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Originally published In Press as doi:10.1074/jbc.M408609200 on September 22, 2004

J. Biol. Chem., Vol. 279, Issue 52, 54552-54557, December 24, 2004
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lacP1 Promoter with an Extended – 10 Motif

PLEIOTROPIC EFFECTS OF CYCLIC AMP PROTEIN AT DIFFERENT STEPS OF TRANSCRIPTION INITIATION*

Mofang Liu, Susan Garges{ddagger}, and Sankar Adhya§

From the Laboratory of Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892-4264

Received for publication, July 29, 2004 , and in revised form, September 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cyclic AMP receptor protein (CRP), which activates transcription from the wild-type lacP1 promoter and most of its mutants, represses productive RNA synthesis from a lacP1 promoter variant that contains an extended -10 element, although CRP enhances RNA polymerase binding as well as open complex formation in both promoters. Moreover, abortive RNA synthesis, which is already higher in the extended -10 variant compared with the parent promoter, was further enhanced by CRP. These results, together with the observed decrease in productive RNA synthesis, indicate that CRP, while facilitating the earlier steps of initiation, inhibits transcription from the extended -10 lacP1 by hindering promoter clearance. We propose that CRP decreases energetic barriers to RNA polymerase binding, isomerization, and abortive RNA synthesis but stabilizes the abortive RNA initiating complex, which results in increasing the activation energy of the transition state before the elongation complex. The results demonstrate for the first time that a DNA-binding regulatory protein acts as an activator or a repressor in different steps of the transcription initiation pathway because of the energetic differences of the intermediate complex in the same promoter.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The premier step of gene regulation occurs at the level of transcription in which a gene regulatory DNA-binding protein acts as an activator or a repressor causing enhancement or inhibition of the transcription initiation process, respectively. Activators could act by helping the binding of RNA polymerase (RNAP)1 or other associated factors at the promoter and/or by influencing a post-binding step, such as isomerization or promoter clearance. On the other hand, repressors could sterically hinder the binding of RNAP or other essential transcription factors to the promoter and/or influence one or more post-binding steps of the initiation process. Although classified as activators or repressors of transcription, some of these regulatory proteins have been found to be bifunctional, acting as an activator at one promoter and as a repressor at another (1–8).

The cyclic AMP receptor protein (CRP), an important paradigm of the positive control of gene expression, activates transcription at more than 100 promoters in Escherichia coli (9, 10). CRP functions by enhancing the binding of RNAP, stimulating isomerization to the open complex at the lacP1 and galP1 promoters (3, 11–15), and accelerating the rate of promoter clearance at the malT promoter (16, 17). However, CRP turns out to be bifunctional; it represses transcription initiation at several promoters. It has been suggested that, by sterically hindering the access of RNAP, CRP inhibits RNA synthesis from the galP2, lacP2, lacP3, and cyaP2 promoters (18–22). CRP also acts as a co-repressor in the CytR regulon (23, 24).

CRP enhances the transcription of lacP1, which is an intrinsically weak -35 element-containing promoter, by increasing both RNAP binding and isomerization, both of which involve a specific contact between CRP and the carboxyl-terminal domain of the {alpha} subunit ({alpha}CTD) of RNAP (10, 12, 14, 15, 25). We described previously a lacP1 promoter mutant that showed a very high level of transcription in the absence of CRP (26). Here we report that in an extended -10 sequence-containing derivative of this CRP-independent lacP1 promoter, the regulator bound to the original -61.5 activation site acts as a repressor. CRP enhances RNAP binding and isomerization as well as abortive RNA synthesis but decreases full-length RNA transcription from the extended -10 promoter.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Proteins and DNA—RNAP was purified from E. coli K-12 cells (MG1655) as described (27). The purified RNAP was >95% pure by Coomassie staining of the protein resolved by SDS-polyacrylamide gel electrophoresis. RNAP concentration was determined as described (28). The {alpha}1–235 mutant RNAP ({Delta}{alpha}CTD), with 94 carboxyl-terminal amino acid residues deleted from the {alpha} subunit, was a gift from Szabolcs Semsey (Laboratory of Molecular Biology, NCI, National Institutes of Health). CRP was purified to 98% homogeneity by fast protein liquid chromatography (Amersham Biosciences) from an E. coli strain carrying the wild type crp gene in the multicopy plasmid pHA5 (29). The sequences of the promoters used in the study are listed in Table I. The promoters were 390-bp PCR-amplified DNA fragments that contained 314 bp upstream of the transcription start point (+1) of lacP1 and 76 bp downstream of +1.


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  		TABLE I
Sequence of the promoters used in this study

 
In Vitro Transcription Assays—The experiments followed the protocol described previously (15, 26). All promoters were tested for function by runoff transcription of PCR-amplified fragments as templates. The templates were 390-bp long, which contained 314 bp upstream of the transcription start point (+1) of lacP1 and 76 bp downstream of +1. Reactions were carried out at 37 °C and the runoff transcripts were analyzed through an 8% polyacrylamide, 7 M urea sequencing gel. For abortive transcripts assays, samples were analyzed on a 20% polyacrylamide, 7 M urea gel. The transcripts were quantified with a Amersham Biosciences ImageQuantTM StormTM imaging system; the data for each species were corrected for background.

Electrophoresis Mobility Assays—Electrophoresis mobility assays were carried out as described (26, 30). Various amounts of RNAP and 10 nM 32P-labeled, 390-bp, PCR-amplified DNA fragments (also used in transcription) were incubated with or without 30 nM CRP and 0.1 mM cAMP at 37 °C for 30 min in 1x transcription buffer with 5% sucrose and 0.1 mg/ml bovine serum albumin. Heparin, when added, was at a concentration of 50 µg/ml. Reaction mixtures were loaded on a 4% polyacrylamide gel for electrophoresis at room temperature with 0.5x Tris borate-EDTA buffer and 0.2 mM cAMP in the top reservoir and 1x Tris borate-EDTA in the bottom.

KMnO4 Footprinting—The experiments followed a modification of a protocol described previously (15, 26, 31). DNA (10 nM 390-bp PCR-amplified fragments) and RNAP (30 nM) were incubated with or without 50 nM CRP and 0.1 mM cAMP in 1x transcription buffer with 5% glycerol and 50 µg/ml bovine serum albumin for 30 min at 37 °C before KMnO4 (12.5 mM) was added. The reactions were quenched by 2-mercaptoethanol. The primers were extended using a PCR cycle to amplify signals. The products were electrophoresed in an 8% polyacrylamide and 7 M urea sequencing gel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CRP-independent lac Promoter Variants Are Further Stimulated by the Activator—The E. coil lacP1 promoter is intrinsically weak because of its non-consensus -35 and -10 elements and 18-bp spacer length. Mutations that strengthen RNAP binding or isomerization make the promoter CRP-independent. For example, the CRP-independent lacP1 mutants, lacPs and lacPUV5, have base pair changes that make the -10 region consensus or near consensus (32). We isolated previously a lacP1 promoter variant (lacP1-6) in which an 8-bp GC-rich sequence (-20CCGGCTCG-13) was replaced by an AT-rich sequence (TTTATGTT) in the "spacer" region between the -35 and -10 elements. lacP1-6 showed extraordinary promoter activity and, like lacPs and lacPUV5, CRP independence (26). All of these CRP-independent lac variants were -35 element-dependent, and their transcription was further stimulated by CRP (26, 32).

A CRP-independent lac Mutant Is Repressed by CRP—When the CRP-independent lacP1-6 was changed to become -35 element-independent by converting it to an extended -10 element-containing promoter, lacP1-6S1, it remained highly active without CRP and a -35 element (Fig. 1A, lane 13 and Fig. 1B, lane 3). However, it showed significant reduction of transcription in the presence of CRP (Fig. 1A, lanes 13–18). Interestingly, CRP repression at the promoter was dependent upon the presence of the -35 element; mutation of the -35 element abolished CRP repression at the promoter. If anything, CRP increased transcription in the latter template (Fig. 1B, lane 4). In addition, we found the -10 and extended -10 elements were also important in lacP1-6S1. The promoter activity was reduced by the single base pair substitutions at the -10 and extended -10 elements, and CRP repression was switched back to activation by the mutations (Table II).



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  		FIG. 1.
In vitro transcription with lacP1-6 and lacP1-6S1 template. The DNA sequences of the promoters are shown in Table I. A, 10 nM DNA, 30 nM wild-type RNAP, and 0–200 nM CRP. B, 10 nM DNA and 30 nM wild-type RNAP, in the absence (-) or presence (+) of 50 nM CRP. C, 10 nM DNA and 30 nM RNAP in the absence (-) or presence (+) of 50 nM CRP. Lanes 1–4, wild-type (WT) RNAP; lanes 5–8, {Delta}{alpha}CTD mutant RNAP.

 


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  		TABLE II
The effect of single base-pair substitutions at the extended -10 motif and -10 element on promoter activity and CRP regulation

 
CRP Repression Is {alpha}CTD-dependent—The interaction of CRP with the {alpha}CTD of RNAP is the basis of transcription activation in wild-type lacP1 and the CRP-independent lacP1 variants mentioned above (10, 25). As shown in Fig. 1C, when using wild-type RNAP, the full-length transcripts from the CRP-independent lacP1-6 promoter were enhanced by CRP, and those from lacP1-6S1 were reduced by CRP (lanes 1–4). However, when using a {alpha}CTD deletion mutant RNAP ({alpha}1–235), CRP neither activated lacP1-6 nor repressed lacP1-6S1 (lanes 5–8), suggesting that CRP-{alpha}CTD interaction is also needed for CRP repression at lacP1-6S1. We also noted that the intrinsic promoter activity of lacP1-6 was stronger than that of lacP1-6S1 when wild-type RNAP was used; however, the opposite was true when {Delta}{alpha}CTD RNAP transcribed the templates, suggesting that {alpha}CTD supports the lacP1-6 activity but inhibits lacP1-6S1 activity even in the absence of the regulator. Although {alpha}CTD is not absolutely required for transcription initiation at promoters lacking an UP element, it contributes to transcription initiation, which is independent of its ability to form a specific protein-DNA contact (33).

CRP Enhances RNAP Binding lacP1-6S1—We used electrophoretic mobility assays to determine the strength of RNAP binding to the promoters and observed that lacP1-6S1 had a higher affinity (~30% higher) for RNAP compared with the parental lacP1-6 promoter at equilibrium (Fig. 2A and Table III), although the latter was stronger as judged by RNA synthesis (Fig. 1A, lane 7 versus lane 13). However, CRP also stimulated the binding of RNAP to both lacP1-6S1 (~1.4-fold) and lacP1-6 (~1.8-fold) (Fig. 2B and Table III). Interestingly, the lacP1-6 mutant with a defective -35 element, lacP1-6M(-35), formed at least three types of weak complexes with RNAP (Fig. 2C, lanes 1–8), most of which were not observed if the binding reactions were terminated by heparin before loading onto the gel (Fig. 2E, lanes 1–8). These complexes are likely different forms of closed complexes formed at the promoter. On the other hand, lacP1-6S1M(-35), with a defective -35 element, formed the same type of complex as with lacP1-6S1 (Fig. 2, panel C, lanes 9–16 versus panel A, lanes 9–16). These complexes were unaffected by adding heparin (Fig. 2E, lanes 9–16). CRP stimulated the binding of RNAP to both of the -35 mutant templates (~3-fold at lacP1-6M(-35) and 1.4-fold at lacP1-6S1M(-35)) and changed the pattern of bands at lacP1-6M(-35) (Fig. 2D and Table III). Under these conditions, CRP made the DNA· RNAP·CRP ternary complexes the major species observed on a gel, the other species being reduced at lacP1-6M(-35) (Fig. 2D, lanes 1–9). Unlike at lacP1-6S1M(-35) (lanes 10–18), the DNA· RNAP binary open complex, which migrates close to the DNA·RNAP·CRP, was not observed at lacP1-6M(-35) even with excess RNAP, suggesting that the DNA·RNAP binary open complex could not form at the promoter without CRP. However, except for dissociation of the DNA·CRP complex, heparin appeared not to alter the pattern of bands in the other three promoters (lacP1-6, lacP1-6S1, and lacP1-6S1M(-35)), suggesting that the complexes detected in these three are mostly open complexes (Fig. 2F, lanes 10–18) (26).



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  		FIG. 2.
Electrophoresis mobility assay of RNAP binding at various promoters. Binding reactions contained 10 nM DNA (32P-labeled lacP1-6, lacP1-6S1, lacP1-6M(-35), or lacP1-6S1M(-35)) and 0–60 or 0–90 nM wild-type RNAP as shown at the top. A, binding in the absence of CRP. Lanes 1–8, lacP1-6; lanes 9–16, lacP1-6S1. B, binding in the presence of 30 nM CRP. Lanes 1–9, lacP1-6; lanes 10–18, lacP1-6S1. C, binding in the absence of CRP. Lanes 1–8, lacP1-6M(-35); lanes 9–16, lacP1-6S1M(-35). D, binding in the presence of 30 nM CRP. Lanes 1–9, lacP1-6M(-35); lanes 10–18, lacP1-6S1M(-35). E, binding in the presence of 50 µg/ml heparin. Lanes 1–8, lacP1-6M(-35); lanes 9–16, lacP1-6S1M(-35). F, binding in the presence of 30 nM CRP and 50 µg/ml heparin. Lanes 1–9, lacP1-6M(-35); lanes 10–18, lacP1-6S1M(-35).

 


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  		TABLE III
Relative RNA polymerase affinity to the promoters

 
KMnO4 Footprinting—KMnO4 has been widely used as a probe to detect open complex formation by RNAP binding (31, 34). The salt reacts preferentially with pyrimidines when they are distorted in the DNA duplex. KMnO4 footprinting provides direct information as to the location and degree of the DNA distortions in the open complex. The results of KMnO4 probing of the different promoters in the absence and presence of CRP are shown in Fig. 3. As expected, the wild-type lacP1 DNA was distorted weakly at positions +1, -11, and -12, whereas the lacP2 DNA was strongly distorted at its -10, -11, and -12 positions (corresponding to -31, -32, and -33 of lacP1) in the absence of CRP. In the presence of the regulator, the distortion at position -11 of lacP1 was significantly enhanced, but the corresponding distortion at lacP2 was inhibited (Fig. 3, lanes 5–7). At lacP1-6, no distortion of lacP2 was detected in the absence or presence of CRP; DNA was distorted with polymerase alone at position -11, which represents the lacP1 locus; the presence of CRP slightly stimulated DNA distortion at this site (Fig. 3, lanes 8–10). At lacP1-6M(-35), formation of open complex was very weak in the absence of CRP, which is consistent with the electrophoresis mobility assay data of this template, but was stimulated in the presence of the regulatory protein (Fig. 3, lanes 11–13). Formation of open complex was very strong at the extended -10 promoters lacP1-6S1 and lacP1-6S1M(-35) in the absence of CRP; slight increases were observed in the presence of CRP (Fig. 3, lanes 14–16 and lanes 17–19, respectively). Quantification of the band at -11 by a PhosphorImager showed that the formation of an open complex was stimulated ~3.3-fold at lacP1-wt, 2.5-fold at lacP1-6M(-35), 50% at lacP1-6, 16% at lacP1-6S1M(-35), and 12% at lacP1-6S1.



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  		FIG. 3.
KMnO4 probing of the promoters open complex formation by RNAP in the presence or absence of CRP. The reaction mixtures were incubated at 37 °C for 30 min before modification by KMnO4 as described under "Experimental Procedures." Lanes 1–4, the sequences of the lacP1 promoter; lanes 5, 8, 11, 14, and 17, KMnO4 treatment in the absence of RNAP; lanes 6, 9, 12, 15, and 18, KMnO4 treatment in the presence of RNAP; lanes 7, 10, 13, 16, and 19, KMnO4 treatment in the presence of RNAP and CRP.

 
In contrast to lacP1-wt, lacP1-6, and lacP1-6M(-35), the following distinguishable modifications were observed at lacP1-6S1 and lacP1-6S1M(-35). (i) Distortion at -15T was unique at the extended -10 promoters, suggesting that the extended -10 motif T-15G-14 at the non-template strand might be specifically distorted when interacting with region 3.0 of the {sigma}70 subunit (35). (ii) Compared with lacP1-6S1, a weaker distortion occurred at +1T, but a stronger one occurred at +13T in lacP1-6S1M(-35). It appears that RNAP has expansive DNA contacts in the downstream region at the extended -10 promoter when the -35 element was defective. (iii) More importantly, the formation of an open complex at the two extended -10 promoters were much stronger than that at lacP1-6, although the latter is a stronger promoter as judged by RNA synthesis (26). The KMnO4 experiments suggest that the extended -10 promoters are quite different from lacP1-wt and lacP1-6 in a DNA distortion profile quantitatively and even qualitatively.

CRP Stimulates Abortive RNA Synthesis—To figure out why the strong binding of RNAP and more open complex formation at the extended -10 promoters results in less amounts of productive RNA synthesis in lacP1-6S1 in the presence of CRP, the transcription products of the promoters were analyzed for abortive RNA synthesis on a 20% gel with 7 M urea. As shown in Fig. 4, 3-mer and 5-mer as well as significant amounts of 4-mer abortive RNA were synthesized from lacP1-6S1 compared with that from lacP1-6 (Fig. 4, lanes 1 and 3). CRP further stimulated the abortive RNA synthesis in both (Fig. 4, lanes 2 and 4). However, mutation of the -35 element in lacP1-6S1 dramatically reduced the abortive RNA synthesis (Fig. 4, lane 7), although CRP restored this synthesis again to the higher level (Fig. 4, lane 8). We noted that very little abortive transcripts were synthesized from lacP1-6M(-35) either in the absence or presence of CRP (Fig. 4, lanes 5 and 6). When using the {Delta}{alpha}CTD mutant RNAP, abortive RNA synthesis was not detected in lacP1-6 and was made in somewhat reduced amounts in lacP1-6S1 either in the absence or presence of CRP (Fig. 4, lanes 9–12). Overall, the amount of abortive RNA synthesis in the presence of CRP is highest in lacP1-6S1. The composition of the abortive RNA in lacP1-6S1 was somewhat different as well.



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  		FIG. 4.
Abortive RNA synthesis. Lanes 1–8, wild type (WT) RNAP; lanes 9–12, {Delta}{alpha}CTD mutant RNAP; lanes 1, 2, 9, and 10, lacP1-6; lanes 3, 4, 11, and 12, lacP1-6S1; lanes 5 and 6, lacP1-6M(-35); lanes 7 and 8, lacP1-6S1M(-35). A plus sign (+) indicates the presence of CRP, and a minus sign (-) indicates the absence of CRP.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Several regulators are known to have dual roles acting at different levels. First, the regulator facilitates binding of RNAP to one promoter but hinders the access of RNAP to another, sometimes overlapping or divergent promoter. Specific examples are as follows: (i) CRP acting as an activator at lacP1 and galP1and as a repressor at lacP2 and gaP2 (18, 21, 22, 32); (ii) LasX acting as an activator of lasA-W transcription and as a repressor of lasXY transcription (36); and (iii) the bacteriophage {Phi}29 p4 protein acting as an activator at the phage promoter A3 and as a repressor at the promoter A2b (37, 38). Second, the regulator stimulates or inhibits transcription by stimulating or disabling RNAP activity at a post-RNAP binding step. By contacting {alpha}CTD to an unfavorable or a favorable state, GalR (and LacI in the case of a hybrid gal-lac promoter) stimulates open complex formation at galP2 and inhibits isomerization at galP1, mediated through {alpha}CTD (2, 4). The {Phi}29 p4 protein also traps the initiating complex at the phage A2C promoter by contacting with {alpha}CTD, leading to repression of promoter clearance by RNAP (5–7). The bacteriophage 434 cI protein activates transcription from the PRM promoter by contacting the {sigma} or/and the {alpha} subunit of RNAP and represses the adjacent but divergent PR promoter by preventing transition of a closed to an open complex (39, 40).

The intrinsically defective lacP1 promoter requires CRP for activation of transcription both in vitro and in vivo (32). Several lacP1 mutants, lacPs, lacP uv5, and lacP1-6, have been reported that are active in the absence of CRP but are also further stimulated by the regulatory protein (26, 32). However, as described above, another variant, lacP1-6S1, which contains an extended -10 element, was repressed by CRP. The repression was -35 element-dependent; mutation of the -35 element abolished CRP-mediated repression. In addition, the -10 and extended -10 elements are important for lacP1-6S1; single base pair substitutions at the two elements decreased the promoter activity and made the promoter regain CRP activation. It is known that interaction between the C-terminal domain of the {alpha} subunit of RNAP, {alpha}CTD, and CRP is the sole basis of activation at lacP1 (10, 25). An {alpha}CTD-deleted mutant RNAP abolished not only the enhancement of transcription from the parental lacP1-6 promoter but also CRP-mediated repression of transcription at lacP1-6S1, suggesting that an CRP-{alpha}CTD interaction is also the basis of repression at lacP1-6S1.

Detailed biochemical analysis of the steps of transcription initiation at lacP1-6 and lacP1-6S1 described above showed that CRP increased the binding of RNAP, enhanced formation of an open complex, and made large amounts of abortive RNA synthesis. The accumulation of abortive RNA in the presence of CRP suggested that CRP provides a barrier to promoter clearance in both promoters. Our results also showed that the contact(s) between CRP and {alpha}CTD of RNAP is obligatory to the stimulatory or inhibitory effect of CRP at different steps of transcription in both lacP1-6 and lacP1-6S1. Moreover, lacP1-6S1 has four potential target sites (the -35 element, the -15 element, the extended -10 motif, and the -10 element), which is one more than lacP1-6 (the -35, -15, and -10 elements) has, for RNAP contact. The CRP-{alpha}CTD contact may further increase the strength of one or more of the RNAP-promoter contacts. We hypothesize that by stabilizing the corresponding transition states, the CRP-{alpha}CTD interaction(s) decreases the energetic barrier to closed, open, and initiating complex formation and helps increased RNAP binding, open complex formation, and abortive RNA synthesis. In this model, the (R.P)i intermediate is more stable in lacP1-6S1, thereby increasing the activation energy of promoter clearance. The net effect is in reduced frequency of transition from the initiating to the elongating complex and decreased full-length RNA synthesis. This differential CRP action at lacP1-6S1 is illustrated in the energy diagram (Fig. 5) (8). In this connection, we note that CRP has been proposed to stimulate the promoter clearance step at another promoter, PmalT, by differentially stabilizing the appropriate intermediates of initiation (41). By these criteria, CRP, a paradigm of activator of transcription, becomes bifunctional; it activates and represses different steps of the initiation pathway. More interestingly, CRP, bound at the same DNA site, acts either as an activator or as a repressor at different steps of transcription initiation, depending on the energy differentials of the steps of initiation at the same promoter.



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  		FIG. 5.
Free energy diagram of CRP function in transcription initiation in lacP1-6S1. Blue line, in the absence of CRP; red line,inthe presence of CRP; R, RNAP; P, promoter DNA; (R.P){ddagger}, the transition state; (R.P)C, closed complex; (R.P)O, open complex; (R.P)i, initiating complex; and (R.P)e, elongating complex.

 
It was demonstrated previously that intrinsic promoter strength is determined by various rate-limiting steps of the overall process. The efficiency of each step is determined by a given DNA sequence element of the promoter (42–44). The net promoter activity depends upon the local DNA sequence elements that guide the corresponding steps. Consequently, promoters of identical intrinsic strength may exhibit different efficiencies at different steps because of the difference in DNA sequence. We propose here that even a regulator can also exert varying effects at the energetics of different steps in the same promoter.


   FOOTNOTES
 
* 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 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Office of Biodefense Research Affairs, Division of Microbiology and Infectious Diseases, NIAID, National Institutes of Health, Bethesda, MD 20892-6605. Back

§ To whom correspondence should be addressed: Laboratory of Molecular Biology, NCI, National Institutes of Health, 37 Convent Dr., Rm. 5138, Bethesda, MD 20892-4264. Tel.: 301-496-2495; Fax: 301-480-7687; E-mail: sadhya{at}helix.nih.gov.

1 The abbreviations used are: RNAP, RNA polymerase; CRP, cyclic AMP receptor protein; {alpha}CTD, the carboxyl terminal domain of the {alpha} subunit; {Delta}{alpha}CTD, the {alpha}1–235 mutant RNAP. Back


   ACKNOWLEDGMENTS
 
We thank Szabolcs Semsey for the gift of {alpha}1-235 mutant RNAP and Anna Mazzuca for editing.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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