demonstrated instantaneous inactivation of free RNAP by heparin.

lac UV5 promoter fragment formed in the absence or in the presence of LacR were incubated under the -C reaction conditions in the presence of heparin, which sequesters free RNAP but does not attack the enzyme engaged in the open complex. A single round of elongation was then allowed to occur by adding radioactive CTP with or without IPTG. The result is presented in Figure 1 for the reactions primed with pppA (Figure 1A) and ApA (Figure 1 B). Obviously, the presence of LacR substantially inhibited the single-round transcription (lanes 2) as compared with the control without LacR (lanes l), while the addition of IPTG led to a dramatic induction (lanes 3). Since IPTG was added after heparin, its effect can be attributed only to the activation of RNAP molecules engaged with the promoter in the joint complex with LacR. The result unequivocally proves the existence of a heparin-resistant RNAP-LacR promoter complex responsive to IPTG induction, in complete agreement with the early rifampicin challenge data (de Crombrugghe et al., 1971; Chen et al., 1971). The heparin resistance suggests that the joint complex is equivalent to the stable, open complex step of the normal transcription cycle. This result disagrees with the conclusion of Straney and Crothers (1987b) that the joint RNAP-

in their gel retardation

assay, is

Aberrant Abortive Initiation by the RNAP-LacR Complex Resembles the Effect of a p Subunit Mutation We next addressed the question of whether the joint RNAP-LacR-promoter complex is catalytically active. For this purpose we analyzed the products accumulated in the steady-state -C reaction at the lac UV5 promoter in the presence or absence of LacR (Figure 2). We included in this analysis a mutant RNAP carrying the substitution Gl~~‘~+Lys in the 8 subunit (E813K). Elsewhere we have shown that this lethal mutation diminishes the rate of promoter clearance but does not interfere with the formation of catalytically active ITC (Lee et al., 1991). As the control for the E813K enzyme, which carries a background rifampicin-resistant marker, the isogenic Rip18 RNAP was used. Figure 2 documents the effect of LacR on the wildtype (lanes 1 and 2), RiPl8 (lanes 3 and 4) and E813K (lanes 5 and 8) enzymes in the -C reaction primed with pppA (Figure 2A), ApA (Figure 2B), and GpA (Figure 2C). It can be seen that both the wild-type and the Rifdl8 enzymes engaged in the RNAP-LacR-promoter complex (Figure 2, lanes 2 and 4, respectively) efficiently transcribed the proximal part of the ITS, up to 6U, although at a lower rate than without LacR (lanes 1 and 3). However, the joint promoter complexes failed to make longer abortive products, 7G, 8A, and 9G. The qualitative difference of the product patterns with and without LacR rulesout the possibility that activity in the presence of LacR was due to a few contaminating complexes lacking LacR. The generation of the shorter abortive products by the joint complex followed linear kinetics in the presence of heparin (data not shown). We conclude that the RNAP-LacR-promoter complex is catalytically active even though it cannot extend into the distal section of the ITS. The second principal observation from Figure 2 is that the E813K mutant RNAP in the absence of LacR (lanes 5) displays the product pattern qualitatively similar to that of two other enzymes in the joint complex with LacR (lanes 2 and 4). Thus, the malfunctioning of the ITC caused by the mutation and that induced by LacR seem to be of the same nature. The combination of the E813K mutation and LacR almost completely inactivated abortive transcription (lanes 6). RNAP-LacR Complex Displays an Increased ~Nw for Substrates Elsewhere we have shown that the E813K mutation makes RNAP extremely sensitive to a decrease in the concentration of substrates (Lee et al., 1991). Accordingly, we analyzed the performance of the RNAP-LacR-promoter complexes in the ApA-primed -C reaction at different concentrations of ribonucleoside triphosphates (Figure 3). In this experiment, the wild-type (Figure 3A), Rifdl8 (Figure 3B), and E813K RNAP (Figure 3C) were analyzed in the absence (lanes l-3) or in the presence of LacR (lanes 4-6) at the indicated concentrations of NTP. It can be seen that at 5 PM NTP, the formation of even the proximal

/aa6Repressor

Blocks

Promoter

Clearance

123456789: GAAUUGUGAG,CG -,,&.e*i*.~ a

as did the mutant enzyme or the wild-type RNAP-LacR complex at 250 uM NTP. In all these cases the transcription did not proceed beyond 6U. Thus, it appears that the elongation of RNA from 6U to 7G has a naturally high kNTp (see note on terminology in Experimental Procedures) and does not occur efficiently at suboptimal NTP concentrations. Both LacR and E813K seem to enhance this natural kinetic barrier encoded within the ITS of the lac UV5 promoter. Finally, the combination of the LacR effect and the mutation had a cumulative effect that totally abolished abortive initiation at suboptimal substrate concentrations. That the effect of LacR was due to specific interaction with the lac operator is evident from the experiment of Figure 4, which shows the products of the -C reaction on the 8-lactamase (b/a) gene promoter. The experiment was performed in the same format as that of Figure 3, except that UpG was used as the primer. It can be seen that the ITS of the b/a promoter also includes a point of naturally high kNTP, between 6A and 7U. The extension of RNA chains at this junction was particularly sensitive to a decrease in the substrate concentration and was inhibited by the E813K mutation but not by LacR, which does not bind to the b/a promoter.

. _". ,""Q<

PPPAApAGpA--w

B.

A.

c. 123456

123456

123456

9G

9G 8A 7G 6U

PPPA Figure

GPA

APA

2. Abortive

Initiation

at the lac UV5 Promoter

Discussion

The autoradiograms represent sections of the same gel slab and show abortive products generated in the -C reaction at 50 uM NTP primed with the indicated primers (at 1 .O mM). The reactions contained RNAP alone (lanes 1, 3, and 5) or included LacR (lanes 2. 4, and 6) which was preincubated with DNA prior to the addition of RNAP. The enzymes used were the wild type (lanes 1 and 2) Rifd16 (lanes 3 and 4) and E813K (lanes 5 and 8). The products are identified by their 3’terminal residues. [a-“PJUTP (2 Cilmmol) was used as the source of radioactivity in (A) and (B), and (a-“P]ATP (4 Cilmmol) in (C).

Control of Promoter Clearance by LacR On the basis of these results we suggest that the primary effect of LacR on RNAP in the joint complex is the modification of the enzyme’s catalytic function, leading to the apparent increase of kNTpfor the substrate at each step of RNA chain extension. This effect is not sufficient to abolish transcription of the proximal section of the ITS at physiological concentrations of NTP even though the rate of accumulation of the short abortive products is somewhat reduced. However, LacR dramatically inhibits the extension of RNA chains at the 6U17G junction, which is characterized by high kNTpeven under normal conditions.

abortive products by the E813K enzyme and by the RNAPLacR complex was dramatically reduced in almost identical fashion. In the absence of LacR, the wild-type or Rifdl8 enzymes displayed the same product pattern at 5 PM NTP

Figure 3. Abortive moter at Different

9

12345678

GAAUUGUGAG

CG

e

APA

C. 123456

.

123456

1;?5430

^

.

-

^

8A 7G

..

6U

PM us CA:

5

50 250

Repressor:

-

-

-

5

+

50 250

5

50 250

+ +

-

-

-

5 50 250

+ +

+

5

50 250 5

-

-

-

+

50 250

+ +

Initiation atthelac NTP Concentrations

UV5 Pro-

The autoradiogram shows the abortive products made in the -C reaction primed with ApA (1.0 mM) in the presence of the indicated concentrations of each of the three NTPs. [a-“PJUTP was used as the source of the radioactive label, and the specific radioactivity was maintained constant (1.3 Cilmmol) in all samples. Where indicated, LacR was preincubated with DNA before the addition of RNAP. Sections of the same gel slab are shown in (A), (B), and (C), which represent the wild-type, Rifd18, and E813K RNAP, respectively.

Cell 796

12345678 UGAUAAAU$CUU

Figure 4. Abortive moter at Different

x--l

123456

b/a

Pro-

The format of the experiment was the same as in Figure 3, except that the ~2000 bp fragment carrying the b/a promoter was used as template (see Experimental Procedures).

i

UPG

B.

Initiation at the NTP Concentrations

C.

123456

123456

6G 7u 6A 5A 4A 3u

PM ‘J, G, A: Repressor:

5 50 250 5 50 250 - -++-I

5 50 250 5 50 250 --+++

How LacR brings about the apparent increase in the substrate kNTpremains unknown. In principle, it can distort the substrate-binding site in the joint complex. Alternatively, LacR may affect the equilibrium of an intermediate step in the RNAP reaction that is coupled to the substrate binding. For example, the translocation of the nascent RNA 3’terminus in the catalytic center following the phosphodiester bond formation is a reversible step as is demonstrated by the phenomenon of processive pyrophosphorolysis (Rozovskaya et al., 1982). A change in the equilibrium between pre- and posttranslocation states would reduce the rate of RNA chain extension at each step and lead to the effective increase of the enzyme’s kNTPfor elongation substrates. LacR may achieve this effect either through an allosteric mechanism or by direct interference in the catalytic center. The latter possibility is not inconceivable since the binding site of LacR overlaps the +l position of the promoter (Schmitz and Galas, 1979) placing the repressor in the immediate vicinity of the catalytic center of the open complex. All of the above considerations regarding the mechanism of LacR action are equally applicable to the E813K mutation, which mimics the effect of repressor on the lac promoter. The mutation can decrease the substrate binding either directly or through the disruption of a coupled step of the reaction (the latter model is more likely as discussed by Lee et al., 1991). The combination of LacR and the mutation in the same promoter complex leads to the almost total inability of RNAP to accept transcription substrates. High kNTpSites Are Pauses within ITS A principal observation of this work is that the 6U17G junction in the lac UV5 ITS has a particularly high kNTpfor RNA chain extension. Similar high kNTpsites were found within

5 50 250 5 50 250 - _ _ ++ t

the ITSof thebla promoter (the 6A/7U junction; see Figure 4) and T7Al promoter (the 4G15A junction; see Lee et al., 1991). In all three cases these were the positions that the mutant E813K RNAP could not overcome. Thus the E813K RNAP can serve as a diagnostic enzyme to detect high kNTpsites in the ITS of other promoters, and we indeed found such sites in two other cases (data not shown). The fact that the E813K RNAP is generally inactive in standard transcriptional assays suggests that all promoters encode similar kinetic barriers that may control the rate of promoter clearance (Gralla et al., 1980; Kammerer et al., 1986). From the location of the high kNTpsites within the ITS of T7A1, b/a, and lac UV5 promoters, and from the fact that in the latter case the block occurred at the same position regardless of whether RNA was initiated with pppA, GpA, or ApA (Figure 2), we conclude that the high kNTpsites are not dictated by the length of the abortive product or the nature of the preceding or the subsequent residue. Rather, it seems that a larger element of the promoter is responsible for placing the kinetic barrier in a particular position within each promoter’s ITS. The high kNTp sites in ITS resemble some elongation pause sites, particularly those that are not associated with RNA hairpins (Levin and Chamberlin, 1987; reviewed in Yager and von Hippel, 1987) where the extension of RNA chains is almost totally blocked at suboptimal NTP concentrations. As we reported earlier, the E813K RNAP, which eventually clears the promoter after prolonged incubation with high concentrations of NTP, displays unusually long pausing times. This defect could be partially compensated by increasing the concentration of substrates (Lee et al., 1991). On this basis we suggested that a common mechanism, which is affected by the E813K mutation, determines the rate of promoter clearance and the rate of release from

lac Repressor 797

Blocks

Promoter

Clearance

pause sites. In other words, the high kNTPsites in ITS can be viewed simply as pause sites embedded within the promoter. Extending this logic to the case of LacR, its seems reasonable to speculate that the structure-functional target for its action within the RNAP molecule is the same one that accepts the input of termination factors, such as the NusA protein, which enhances pausing during the elongation stage (Kassavetis and Chamberlin, 1981; Schmidt and Chamberlin, 1984; Levin and Chamberlin, 1987). RNAP Catalytic Function as Regulation Target It is tempting to speculate that factor-mediated modulation of kinetic parameters of the abortive initiation reaction may be a general mechanism of transcription control. We demonstrate that LacR uses this mechanism to exert negative regulation. At least in one instance of positive regulation, the cyclic AMP receptor protein (CRP) acting on the ma/T promoter, it was shown that the activation occurs at the promoter clearance step (Menendez et al., 1987) and is accompanied by a change of abortive product pattern. By analogy with LacR, it could be imagined that CAMP-CRP stimulates promoter clearance by effectively decreasing kNTpof the joint ITC. In the case of CAMP-CRP action on the lac Pl promoter, two effects were shown to contribute to the activation: a direct stimulation of open complex formation and the competitive inhibition of repression caused by RNAP binding at an upstream site (Malan and McClure, 1984; Spas&yet al., 1984). However, a8 subunit mutation (rpoS77) that abolished RNAP dependence on CAMPCRP stimulation did not improve promoter binding, suggesting that another mechanism, e.g., the enhancement of promoter clearance, could be involved (Guidi-Rontani and Spassky, 1985). Our results warrant careful reexamination of abortive initiation parameters in these and other cases of joint complexes between RNAP and transcription control factors. Experimental

Procedures

Proteins Wild-type core RNA polymerase was isolated from MREBOO cells (Malik and Goldfarb, 1984) and was saturated with o factor obtained from an overexpression strain (Gribskov and Burgess, 1983). The RiP18 and E813K enzymes were assembled from individually overexpressed subunits (Zalenskaya et al., 1990). The same preparations were used that were characterized in detail by Lee et al. (1991). LacR (Pace et al., 1990) was a gift of Helen Pace and Ponzy Lu. Templates The 123 bp DNA fragment carrying the lac UV5 promoter and the -2000 bp fragment carrying the b/a promoter were isolated by extraction from an 8% polyacrylamide gel after digestion of the pHC624lac plasmid (Chan et al., 1989) with EcoRl and Hindlll. It should be noted that the 123 bp fragment did not carry the upstream (P2) lac promoter (Malan and McClure, 1964). Transcription Reactions were carried out under general conditions described by Lee et al. (1991). The volume of the standard reaction mixture was 12 fd. The-Cabortiveinitiation reactions(Figures2-4)containedO.l25pmol of template DNA fragment, 1.25 pmol of RNAP, and, where indicated, 6.25 pmol of LacR tetramer, resulting in the 5O:lO:l molar ratioof LacR to RNAP to DNA. The single-round reaction (Figure 1) contained 0.625 pmol of DNA, 3.5 pmol of RNAP. and 7.0 pmol of LacR tetramer. The

order of addition and the concentrations of variable components are indicated in the figure legends. The analysis of the products was performed as described by Lee et al. (1991) using a 15% polyacrylamide gel in the experiment of Figure 1, and a 23% gel in all other cases. Terminology The use of the term kNTP instead of the conventional kinetic constant K,,, is dictated by the following considerations. RNA chain elongation by RNAP does not obey a simple steady-state kinetic mechanism, since the efficiency of interaction of the enzyme at different template bases with the NTP substrate can vary over a factor of about lO’(Levin and Chamberlin, 1987). This invalidates the assumption, needed for a steady-state mechanism, that transcription fits a ping-pong kinetic pathway with only four states, corresponding to polymerase at bases A, T, G, and C of the template (Rhodes and Chamberlin, 1974) and means that a true K, cannot be defined for an elongation step. However, it is important to be able to refer to the concentration of a particular NTP needed to allow efficient synthesis by RNAP at a particular point along the template, for example at position 6 of the lac promoter. This concentration can be experimentally defined by determining the concentration of NTP needed to eliminate the pause at such a site, and we refer to this as the “kNTpn for that site. Acknowledgments We are grateful to Helen Pace and Ponzy Lu for the gift of the lac repressor, to John Sullivan and Jacob Lebowitz for the pHC624lac plasmid, to Sergei Borukhov for advice on RNAP reconstitution, and to Max Gottesman for comments. This work was supported by NIH grant GM30717 to A. G. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

May 21, 1991; revised

June

18, 1991.

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initiafor CRP

Metzger, W., Schickor, P., and Heumann, H. (1989). A cinematographic view of Eschericbia co/i RNA polymerase translocation. EMBO J. 8, 2745-2754. Miller, J. H., and Reznikoff, W. S., eds. (1960). The Operon Spring Harbor, New York: Cold Spring Harbor Laboratory).

(Cold

Pace, H. C., Lu, P., and Lewis, M. (1990). lac repressor: crystallization of intact tetramer and its complexes with inducer and operator DNA. Proc. Natl. Acad. Sci. USA 87, 1670-1873. Rhodes, G., and Chamberlin, M. J. (1974). Ribonucleic acid chain elongation by Escherichia co/i RNA polymerase. I. Isolation of ternary complexes and the kinetics of elongation. J. Biol. Chem. 249, 66756683. Rozovskaya, T. A., Chenchik, A. A., and Beabealashvilli, Processive pyrophosphorolysis of RNA by Eschefichia merase. FEES Lett. 137, 100-104.

R. S. (1962). co/i RNA poly-

Spassky, A., Busby, S., and But, H. (1964). On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia co/i lactose and galactose promoter regions. EMBO J. 3, 43-50. Schmidt, M. C., and Chamberlin, M. J. (1964). Amplification and isolation of Escherichia co/i NusA protein and studies of its effects on in vitro RNA chain elongation. Biochemistry 23, 197-203. Schmitz, A., and Galas, D. J. (1979). The interaction of RNA polymerase and lac repressor with the lac control region. Nucl. Acids Res. 6, 111-137. Straney, S. B., and Crothers, D. M. (1967a). Kinetics of the stages of transcription initiation at the Escherichia co/i lac UV5 promoter. Biochemistry 26, 5063-5070. Straney, S. B., and Crothers, D. M. (1987b). gene-activating protein. Cell 51, 699-707.

Lac repressor

is a transient

von Hippel, P. H., Bear, D. G., Morgan, W. D., and McSwiggen, J. A. (1984). Protein-nucleic acid interactions in transcription: a molecular analysis. Annu. Rev. Biochem. 53, 389-446. Yager, T. D., and von Hippel, P. H. (1987). Transcript elongation and termination in Escherichia co/i. In Escherichia coliand Salmonella typnimunum: Cellular and Molecular Biology, F. C. Neidhardt, ed. (Washington, DC: American Society for Microbiology), pp. 1242-1275. Zalenskaya, K., Lee, J., Gujuluva, C. N., Shin, Y. K., Slutsky, ht., and Goldfarb, A. (1990). Recombinant RNA polvmerase: inducible overexpression, purification and assembly of ikscherichia colirpo gene products. Gene 89, 7-12.