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Studies with the Escherichia Coli Galactose Operon Regulatory Region Carrying a Point Mutation That Simultaneously Inactivates T

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Studies with the Escherichia Coli Galactose Operon Regulatory Region Carrying a Point Mutation That Simultaneously Inactivates T CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Volume 219, number 1, 189-196 FEB 04899 July 1987 Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein Sreenivasan Ponnambalarn, Annick Spassky* and Stephen Busby Department of Biochemistry, University of Birmingham, PO Box 363, Birmingham B15 2TT, England and *D~partement de Biologie Molbculaire, lnstitut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France Received 22 April 1987 We report in vitro studies of the interactions between purified E. coli RNA polymerase and DNA from the regulatory region of the E. coli galactose operon which carries a point mutation that simultaneously stops transcription initiation at the two normal start points, $1 and $2. In the presence of this point muta- tion, transcription initiates at a third start point 14/15 bp downstream of $1, showing that inactivation of the two normally active promoters, P1 and P2, unmasks a third weaker promoter, P3. Transcription initia- tion in the gal operon is normally regulated by the cyclic AMP receptor protein, CRP, that binds to the gal regulatory region and switches transcription from P2 to PI. With the point mutation, CRP binding swit- ches transcription from P3 to P1, although the formation of transcriptionally competent complexes at P1 is very slow. The results are discussed with respect to the mechanism of transcription activation by the CRP factor and the similarities between the regulatory regions of the galactose and lactose operons. Galactose operon; Tandem promoter; Promoter mutation; RNA polymerase; cyclic AMP receptor protein; Protein interaction; (E. coli) 1. INTRODUCTION complex binds to the gal promoter region and switches transcription from P2 to P1 [2]. The regulatory region of the Escherichia coli In recent work, we and others have measured the galactose operon is unusual as it contains two effects of over 60 different point mutations in the overlapping but distinct promoters, P1 and P2, gal operon promoter region [3-8]. Surprisingly, that are regulated by the cyclic AMP receptor pro- with one exception, none of the mutations cause tein, CRP [1]. The transcription start point for the drastic reductions in expression from the gal pro- P2 promoter is at $2, 5 bp upstream of $1, the moter region. This can be readily explained as the transcription start point of the P1 promoter DNA sequences necessary for P1 and P2 function (fig.l). In vivo, gal operon transcription usually are distinct and, thus, mutations that knock out P1 starts at $2. However in conditions where the in- leave P2 active and vice versa. The exception is a tracellular level of cAMP rises, the cAMP-CRP point mutation at - 12 that falls at the intersection of the P1 and P2 -10 hexamer sequences (see Correspondence address: S. Busby, Department of fig. 1): in vivo, this mutation causes a > 95°70 reduc- Biochemistry, University of Birmingham, PO Box 363, tion in expression from the gal operon regulatory Birmingham BI5 2TT, England region [6]. Here we report the properties, in vitro, Published by Elsevier Science Publishers B.V. (Biomedical Division) 00145793/87/$3.50 © 1987 Federation of European Biochemical Societies 189 Volume 219, number l FEBS LETTERS July 1987 of the gal operon regulatory region DNA carrying incubated in the presence or absence of 100 nM this unique mutation that simultaneously knocks CRP and 2001tM cAMP in 40 mM Tris, pH 8, out both promoters. We show that the absence of 10 mM MgC12, 100 mM KC1, 0.1 mM P1 and P2 unmasks a third overlapping promoter dithiothreitol, 5°7o glycerol. After 15 min, 150 nM to which RNA polymerase can bind and initiate RNA polymerase was added and after a further transcription 14-15 bp downstream of S1. Fur- 15 rain the footprint was determined by adding ther, we have investigated the interactions between 75 ng/ml DNase I or the copper-ortho- CRP and RNA polymerase at the gal regulatory phenanthroline mix described by Sigman et al. region carrying this mutation at -12. [12]. Digestion was stopped after 20 s by phenol extraction and alcohol precipitation. Samples were analysed on 10°70 sequencing gels followed by 2. MATERIALS AND METHODS autoradiography to visualise the footprint. The gal operon regulatory region was cloned on a 144 bp DNA fragment between the EcoRI and 3. RESULTS HindIII sites of pBR322 as described [9]. The gal fragment carried either the wild type promoter se- 3.1. Inactivation of gal P1 and gal P2 reveals a quence [4], point mutations at - 12 or - 19 [6] or new transcription start point the A420 deletion [10]. Transcription and footprint The sequence of the gal operon promoter region experiments were performed on PstI-HindIII or is shown in fig.1 together with the transcription PstI-BstEII fragments isolated from these con- start points and corresponding -10 hexamer se- structions as before [9,11]. quences for the P1 and P2 promoters. The position 'Run-off' transcription assays were performed of the mutation at - 12 that disrupts both the P1 exactly as previously described by us [9]. Footprint and P2 -I0 hexamer sequences is also indicated. experiments were as described by Spassky et al. The 890 bp PstI-HindIII fragment, illustrated in [11]. 1-2 nM end-labelled DNA fragments were fig. 1, was isolated carrying either the wild type gal Psf I pBR 322 P-GAL Hin d III T T TT TC ITAC CATA A[iCC TA AT GG AG"~ P2 T AT GC T I...... AUU- 53n P1 ITATGGT ...... AUA- 48n,.~ P3 tTACCAT~...... AA_U- 35n Fig. 1. Diagram of the PstI-HindIII fragments used in this study. The bar represents the DNA sequence consisting of 750 bp from the Pstl site to the EcoRI site of pBR322 (open) and a 144 bp EcoRI-HindIII fragment carrying the gal operon promoter region (shaded). The position of the BstEII site just upstream of the HindIII site is marked. Below the bar is shown in detail an expanded segment of the gal promoter region with the nucleotide sequence of the upper strand of the DNA around the transcription start points. For gal P1, P2, and the P3 promoter described here, the - l0 hexamer sequences are indicated by brackets and the tr~/nscr~ption start points are shown by the end of a wavy arrow. Above each arrow is marked the RNA sequence at the 5 '-end of the transcript and the distance in bases from that start point to the HindIII end of the fragment. The gal sequence is numbered ~vith respect to the P1 transcription start point: the position of the transition at - 12 that inactivates both P1 and P2 is indicated. 190 Volume 219, number 1 FEBS LETTERS July 1987 promoter sequence or the mutation at - 12. After $2 and runs 53 bases to the end of the fragment: addition of purified RNA polymerase, run-off a minor band 48 bases long is due to some initia- transcripts were made and analysed on tion at S1 (fig.2a). With the fragment carrying the polyacrylamide gels (fig.2). With the wild type gal mutation at - 12, no transcripts initiate at S1 or $2 promoter sequence, the major transcript starts at but a new band appears that is 35 bases long cz b c d e 53n P2 P2 53 n "~,----- P 1 P1 ~ ~ 48n 43n 38n 35n P3 -- 35n P3 25n -- 25n Fig.2. In vitro transcription experiments. The figure shows autoradiograms of gels run to analyse RNA made in run off transcription assays. The gels were calibrated and the length in bases of each band is indicated together with the promoter that is responsible for each band. The DNA fragments used were (a) PstI-HindIII fragment carrying the wild type ga! promoter sequence, (b) PstI-HindIII fragment carrying the mutation at - 12, (c) PstI-BstEII fragment carrying the mutation at - 12, (d) PstI-HindIII fragment carrying the wild type gal promoter sequence but a deletion from - 29 to -92, (e) PstI-BstEII fragment carrying the deletion from -29 to -92. 191 Volume 219, number 1 FEBS LETTERS July 1987 (fig.2b). To identify the origin of this transcript the restriction, the 35 base transcript is reduced to 25 PstI-HindIII fragment carrying the mutation at bases (fig.2c), showing that this transcript is due to - 12 was shortened by restriction with BstEII; this RNA polymerase initiating transcription at + 14 or cuts l0 bp before the HindIII site (fig.l). After + 15 and moving rightwards to the end of the frag- -12 -12 -19 mutation ~ I I I 1 I lane a b c d e f g h i j k t m n o CRP + ++ + + + -+ time (rains) 1/2 2 5 15 30 60 kz 2 S 15 30 60 2 2 P1 = ~.8n P3----~ 35n Fig.3. Kinetics of transcription initiation. RNA polymerase was incubated with the PstI-HindlII fragment carrying the mutation at - 12 in the absence (lanes a-f) or presence (lanes h-m) of cAMP-CRP. After the different times shown, nucleoside triphosphates and heparin were added and the transcripts were analysed by gel electrophoresis as shown in the figure. Lanes n and o show an experiment with DNA carrying a mutation at - 19 that gives transcription from P1 under all conditions. 192 Volume 219, number 1 FEBS LETTERS July 1987 RNP + + ment. Fig.1 shows the position and orientation of CRP -- 4" -" + the transcription start point and indicates a plausi- ble -10 sequence, 5'-TACCAT-3', from +2 to +7.
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