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Journal of Bacteriology, May 2000, p. 2982-2984, Vol. 182, No. 10
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Positioning of Region 4 of the Escherichia coli RNA Polymerase ⁷⁰ Subunit by a Transcription Activator

School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom¹; Institut Pasteur, Unité de Physico-chimie des Macromolécules Biologiques, 75724 Paris Cedex 15, France²; Department of Chemistry, University of California, Davis, California 95616³; and Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan⁴

Received 10 September 1999/Accepted 12 January 2000

	ABSTRACT

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A DNA cleavage reagent, specifically tethered to residue 581 of the Escherichia coli RNA polymerase ⁷⁰ subunit, has been used to investigate the location of ⁷⁰ region 4 in different complexes at the galp₁ promoter and the effect of the cyclic AMP receptor protein. The positions of DNA cleavage by the reagent are not affected by the cyclic AMP receptor protein. We conclude that transcription activation at the galp₁ promoter by the cyclic AMP receptor protein does not involve major conformation changes in or repositioning of ⁷⁰ region 4.

	TEXT

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Many bacterial transcription activators bind to DNA sites that overlap the 35 region of target promoters and interact directly with holo-RNA polymerase (RNAP). In most cases, these activators have specific interactions with region 4 of the principal RNAP  subunit, ⁷⁰, that appear both to recruit RNAP to the promoter DNA and to accelerate the transition from the closed to the open complex (4, 8, 16; see also reference 11). Although it is well known that a major function of ⁷⁰ region 4 is to contact promoter 35 elements, little is known about activator-⁷⁰ interactions (7). For example, does the activator-⁷⁰ interaction merely recruit ⁷⁰ region 4 to the target promoter or does it reposition region 4? To investigate this point, we have exploited previous work in which ⁷⁰ region 4 was specifically labeled with a DNA cleavage reagent. DNA cleavage by this reagent was then used to investigate the position of region 4 of ⁷⁰ in different RNAP-promoter complexes.

Previous publications have described the cloning of the rpoD gene (encoding ⁷⁰) that had been mutated to remove all three cysteine codons and the subsequent introduction of a cysteine codon at position 581 in ⁷⁰ region 4 (13, 14). We previously overexpressed and purified [Cys581] ⁷⁰, showed that it was functional, and described how Cys581 could be specifically tagged with the DNA cleavage agent p-bromoacetamidobenzyl-EDTA-Fe (FeBABE). It was shown previously that RNAP, reconstituted with FeBABE-tagged [Cys581] ⁷⁰, could form open complexes at different activator-independent promoters (3, 13). Activation of the tagged FeBABE reagent by addition of sodium ascorbate leads to cleavage of specific bases in the 35 region of the promoter DNA.

In this work, we have studied the interactions of RNAP containing FeBABE-tagged [Cys581] ⁷⁰ with the Escherichia coli gal operon promoter region, which contains two overlapping promoters, P1 and P2, that are regulated by the cyclic AMP receptor protein (CRP) (Fig. 1). It is well established that, in the absence of CRP, RNAP initiates transcripts at P2. CRP binds to a DNA target that overlaps the 35 element of both promoters and switches RNAP from P2 to P1 by repressing P2 and activating P1 (12). Here we have exploited the galp19T mutation that totally inactivates P2 (Fig. 1). In previous studies with galp19T, it was shown that P1 can function in the absence of CRP but that transcription initiation at P1 is stimulated by CRP (2, 9, 10). Thus, the use of galp₁ allows us to study the position of ⁷⁰ region 4 in open complexes at the same promoter either with or without CRP. In this work, we used previously established protocols to purify wild-type and mutant CRP derivatives (6, 15) and DNA fragments carrying gal promoter region sequences that were labeled on either strand (1). Since very similar results were obtained with fragments labeled on either strand, Fig. 2 and 3 show experiments solely with fragments labeled on the template strand.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Base sequence of the wild-type E. coli gal operon regulatory region, showing the two overlapping promoters P1 and P2. The sequence is numbered with the P1 transcript start as +1. Above the sequence are shown the various P1 elements: the 22-bp DNA site for CRP and the 35 and extended 10 elements. Below the sequence are shown the P2 35 and extended 10 element and the P2 start point. The location of the p19T substitution, which completely inactivates P2, is indicated. With the gal regulatory region carrying the p19T substitution, RNAP initiates transcription at P1 in both the absence and the presence of CRP.

Figure 2 shows the pattern of DNA cleavage that results when open complexes were formed by RNAP containing FeBABE-tagged [Cys581] ⁷⁰ at galp₁, using DNA carrying the galp19T mutation. Consistent with previous reports (3, 13), the FeBABE tag results in cleavage of promoter DNA near positions 29 and 40. Our results show that the pattern of cleavage is identical in both the absence and the presence of CRP, implying that CRP does not reposition ⁷⁰ region 4 during transcription activation. To confirm this conclusion, we exploited two CRP derivatives that are altered in their ability to activate transcription at promoters such as galp₁, where the DNA site for CRP overlaps the target promoter 35 element (class II CRP-dependent promoters: reviewed in reference 5). First, [Leu159 Glu101] CRP carries the His159Leu and Lys101Glu substitutions that inactivate the two principal activating regions of CRP that interact with RNAP at class II CRP-dependent promoters (5, 15). Second, [Leu159 Glu101 Asn52] CRP carries an additional substitution, Lys52Asn, that improves the function of a third activating region that is known to interact directly with ⁷⁰ region 4 (11). Previous studies have shown that the Lys52Asn substitution in CRP greatly increases the ability of CRP to activate transcription at class II CRP-dependent promoters (11, 17). The results in Fig. 2 show that these substitutions have little or no effect on the pattern of digestion due to FeBABE-tagged [Cys581] ⁷⁰ at galp₁.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   DNA cleavage of galp19T by RNAP containing FeBABE-tagged [Cys581] 70. Purified wild-type (wt) CRP (150 nM) or mutant derivatives were mixed, as indicated, with 32P-end-labeled AatII-HindIII DNA fragments (0.4 nM, labeled on the template strand) carrying the gal promoter region with the p19T mutation in a reaction volume of 35 µl [20 mM HEPES (pH 8.0), 5 mM MgCl2, 200 µM cyclic AMP, 50 mM potassium glutamate, 50 µg of bovine serum albumin per ml, 5 µg of poly(dI-dC)]. RNAP holoenzyme (30 nM) reconstituted with FeBABE-tagged [Cys-581] 70 was then added and incubated at 37°C for 20 min. Complexes were challenged with heparin (200 µM for 5 min), and DNA cleavage was initiated by the addition of sodium ascorbate (2 mM) followed by incubation at 37°C for 20 min. Modified DNA was extracted with phenol-chloroform and analyzed on a 6% polyacrylamide sequencing gel, which was calibrated with Maxam-Gilbert G+A sequence ladders and processed and scanned using a Molecular Dynamics PhosphorImager (full protocols are given in reference 3). Bands due to DNA cleavage near positions 40 and 29 upstream of the galp1 transcription start point are indicated. Lane 1, no CRP; lane 2, wild-type CRP; lane 3, [Leu159 Glu101] CRP; lane 4, [Leu159 Glu101 Asn52] CRP; lane 5, G+A sequencing ladder.

As a control, the experiment with galp19T DNA shown in Fig. 2 was repeated in exactly the same conditions, using the wild-type gal regulatory region. Figure 3 shows the DNA cleavage due to the FeBABE tag when RNAP containing FeBABE-tagged [Cys581] ⁷⁰ was incubated with wild-type gal DNA either with or without different CRP derivatives. In the absence of CRP, the FeBABE tag results in cleavage of promoter DNA near positions 34 and 45, due to occupation of P2 (see Fig. 1 for the location of P2). The presence of CRP shifts the sites of DNA cleavage downstream to positions 29 and 40, as P2 is repressed and P1 is activated (this pattern of cleavage is similar to that seen with galp19T DNA). Further results in Fig. 3 show that [Leu159 Glu101 Asn52] CRP but not [Leu159 Glu101] CRP is able to shift RNAP from P2 to P1. The result with [Leu159 Glu101] CRP was unexpected and implies that, in our conditions, mere occupation of the DNA site for CRP is insufficient to shift RNAP from P2 to P1. However, since with [Leu159 Glu101 Asn52] CRP the sole functional activating region of CRP interacts with ⁷⁰ region 4, we can infer that this interaction is sufficient to shift RNAP from P2 to P1 at the wild-type gal operon regulatory region.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   DNA cleavage of wild-type (wt) gal by RNAP containing FeBABE-tagged [Cys581] 70. Purified wild-type CRP (150 nM) or mutant derivatives were mixed, as indicated, with 32P-end-labeled AatII-HindIII DNA fragments (0.4 nM, labeled on the template strand) carrying gal promoter DNA in a reaction volume of 35 µl, and RNAP holoenzyme (30 nM) reconstituted with FeBABE-tagged [Cys581] 70 was then added. Samples were processed exactly as described in the legend to Fig. 2 (full protocols are given in reference 3). Bands due to DNA cleavage at different positions upstream of the galp1 transcription start point are indicated. Lane 1, wild-type gal, no CRP; lane 2, wild-type gal, wild-type CRP; lane 3, wild-type gal, [Leu159 Glu101] CRP; lane 4, wild-type gal, [Leu159 Glu101 Asn52] CRP; lane 5, G+A sequencing ladder; lane 6, galp19T, no CRP; lane 7, galp19T, wild-type CRP.

The main conclusion from this work comes from the study with galp19T, where RNAP can form open complexes at P1 both in the absence and in the presence of CRP. Our study shows that both [Leu159 Glu101 Asn52] CRP and wild-type CRP, in conditions where they can stimulate transcription and participate in ternary CRP-RNAP-galp₁ complexes, have no effect on the location of ⁷⁰ region 4. Thus, we conclude that the primary function, at this particular promoter, of the CRP-⁷⁰ interaction is not to trigger a major conformation change or repositioning of ⁷⁰ region 4 but rather to recruit region 4 to the promoter 35 element. It will be interesting to see if this conclusion holds for other transcription activators that bind to targets that overlap promoter 35 elements and interact with ⁷⁰.

	ACKNOWLEDGMENTS

This study was funded by a project grant from the Wellcome Trust and an Anglo-Japanese Scientific Collaboration Award from the Royal Society.

	FOOTNOTES

^* Corresponding author. Mailing address: School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. Phone: 44-121-414-5439. Fax: 44-121-414-7366. E-mail: S.J.W.Busby{at}bham.ac.uk.

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