Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Region 2.5 of the Escherichia coli RNA polymerase σ70 subunit is responsible for the recognition of the ‘extended −10’ motif at promoters

Region 2.5 of the Escherichia coli RNA polymerase σ70 subunit is responsible for the recognition... The EMBO Journal Vol.16 No.13 pp.4034–4040, 1997 Region 2.5 of the Escherichia coli RNA polymerase σ subunit is responsible for the recognition of the ‘extended –10’ motif at promoters Sequence analysis of bacterial σ factors has revealed Kerry A.Barne, Jon A.Bown, four conserved important regions, regions 1, 2, 3 and 4 Stephen J.W.Busby and (reviewed by Helmann and Chamberlin, 1988). The Stephen D.Minchin analysis argues that region 2 is subdivided into four School of Biochemistry, The University of Birmingham, Edgbaston, segments (2.1–2.4) and region 4 is divided into two (4.1 Birmingham B15 2TT, UK and 4.2). A series of studies, based mainly on suppression Corresponding author genetics, has shown that sub-region 2.4 (located at the C- e-mail: [email protected] terminal end of region 2) interacts directly with promoter – 10 hexamer elements, whilst sub-region 4.2 (located at At some bacterial promoters, a 5-TG-3 sequence the C-terminal end of region 4) interacts directly with element, located one base upstream of the –10 hexamer promoter –35 hexamer elements. For example, Waldburger element, provides an essential motif necessary for et al. (1990), found that a Gln to His change at position transcription initiation. We have identified a mutant 70 437 of σ , within region 2.4, suppressed the effects of a of the Escherichia coli RNA polymerase σ subunit T–A to C–G ‘down’ mutation at position –12 in the –10 that has an altered preference for base sequences in hexamer of the ant and lac promoters. Similarly, changing this ‘extended –10’ region. We show that this mutant Thr to Ile at position 440 increases the activity of promoters σ subunit substantially increases transcription from containingaGorC instead of T at this position (Siegele promoters bearing 5-TC-3 or 5-TT-3 instead of a et al., 1989; see also Zuber et al., 1989; Daniels et al., 5-TG-3 motif, located one base upstream of the –10 1990; Tatti et al., 1991). Recently, the crystal structure of hexamer. The mutant results from a single base pair 70 a σ fragment that includes region 2.4 was solved substitution in the rpoD gene that causes a Glu to Gly (Malhotra et al., 1996), showing that region 2.4 contains an change at position 458 of σ . This substitution identifies amphipathic α-helix, with residues 437 and 440 positioned a functional region in σ that is immediately adjacent such that they can both contact bases at the –12 position. to the well-characterized region 2.4 (positions 434–453, A number of activator-independent promoters have now previously shown to contact the –10 hexamer). From been reported where specific –35 hexamer contacts are these results, we conclude that this region (which we not required for transcription initiation (reviewed by Bown name region 2.5) is involved in contacting the 5-TG-3 et al., 1997). Transcription initiation at these promoters is motif found at some bacterial promoters: thus, dependent on a supplementary sequence element, 5-TG-3, extended –10 regions are recognized by an extended located one base upstream of the –10 hexamer. This results region 2 of the RNA polymerase σ subunit. in an ‘extended’ –10 element, 5-TGnTATAAT-3, which Keywords: promoter recognition/RNA polymerase/sigma appears to create alternative contact points for RNAP, subunits/transcription initiation most likely via the σ subunit, such that transcription can be initiated in the absence of specific –35 region contacts (Ponnambalam et al., 1986; Keilty and Rosenberg, 1987; Introduction Burns et al., 1996). In support of this, Kumar et al. (1993) showed that RNAP containing an altered form of σ lacking Transcription initiation is a complex process which the C-terminal 84 amino acids (including region 4.2, involves rallying many diverse molecular interactions which is responsible for contacting the –35 hexamer) to facilitate promoter recognition by RNA polymerase could initiate transcription at a consensus extended –10 (RNAP) and DNA unwinding around the transcription promoter (but was unable to initiate transcription at a start-point. In bacteria, one RNAP with subunit composi- typical promoter with –10 and –35 regions resembling the tion α ββ (E) is responsible for all transcription. However, consensus but without the 5-TG-3 extension). In this although this core enzyme is capable of transcript elonga- work we describe an experiment designed to identify the tion, it cannot initiate transcription without enlisting a segment of the σ subunit involved in making contact specific transcription factor, σ.Itis σ that directs RNAP with the 5-TG-3 extension. Our strategy was to start to specific promoters, and σ is also involved in promoter with a promoter where activity was completely dependent melting. Escherichia coli contains many classes of pro- on a 5-TG-3 extension, to inactivate the promoter by moter, all of which are recognized by separate σ factors. making base changes in the extension, and then to search The major σ factor, σ , makes sequence-specific contacts for mutant σ derivatives that allowed transcription initi- with two hexamer elements at promoters, the –10 and –35 sequences, that are separated by an optimal spacing of ation at the inactivated promoters. This experiment identi- 17 bp (Hawley and McClure, 1983; Harley and Reynolds, fies a region of σ immediately adjacent to region 2.4 1987): the consensus sequences for –10 and –35 hexamers which is involved in the recognition of the 5-TG-3 motif. are 5-TATAAT-3 and 5-TTGACA-3, respectively We report both in vivo and in vitro data to support our (Figure 1). conclusions. 4034 © Oxford University Press 70 E.coli RNA polymerase σ subunit Fig. 1. Base sequence of the KAB-TG, KAB-TC and KAB-TT promoters used in this work. The –10 and –35 hexamers are highlighted in bold: the 5-TG-3 extension at position –14/–15 is underlined. Results Isolation and characterization of σ mutants The E.coli galP1 promoter is an example of an extended –10 promoter, having a –35 region with little homology to the consensus (Ponnambalam et al., 1986) and mutations in the 5-TG-3 motif render galP1 inactive (Chan et al., 1990). Both methylation protection and interference studies suggest that RNAP makes direct contact with the G–C base pair at position –14 of galP1 (within the 5- TG-3 motif) (Minchin and Busby, 1993). The starting point of this work was the KAB set of three semi-synthetic promoters, based on the galP1 sequence (Figure 1). Activity of the KAB-TG promoter is dependent on a 5- TG-3 motif (at positions –14 and –15) upstream of the –10 hexamer, 5-TATGGT-3. In the KAB-TC and KAB- TT promoters, the 5-TG-3 motif is changed to 5-TC- 3 and 5-TT-3 respectively, and this greatly reduces promoter activity. Our aim was to identify σ mutants that would permit RNAP to serve the KAB-TC and KAB-TT promoters. To do this, the rpoD gene (cloned in plasmid pKBσ ) was amplified by error-prone PCR, using conditions such that, Fig. 2. The activity of the KAB-TG, KAB-TC and KAB-TT promoters 70 70 on average, each amplified DNA fragment encoding rpoD with σ (shown in black) and σ -EG458 (shown in grey) in trans. EcoRI–HindIII fragments carrying the promoters were cloned into the contained one base pair substitution. These fragments 70 70 lacZ expression vector, pRW50 and β-galactosidase levels are taken as were then cloned into pKBσ , generating a library of σ a direct measure of the promoter activity. In the histogram, activity mutants, which was then screened for σ derivatives that levels are expressed as a percentage of the wild type activity at each permitted increased transcription initiation at the KAB- promoter. The activity (nmole ONPG hydrolysed per min per mg dry TC and KAB-TT promoters. To facilitate this screen, the weight bacteria) of Eσ at KAB-TG, KAB-TC and KAB-TT was 2570, 51 and 49 respectively. three promoters KAB-TG, KAB-TC and KAB-TT were each fused to lacZ. As expected, cells carrying the KAB- TG::lacZ fusion exhibited a Lac phenotype (red colonies KAB-TG, KAB-TC and KAB-TT was assessed, exploiting on MacConkey indicator plates) whilst cells carrying the the lac fusions described above. Measurements were made – 70 KAB-TC::lacZ or KAB-TT::lacZ fusions exhibited a Lac in the strain CAG20177 which carries host σ under the phenotype (white colonies on MacConkey indicator control of the repressible trp promoter. Plasmid pKBσ 70 70 plates). Candidates from the σ mutant library were encoding either wild type or σ -EG458 was introduced screened for the ability to confer a red/pink Lac phenotype and β-galactosidase assays were performed on cells grown on cells carrying the KAB-TC::lacZ or KAB-TT::lacZ in conditions in which the host-encoded σ was repressed. fusions. Thirty thousand colonies from 10 independent Thus, the level of transcription from each promoter as a 70 70 PCR libraries were screened and two mutant σ candidates result of specific initiation by either Eσ or Eσ -EG458 (from different libraries) were isolated. We confirmed that could be determined. The results, presented in Figure 2, both of these candidates made stable functional sigma by show that the EG458 substitution significantly affects the checking their ability to complement the rpoD tempera- activity of all three promoters. The activity of promoter ture-sensitive mutation in the E.coli strain 285c.recA, KAB-TG is reduced by 15% with Eσ -EG458. In contrast, during growth at 42°C. The complete nucleotide sequence the activity of the mutant promoter derivatives, KAB-TC of rpoD for both candidates was determined. Both con- and KAB-TT is increased by 2.5- to 3-fold with Eσ - tained the same single base change, an A–T to G–C EG458 (compared with Eσ ). As a control, we made two transition at position 1373 of the rpoD sequence, which further derivatives of promoter KAB-TG. In one, the alters a GAG codon to GGG, and results in the substitution activity of the promoter was reduced 50-fold by the of a glutamic acid for glycine at position 458 of σ . introduction of a single change in the –10 hexamer (from 5-TATGGT-3 to 5-CATGGT-3) and, in the other, a Specificity of Eσ -EG458 similar effect was produced by altering the –35 hexamer The activity of RNAP holoenzyme carrying σ with the element (from 5-TAGACA-3 to 5-TAGGTA-3). The EG458 substitution (Eσ -EG458) at the three promoters effects of these promoter mutations could not be sup- 4035 K.A.Barne et al. Fig. 4. An autoradiograph of a gel to show the pattern of cleavage resulting from potassium permanganate and piperidine treatment of PstI–HindIII fragments carrying KAB-TG (lanes 1, 4, 7 and 10), KAB-TC (lanes 2, 5, 8 and 11) and KAB-TT (lanes 3, 6, 9 and 12). Fig. 3. An autoradiograph of gel mobility shift assays to separate Experiments were performed with 100 nM core enzyme (lanes 1–3), promoter fragments bound to RNA polymerase from free fragments. 70 70 100 nM Eσ (lanes 4–6), 100 nM Eσ -EG458 (lanes 7–9) and RNA polymerase was pre-incubated with labelled fragment carrying labelled fragment alone (lanes 10–12). Lane M is a calibration marker KAB-TG (lanes 1–4), KAB-TC (lanes 5–8) and KAB-TT (lanes 9–12). made by the Maxam–Gilbert G-specific sequence reaction on one of Experiments were performed with 100 nM core enzyme (lanes 1, 5 70 70 the promoter constructions. The gels are calibrated with the and 9), 100 nM Eσ (lanes 2, 6 and 10), 100 nM Eσ -EG458 (lanes transcription start point as 1. 3, 7 and 11) and labelled fragment alone (lanes 4, 8 and 12). The samples were heparin challenged before loading on the gel. The faint retarded bands seen with core enzyme are likely to be due to a trace contamination of σ . 70 70 with E, Eσ and Eσ -EG458. Single-stranded T residues in the open complexes could be modified by KMnO and pressed by the introduction of plasmid pKBσ encoding the points of modification were detected by cleavage with σ -EG458. This shows that the effect of the EG458 piperidine and analysis on a denaturing polyacrylamide substitution is position specific. gel. The results (Figure 4) show that all open complexes form at the same position on the DNA, confirming that Improved interaction of Eσ -EG458 with the RNAP is binding to the promoters as expected. Moreover, KAB-TC and KAB-TT promoters the pattern and location of unwinding in the open com- 70 70 70 70 The binding of Eσ and Eσ -EG458 to promoters can plexes formed by both Eσ and Eσ -EG458 are the same be assessed simply using gel mobility shift assays. Labelled at the KAB-TG, KAB-TC and KAB-TT promoters, with DNA fragments carrying the promoters, KAB-TG, KAB- DNA opening confined to positions –11 to 3. The TC or KAB-TT were incubated with either purified core degree of promoter opening by the two forms of RNAP 70 70 enzyme (E), purified Eσ or purified Eσ -EG458 at 37°C holoenzyme, as detected by KMnO , corroborates the for 30 min to allow open complex formation. The resulting β-galactosidase and gel mobility shift assay data. Wild complexes were challenged with heparin (to test the type holoenzyme, Eσ , readily forms open complexes at stability of the RNAP–DNA complexes) before separation KAB-TG, a very small amount of opening is observed at on a native polyacrylamide gel. The assays (Figure 3) KAB-TC, and no opening is found at KAB-TT (compare clearly show that core enzyme (E) is unable to form stable lanes 4, 5 and 6). In contrast, with Eσ -EG458, open complexes with any promoter DNA (lanes 1, 5 and 9) complexes form at all three promoters, confirming that and that the binding of holo-RNAP (Eσ ) to KAB-TG is the EG458 substitution confers a relaxed specificity on greater than to KAB-TC or KAB-TT (lanes 2, 6 and 10). σ and allows RNAP to initiate transcription at promoters 70 70 Comparing the binding of Eσ and Eσ -EG458, at both carrying ‘down’ mutations in the 5-TG-3 motif of KAB-TC and KAB-TT in this assay, Eσ binds relatively extended –10 promoters. poorly, whereas Eσ -EG458 has a greater affinity (com- pare lanes 6 and 10 with lanes 7 and 11). The assay shows The region between conserved regions 2.4 and 3 70 70 that Eσ -EG458 can still bind to the KAB-TG promoter in σ : effects of the HA455 substitution (compare lanes 2 and 3), suggesting that EG458 must be Figure 5 shows an alignment of amino acid sequences 70 70 a relaxed specificity mutant of σ . around E458 in σ with the corresponding sequence in nine bacterial σ factors. E458 is located in the spacer Characterization of open complexes formed by between conserved regions 2.4 and 3. However, it is clear 70 70 Eσ and Eσ -EG458 from the figure that a number of amino acids in this region Since DNA unwinding is an important step in transcription (including E458) are well conserved between different σ 70 70 initiation, we studied the interactions of Eσ and Eσ - factors. For example, the histidine residue at position 455 EG458 in the open complexes by probing with potassium is particularly conserved. We investigated the effect of permanganate (as described by Chan et al., 1990). Open changing this residue in σ to alanine on the activity of complexes were formed by preincubating DNA fragments the three KAB promoters, together with the effects of carrying the KAB-TG, KAB-TC and KAB-TT promoters alanine substitutions at the neighbouring less-conserved 4036 70 E.coli RNA polymerase σ subunit Fig. 5. A PILEUP [Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin] of amino acid sequences of bacterial σ factors between region 2.4 and the start of region 3. The location of E458 and H455 is indicated by open boxes. The position of region 2.4 and the 70 70 start of region 3 is indicated by shaded boxes. The lineup includes: E.coli σ (RP70_ECOLI), Salmonella typhimurium σ (RP70_SALTY), 70 80 A Pseudomonas aeruginosa σ (RP70_PSEAE), Myxococcus xanthus σ (RP80_MYXXA), Bacillus subtilis σ (RPSA_BACSU), Chlamydia 70 HrdB A trachomatis σ (RP70_CHLTR), Streptomyces coelicolor σ (HRDB_STRCO), Anabaena sp. σ (RPSA_ANASP) and Streptomyces aureofaciens σ (RPSA_STRAU). isoleucine 457 and threonine 459. The mutants σ -HA455, 70 70 σ -IA457 and σ -TA459 were constructed in plasmid pKBσ and their ability to drive transcription initiation at the KAB-TG, KAB-TC and KAB-TT promoters was assayed in strain CAG20177 as described above. The results (Figure 6) show that alanine substitutions at either isoleucine 457 or threonine 459 have little or no effect on the ability of RNA polymerase to initiate transcription. In contrast, substitution of histidine 455 results in a 50% reduction in expression from the KAB-TG promoter and smaller effects at the KAB-TC and KAB-TT promoters. We conclude that histidine 455 of σ is also involved in interactions with the 5-TG-3 motif at extended –10 promoters. Discussion It is well documented that the σ subunits of bacterial RNA polymerases are primarily responsible for making sequence-specific contacts with promoter DNA. In pre- vious studies, particular amino acids in regions 2.4 and 4.2 were shown to be responsible for contacting the –10 Fig. 6. The activity of the KAB-TG, KAB-TC and KAB-TT promoters and –35 hexamers respectively. At extended –10 pro- 70 70 with Eσ (shown in dark grey), Eσ -HA455 (shown in black), moters, contacts between region 4.2 of σ and the –35 70 70 Eσ -IA457 (shown in white) and Eσ -TA459 (shown in light grey). region appear to be unimportant for promoter activity, and EcoRI–HindIII fragments carrying the promoters were cloned into the lacZ expression vector, pRW50. The β-galactosidase level was these promoters function because σ makes supplementary measured and is taken to be a direct measure of the activity of the contacts with a 5-TG-3 motif just upstream from the cloned promoter. In the histogram, levels are expressed as a percentage –10 hexamer. To identify the part of σ that interacts of the wild type activity at each promoter. with the 5-TG-3 motif of extended –10 promoters, we developed a genetic screening system for selecting and analysing a mutant form of σ that suppressed ‘down’ motif. This conclusion was corroborated by the gel mobil- mutations in the G–C base pair at position –14 of the ity shift assays shown in Figure 3, and potassium perman- extended –10 region of the KAB-TG promoter. The entire ganate footprints shown in Figure 4. rpoD gene which encodes a product of 613 amino acids Glu458 of σ lies in a region just downstream of was randomly mutated, and yet the screening system twice conserved region 2.4 (amino acids 434–453) which is generated the same σ mutant carrying a single amino known to be involved in recognition of the –10 hexamer acid substitution at position 458. This substitution partially of promoters (Helmann and Chamberlin, 1988; Siegele restores the activity of the KAB-TC and KAB-TT pro- et al., 1989; Daniels et al., 1990). Since Glu458 and other moters, containing a C–G or T–A base pair at position – residues in this region are well conserved between different 14 respectively. The activities of these promoters is σ factors (Figure 5), we suggest that this region, which increased 2.5- to 3-fold in the presence of Eσ -EG458 we propose to call region 2.5, has a function, which is to compared with Eσ , whilst, at the starting KAB-TG recognize 5-TG-3 elements upstream of promoter –10 promoter, activity is reduced to ~85% of that with Eσ . hexamer elements. We conclude that extended –10 regions The mutant σ was stable, retained its core binding affinity are recognized by an extension of region 2 of σ (region and no difference in electrophoretic mobility could be 2.5). One interesting exception, where Glu458 is not 70 32 detected. The simplest conclusion is that Glu458 of σ conserved, is σ , the heat shock σ factor, where this is involved in recognition of the G–C bp of the 5-TG-3 region has a different function and is involved in 4037 K.A.Barne et al. Fig. 7. The amino acid sequence of σ with the four conserved regions highlighted by shading. Regions 1, 2, 3 and 4 are divided into sub-regions. The figure also shows a consensus promoter and indicates that the –10 and –35 promoter elements are recognized by regions 2.4 and 4.2 respectively. Region 2.5 is shown making direct interaction with the 5-TG-3 motif located one base pair upstream of the –10 element. Note that the precise boundaries of different regions are arbitrary: here, we define region 2.5 as the entire sequence between region 2.4 and region 3.1. chaperone-mediated post-translational regulation of heat backbone, accounting for how Eσ -EG458 can drive shock σ levels (Yura, 1996). transcription at the KAB-TC and KAB-TT promoters. It should be noted that a mutant σ subunit carrying Malhotra et al. (1996) recently solved the crystal structure an amino acid change at position 458 had previously been of a σ fragment from amino acid 114 to 448. This isolated by Waldburger et al. (1990), as a suppressor of fragment contains part of conserved region 1.2 and all but mutant ant promoters. The P promoter contains a the C-terminal five residues of conserved region 2. Region ant consensus –35 hexamer and a –10 hexamer that differs 2.4 was found to form an α-helix with the amino acids from consensus at only one position. The σ mutant, rpoD- involved in recognition of the –10 hexamer being solvent EK458, was isolated as a suppressor of a down mutation exposed on one face of the helix. Figure 7 is a model at –12, and also as a suppressor of down mutations at showing the orientation of region 2.4 with respect to the –10 and –9. EK458 causes a small increase in the activity DNA (Malhotra et al., 1996), and also the predicted of 16 P promoters with down mutations at different orientation of region 4.2. The model predicts that amino ant positions in both the –10 and –35 hexamers, and it acids involved in contacting the 5-TG-3 motif would be was suggested that the effects of EK458 on promoter in the region defined by EG458 (which we dub region recognition were position independent. Our result provides 2.5). Finally, extended –10 promoters are ubiquitous in a possible explanation for the observations of Waldburger nature, occurring in most bacteria. Indeed, nearly half of et al. (1990). The P promoter is not an extended –10 the total promoters in some Gram-positive microorganisms ant promoter, and all the promoters used in their study carried can be classified as extended –10 promoters (Helmann, a5-TA-3 at position –15/–14. It is likely that the EK458 1995). We conclude that region 2.5 of σ plays an important substitution is ‘suppressing’ the effect of having 5-TA- role in setting the promoter preferences of many bacterial 3 rather than 5-TG-3 upstream of the –10 hexamer. RNA polymerases. How could region 2.5 of σ make contact with the 5- TG-3 element? Analysis of protein–DNA hydrogen bonds Materials and methods in 28 regulatory protein complexes solved by X-ray crystallography revealed that nine out of ten hydrogen Strains, promoters and plasmids bonds involving glutamic acid involved interactions with The E.coli K12 host strains for this work were: the Δlac, recA strain DH5α (Hanahan, 1983), the rpoD (ts) strain 285c.recA donated by cytosine (Mandel-Gutfreund et al., 1995). This preferred R.Hayward (Harris et al., 1978), the rpoD repressible strain CAG20177 binding of glutamic acid to cytosine suggests that the (donated by M.Lonetto) and the BL21 (λDE3) strain which carries a protein side chain of Glu458 of σ interacts with cytosine copy of the inducible gene for T7 RNAP on its chromosome (Studier rather than the guanine at position –14 of our test pro- and Moffatt, 1986). The promoters KAB-TG, KAB-TC and KAB-TT were cloned as EcoRI–HindIII fragments in either the low copy number moters. Presumably the substitution of other bases, as at lacZ reporter vector pRW50 (Lodge et al., 1992) or the galK fusion the KAB-TC and KAB-TT promoters, leads to electrostatic vector, pAA121 (Kelsall et al., 1985). These promoters, shown in Figure repulsion that is alleviated by the EG458 substitution. 1, are described in detail by Chan and Busby (1989). Derivatives of Note that interactions between Gly458 and other base KAB-TG carrying further substitutions in the –10 or –35 hexamer pairs could occur through interactions involving the protein were made by site-directed mutagenesis and cloned as EcoRI–HindIII 4038 70 E.coli RNA polymerase σ subunit fragments into pRW50. By convention, promoter sequences are numbered given by Akira Ishihama. Plasmid pGEMD-EG458, encoding σ - with the transcription start site as 1, with upstream sequences prefixed EG458, was constructed by digesting the pKBσ derivative encoding with a ‘–’ sign. The plasmid, pKBσ, encoding σ was constructed rpoD-EG458 with BamHI and XhoI and cloning the resulting 444 bp using megaprimer PCR (Barne, 1997: details can be found on http:// fragment carrying rpoD-EG458 into pGEMD. E.coli BL21 (λDE3) cells www.biochemistry.bham.ac.uk/sdm/pkbsigma.html). The construct was were transformed with the different plasmids for overproducing RNAP checked by its ability to complement the E.coli rpoD temperature- subunits: over-expression and purification of the individual subunits and sensitive strain 285c.recA for growth at 42°C. To confirm the construct, reconstitution into functional RNAP was performed as detailed in the complete base sequence of the insert was determined using the Igarashi and Ishihama (1991). Pharmacia T7 Sequencing Kit. All preparations of plasmid DNA, restriction endonuclease treatments and ligations were performed as Electromobility shift assays described by Sambrook et al. (1989). Synthetic oligos were purchased Band shift assays were performed on labelled EcoRI–HindIII promoter from Alta Bioscience at The University of Birmingham. fragments carrying the KAB-TG, KAB-TC and KAB-TT promoters (Figure 1). Typically, labelled fragments (1 nM) were incubated at 37°C In vitro mutagenesis of rpoD and isolation of altered with different preparations of RNAP (100 nM) in transcription buffer specificity mutants of σ (200 mM HEPES pH 8.0, 50 mM MgCl , 500 mM K-glutamate, 10 mM PCR reaction mixes were set up in a final volume of 50 μl with various DTT, 250 μg/ml BSA) for 30 min to allow open complex formation. MgCl concentrations (1–10 mM), primers (1 μM) (upstream primer: Loading buffer (3 μl containing 0.1% bromophenol blue, 0.01% xylene 5-AGGCGTATCACGAGGCCCT-3 and downstream primer: 5-GGG- cyanole FF, 50% glycerol and 160 μg/ml heparin in 1 transcription GAAGCTTTTAATCGTCCAGGAAGCTACGC-3), template DNA buffer) was added immediately prior to loading on a 4.5% polyacrylamide (pKBσ) (50–100 pM), dNTPs (200 mM) (Pharmacia) and Taq DNA gel. After electrophoresis, gels were visualized by autoradiography. polymerase (1 unit) (Boehringer Mannheim). The cycle profile was: 39 cycles of 30 s at 94°C, 30 s at 58°C, 2 min at 72°C; 1 cycle of 30 s at DNA footprinting assays using permanganate 94°C, 30 s at 58°C, 4 min at 72°C. The PCR product carrying randomly DNA footprinting experiments were performed on PstI–HindIII promoter mutagenized rpoD was purified on a 2% agarose gel, digested with fragments (purified from pAA121 derivatives) carrying the KAB-TG, EcoRI and HindIII and recloned into pKBσ. Mutants were isolated KAB-TC and KAB-TT promoters. To examine the lower strand, the by screening against KAB-TC/pRW50 or KAB-TT/pRW50 in DH5α. HindIII ends were labelled with [γ- P]ATP (DuPont NEN) using T4 Transformants were selected on MacConkey indicator plates (Difco polynucleotide kinase (New England Biolabs). Typically, labelled DNA Laboratories) supplemented with lactose, ampicillin (80 μg/ml) and fragments (1 nM) were preincubated at 37°C with different RNAP tetracycline (30 μg/ml). Altered or relaxed specificity mutants of σ preparations (100 nM) in transcription buffer (as above) before addition were identified as red/pink colonies on the indicator plates. Plasmid of freshly prepared KMnO (protocols were as previously used by Chan DNA was recovered from candidates and introduced back into the screen et al., 1990). The reaction was stopped after 4 min by the addition of to check phenotypes. The full rpoD nucleotide sequence of each candidate KMnO stop solution (50 μl containing 3 M ammonium acetate, was determined. 0.1 mM EDTA, 1.5 M β-mercaptoethanol). After phenol/chloroform extraction and ethanol precipitation the samples were cleaved by treat- 70 70 70 Construction of σ -HA455, σ -IA457 and σ -TA459 ment with piperidine. Resulting fragments were electrophoresed on For these mutants, the appropriate base changes were introduced into calibrated 6% sequencing gels (Sequagel, National Diagnostics) that plasmid pKBσ using PCR. The base changes were introduced in an were visualized by autoradiography. initial round of PCR during the synthesis of a megaprimer. These megaprimers were then used in a second PCR reaction to incorporate a Acknowledgements downstream restriction site to aid cloning of the DNA fragments into pKBσ. The primers used were 5-GATGGTCTCAATCATAGCCACCG- We would like to thank Richard Hayward, Mike Lonetto and Akira GAATACGGAT-3,5-CTTGTTGATGGTCTCAGCCATATGCACCG- Ishihama for providing E.coli strains and plasmids. We are most grateful GAAT-3 and 5-GTTGAGCTTGTTGATGGCCTCAATCATATG- to the Wellcome Trust for generous funding of this work with a project CAC-3 for the construction of rpoD-HA455, rpoD-IA457 and rpoD- grant, and to the UK BBSRC for a studentship to K.A.B. TA459 respectively (the DNA modifications are highlighted in bold). These primers were used with an upstream primer (5-GCAACATCGGT- CTGA-3) flanking a unique BamHI restriction site in the template References pKBσ. The PCR products were gel purified and then used as primers in a second round of PCR in conjunction with a ‘downstream’ primer 5- Barne,K. (1997) Studies on promoter recognition by Escherichia coli GCTTCACGCGCGGTCAG-3 that flanks a unique XhoI restriction site. RNA polymerase. PhD thesis, The University of Birmingham, UK. The final PCR products were gel purified, restricted with BamHI and Bown,J., Barne,K., Minchin,S. and Busby,S. (1997) Extended –10 XhoI, cloned into plasmid pKBσ and the entire σ sequence was checked. promoters. Nucleic Acids Mol. Biol., 11, 41–52. Burns,H., Belyaeva,T., Busby,S. and Minchin,S. (1996) Temperature Determination of promoter activity in vivo dependence of open complex formation at two E. coli promoters with β-galactosidase expression in cells carrying different promoter::lacZ extended –10 sequences. Biochem. J., 317, 305–311. fusions cloned in pRW50, and different σ mutants cloned in pKBσ, Chan,B. and Busby,S. (1989) Recognition of nucleotide sequences at was measured as described by Lodge et al. (1992) and was taken to the Escherichia coli galactose operon P1 promoter by RNA reflect the activity of the cloned promoters. Assays were performed in polymerase. Gene, 84, 227–236. the E.coli strain CAG20177, carrying rpoD under the control of the Chan,B., Spassky,A. and Busby,S. (1990) The organization of open repressible trp promoter, which allows the level of transcription initiation complexes between Escherichia coli RNA polymerase and DNA in the absence of host σ to be determined (M.Lonetto, V.Rhodius and fragments carrying promoters either with or without consensus –35 C.Gross, personal communication). Pre-cultures were grown in Lennox region sequences. Biochem. J., 270, 141–148. broth plus ampicillin (80 μg/ml), tetracycline (30 μg/ml) and 3-β- Daniels,D., Zuber,P. and Losick,R. (1990) Two amino acids in an RNA indoleacrylic acid (0.2 mM). Assay cultures were grown in Lennox polymerase σ factor involved in the recognition of adjacent base pairs broth plus ampicillin (80 μg/ml) and tetracycline (30 μg/ml) (in the in the –10 region of a cognate promoter. Proc. Natl Acad. Sci. USA, absence of 3-β-indoleacrylic acid, expression of chromosomal rpoD is 87, 8075–8079. suppressed in strain CAG20177). Each assay was performed independ- Hanahan,D. (1983) Studies on transformation of Escherichia coli with ently at least six times and activities were reproducible to within 10%. plasmids. J. Mol. Biol., 166, 557–580. To exclude the possibility that some effects were due to changes in Harley,C.B. and Reynolds,R.P. (1987) Analysis of E. coli promoter plasmid copy number, plasmids were checked by gel electrophoresis. sequences. Nucleic Acids Res., 15, 2343–2361. Assays were also performed in the E.coli DH5α. Assayed in this strain, Harris,J.D., Heilig,J.S., Martinez,I.I., Calendar,R. and Isaksson,L.A. both the mutant σ and the wild type chromosomal σ were present. These (1978) Temperature-sensitive Escherichia coli mutant producing a assays resulted in the same conclusions as assays in strain CAG20177. temperature-sensitive σ subunit of DNA-dependent RNA polymerase. Proc. Natl Acad. Sci. USA, 75, 6177–6181. Reconstitution of RNA polymerase Hawley,D.K. and McClure,W.R. (1983) Compilation and analysis of Plasmids pGEMAX185, pGEMBC and pGEMD, which enable over- Escherichia coli promoter DNA sequences. Nucleic Acids Res., 11, production of the RNAP α, β and β, and σ subunits respectively were 2237–2255. 4039 K.A.Barne et al. Helmann,J.D. (1995) Compilation and analysis of Bacillus subtilis σ - dependent promoter sequences: Evidence for extended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res. 23, 2351–2360. Helmann,J.D. and Chamberlin,M.J. (1988) Structure and function of bacterial sigma factors. Annu. Rev. Biochem., 57, 839–872. Igarashi,K. and Ishihama,A. (1991) Bipartite functional map of the E. coli RNA polymerase α subunit: Involvement of the C-terminal region in transcription activation by cAMP-CRP. Cell, 65, 1015–1022. Keilty,S. and Rosenberg,M. (1987) Constitutive function of a positively regulated promoter reveals new sequences essential for activity. J. Biol. Chem., 262, 6389–6395. Kelsall,A., Evans,C. and Busby,S. (1985) A plasmid vector that allows fusion of the Escherichia coli galactokinase gene to the translation start point of other genes. FEBS Lett., 180, 155–159. Kumar,A., Malloch,R.A., Fujita,N., Smillie,D.A., Ishihama,A. and Hayward,R.S. (1993) The minus 35-recognition region of Escherichia coli sigma 70 is inessential for initiation of transcription at an ‘Extended Minus 10’ promoter. J. Mol. Biol., 232, 406–418. Lodge,J., Fear,J., Busby,S., Gunasekaran,P. and Kamini,N.R. (1992) Broad host range plasmids carrying the Escherichia coli lactose and galactose operons. FEMS Microbiol. Lett., 95, 271–276. Malhotra,A., Severinova,E. and Darst,S.A. (1996) Crystal-structure of a σ subunit fragment from Escherichia coli RNA polymerase. Cell, 87, 127–136. Mandel-Gutfreund,Y., Schueler,O. and Margalit,H. (1995) Compre- hensive analysis of hydrogen bonds in regulatory protein–DNA complexes: In search of common principles. J. Mol. Biol., 253, 370–382. Minchin,S. and Busby,S. (1993) Location of close contacts between Escherichia coli RNA polymerase and guanine residues at promoters either with or without consensus –35 region sequences. Biochem. J., 289, 771–775. Ponnambalam,S., Webster,C., Bingham,A. and Busby,S. (1986) Transcription initiation at the Escherichia coli galactose operon promoters in the absence of the normal –35 region sequences. J. Biol. Chem., 261, 16043–16048. Sambrook,J., Fritsch,E. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Siegele,D.A., Hu,J.C., Walter,W.A. and Gross,C.A. (1989) Altered promoter recognition by mutant forms of the σ subunit of Escherichia coli RNA polymerase. J. Mol. Biol., 206, 591–603. Studier,F.W. and Moffatt,B.A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol., 189, 113–130. Tatti,K.M., Jones,C.H. and Moran,C.P. (1991) Genetic evidence for interaction of sigma-E with the spoIIID promoter in B. subtilis. J. Bacteriol., 173, 7828–7833. Waldburger,C., Gardella,T., Wong,R. and Susskind,M.M. (1990) Changes in conserved region 2 of Escherichia coli σ affecting promoter recognition. J. Mol. Biol., 215, 267–276. Yura,T. (1996) Regulation and conservation of the heat-shock transcription factor σ . Genes to Cells, 1, 277–284. Zuber,P., Healy,J., Carter,H.L., Cutting,S., Moran,C.P.,Jr and Losick,R. (1989) Mutation changing the specificity of an RNA polymerase sigma factor. J. Mol. Biol., 206, 605–614. Received on January 27, 1997; revised on March 18, 1997 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Region 2.5 of the Escherichia coli RNA polymerase σ70 subunit is responsible for the recognition of the ‘extended −10’ motif at promoters

Download PDF
7 pages

Loading next page...
 
/lp/springer-journals/region-2-5-of-the-escherichia-coli-rna-polymerase-70-subunit-is-mwKPf50dEp

References (43)

Publisher
Springer Journals
Copyright
Copyright © European Molecular Biology Organization 1997
ISSN
0261-4189
eISSN
1460-2075
DOI
10.1093/emboj/16.13.4034
Publisher site
See Article on Publisher Site

Abstract

The EMBO Journal Vol.16 No.13 pp.4034–4040, 1997 Region 2.5 of the Escherichia coli RNA polymerase σ subunit is responsible for the recognition of the ‘extended –10’ motif at promoters Sequence analysis of bacterial σ factors has revealed Kerry A.Barne, Jon A.Bown, four conserved important regions, regions 1, 2, 3 and 4 Stephen J.W.Busby and (reviewed by Helmann and Chamberlin, 1988). The Stephen D.Minchin analysis argues that region 2 is subdivided into four School of Biochemistry, The University of Birmingham, Edgbaston, segments (2.1–2.4) and region 4 is divided into two (4.1 Birmingham B15 2TT, UK and 4.2). A series of studies, based mainly on suppression Corresponding author genetics, has shown that sub-region 2.4 (located at the C- e-mail: [email protected] terminal end of region 2) interacts directly with promoter – 10 hexamer elements, whilst sub-region 4.2 (located at At some bacterial promoters, a 5-TG-3 sequence the C-terminal end of region 4) interacts directly with element, located one base upstream of the –10 hexamer promoter –35 hexamer elements. For example, Waldburger element, provides an essential motif necessary for et al. (1990), found that a Gln to His change at position transcription initiation. We have identified a mutant 70 437 of σ , within region 2.4, suppressed the effects of a of the Escherichia coli RNA polymerase σ subunit T–A to C–G ‘down’ mutation at position –12 in the –10 that has an altered preference for base sequences in hexamer of the ant and lac promoters. Similarly, changing this ‘extended –10’ region. We show that this mutant Thr to Ile at position 440 increases the activity of promoters σ subunit substantially increases transcription from containingaGorC instead of T at this position (Siegele promoters bearing 5-TC-3 or 5-TT-3 instead of a et al., 1989; see also Zuber et al., 1989; Daniels et al., 5-TG-3 motif, located one base upstream of the –10 1990; Tatti et al., 1991). Recently, the crystal structure of hexamer. The mutant results from a single base pair 70 a σ fragment that includes region 2.4 was solved substitution in the rpoD gene that causes a Glu to Gly (Malhotra et al., 1996), showing that region 2.4 contains an change at position 458 of σ . This substitution identifies amphipathic α-helix, with residues 437 and 440 positioned a functional region in σ that is immediately adjacent such that they can both contact bases at the –12 position. to the well-characterized region 2.4 (positions 434–453, A number of activator-independent promoters have now previously shown to contact the –10 hexamer). From been reported where specific –35 hexamer contacts are these results, we conclude that this region (which we not required for transcription initiation (reviewed by Bown name region 2.5) is involved in contacting the 5-TG-3 et al., 1997). Transcription initiation at these promoters is motif found at some bacterial promoters: thus, dependent on a supplementary sequence element, 5-TG-3, extended –10 regions are recognized by an extended located one base upstream of the –10 hexamer. This results region 2 of the RNA polymerase σ subunit. in an ‘extended’ –10 element, 5-TGnTATAAT-3, which Keywords: promoter recognition/RNA polymerase/sigma appears to create alternative contact points for RNAP, subunits/transcription initiation most likely via the σ subunit, such that transcription can be initiated in the absence of specific –35 region contacts (Ponnambalam et al., 1986; Keilty and Rosenberg, 1987; Introduction Burns et al., 1996). In support of this, Kumar et al. (1993) showed that RNAP containing an altered form of σ lacking Transcription initiation is a complex process which the C-terminal 84 amino acids (including region 4.2, involves rallying many diverse molecular interactions which is responsible for contacting the –35 hexamer) to facilitate promoter recognition by RNA polymerase could initiate transcription at a consensus extended –10 (RNAP) and DNA unwinding around the transcription promoter (but was unable to initiate transcription at a start-point. In bacteria, one RNAP with subunit composi- typical promoter with –10 and –35 regions resembling the tion α ββ (E) is responsible for all transcription. However, consensus but without the 5-TG-3 extension). In this although this core enzyme is capable of transcript elonga- work we describe an experiment designed to identify the tion, it cannot initiate transcription without enlisting a segment of the σ subunit involved in making contact specific transcription factor, σ.Itis σ that directs RNAP with the 5-TG-3 extension. Our strategy was to start to specific promoters, and σ is also involved in promoter with a promoter where activity was completely dependent melting. Escherichia coli contains many classes of pro- on a 5-TG-3 extension, to inactivate the promoter by moter, all of which are recognized by separate σ factors. making base changes in the extension, and then to search The major σ factor, σ , makes sequence-specific contacts for mutant σ derivatives that allowed transcription initi- with two hexamer elements at promoters, the –10 and –35 sequences, that are separated by an optimal spacing of ation at the inactivated promoters. This experiment identi- 17 bp (Hawley and McClure, 1983; Harley and Reynolds, fies a region of σ immediately adjacent to region 2.4 1987): the consensus sequences for –10 and –35 hexamers which is involved in the recognition of the 5-TG-3 motif. are 5-TATAAT-3 and 5-TTGACA-3, respectively We report both in vivo and in vitro data to support our (Figure 1). conclusions. 4034 © Oxford University Press 70 E.coli RNA polymerase σ subunit Fig. 1. Base sequence of the KAB-TG, KAB-TC and KAB-TT promoters used in this work. The –10 and –35 hexamers are highlighted in bold: the 5-TG-3 extension at position –14/–15 is underlined. Results Isolation and characterization of σ mutants The E.coli galP1 promoter is an example of an extended –10 promoter, having a –35 region with little homology to the consensus (Ponnambalam et al., 1986) and mutations in the 5-TG-3 motif render galP1 inactive (Chan et al., 1990). Both methylation protection and interference studies suggest that RNAP makes direct contact with the G–C base pair at position –14 of galP1 (within the 5- TG-3 motif) (Minchin and Busby, 1993). The starting point of this work was the KAB set of three semi-synthetic promoters, based on the galP1 sequence (Figure 1). Activity of the KAB-TG promoter is dependent on a 5- TG-3 motif (at positions –14 and –15) upstream of the –10 hexamer, 5-TATGGT-3. In the KAB-TC and KAB- TT promoters, the 5-TG-3 motif is changed to 5-TC- 3 and 5-TT-3 respectively, and this greatly reduces promoter activity. Our aim was to identify σ mutants that would permit RNAP to serve the KAB-TC and KAB-TT promoters. To do this, the rpoD gene (cloned in plasmid pKBσ ) was amplified by error-prone PCR, using conditions such that, Fig. 2. The activity of the KAB-TG, KAB-TC and KAB-TT promoters 70 70 on average, each amplified DNA fragment encoding rpoD with σ (shown in black) and σ -EG458 (shown in grey) in trans. EcoRI–HindIII fragments carrying the promoters were cloned into the contained one base pair substitution. These fragments 70 70 lacZ expression vector, pRW50 and β-galactosidase levels are taken as were then cloned into pKBσ , generating a library of σ a direct measure of the promoter activity. In the histogram, activity mutants, which was then screened for σ derivatives that levels are expressed as a percentage of the wild type activity at each permitted increased transcription initiation at the KAB- promoter. The activity (nmole ONPG hydrolysed per min per mg dry TC and KAB-TT promoters. To facilitate this screen, the weight bacteria) of Eσ at KAB-TG, KAB-TC and KAB-TT was 2570, 51 and 49 respectively. three promoters KAB-TG, KAB-TC and KAB-TT were each fused to lacZ. As expected, cells carrying the KAB- TG::lacZ fusion exhibited a Lac phenotype (red colonies KAB-TG, KAB-TC and KAB-TT was assessed, exploiting on MacConkey indicator plates) whilst cells carrying the the lac fusions described above. Measurements were made – 70 KAB-TC::lacZ or KAB-TT::lacZ fusions exhibited a Lac in the strain CAG20177 which carries host σ under the phenotype (white colonies on MacConkey indicator control of the repressible trp promoter. Plasmid pKBσ 70 70 plates). Candidates from the σ mutant library were encoding either wild type or σ -EG458 was introduced screened for the ability to confer a red/pink Lac phenotype and β-galactosidase assays were performed on cells grown on cells carrying the KAB-TC::lacZ or KAB-TT::lacZ in conditions in which the host-encoded σ was repressed. fusions. Thirty thousand colonies from 10 independent Thus, the level of transcription from each promoter as a 70 70 PCR libraries were screened and two mutant σ candidates result of specific initiation by either Eσ or Eσ -EG458 (from different libraries) were isolated. We confirmed that could be determined. The results, presented in Figure 2, both of these candidates made stable functional sigma by show that the EG458 substitution significantly affects the checking their ability to complement the rpoD tempera- activity of all three promoters. The activity of promoter ture-sensitive mutation in the E.coli strain 285c.recA, KAB-TG is reduced by 15% with Eσ -EG458. In contrast, during growth at 42°C. The complete nucleotide sequence the activity of the mutant promoter derivatives, KAB-TC of rpoD for both candidates was determined. Both con- and KAB-TT is increased by 2.5- to 3-fold with Eσ - tained the same single base change, an A–T to G–C EG458 (compared with Eσ ). As a control, we made two transition at position 1373 of the rpoD sequence, which further derivatives of promoter KAB-TG. In one, the alters a GAG codon to GGG, and results in the substitution activity of the promoter was reduced 50-fold by the of a glutamic acid for glycine at position 458 of σ . introduction of a single change in the –10 hexamer (from 5-TATGGT-3 to 5-CATGGT-3) and, in the other, a Specificity of Eσ -EG458 similar effect was produced by altering the –35 hexamer The activity of RNAP holoenzyme carrying σ with the element (from 5-TAGACA-3 to 5-TAGGTA-3). The EG458 substitution (Eσ -EG458) at the three promoters effects of these promoter mutations could not be sup- 4035 K.A.Barne et al. Fig. 4. An autoradiograph of a gel to show the pattern of cleavage resulting from potassium permanganate and piperidine treatment of PstI–HindIII fragments carrying KAB-TG (lanes 1, 4, 7 and 10), KAB-TC (lanes 2, 5, 8 and 11) and KAB-TT (lanes 3, 6, 9 and 12). Fig. 3. An autoradiograph of gel mobility shift assays to separate Experiments were performed with 100 nM core enzyme (lanes 1–3), promoter fragments bound to RNA polymerase from free fragments. 70 70 100 nM Eσ (lanes 4–6), 100 nM Eσ -EG458 (lanes 7–9) and RNA polymerase was pre-incubated with labelled fragment carrying labelled fragment alone (lanes 10–12). Lane M is a calibration marker KAB-TG (lanes 1–4), KAB-TC (lanes 5–8) and KAB-TT (lanes 9–12). made by the Maxam–Gilbert G-specific sequence reaction on one of Experiments were performed with 100 nM core enzyme (lanes 1, 5 70 70 the promoter constructions. The gels are calibrated with the and 9), 100 nM Eσ (lanes 2, 6 and 10), 100 nM Eσ -EG458 (lanes transcription start point as 1. 3, 7 and 11) and labelled fragment alone (lanes 4, 8 and 12). The samples were heparin challenged before loading on the gel. The faint retarded bands seen with core enzyme are likely to be due to a trace contamination of σ . 70 70 with E, Eσ and Eσ -EG458. Single-stranded T residues in the open complexes could be modified by KMnO and pressed by the introduction of plasmid pKBσ encoding the points of modification were detected by cleavage with σ -EG458. This shows that the effect of the EG458 piperidine and analysis on a denaturing polyacrylamide substitution is position specific. gel. The results (Figure 4) show that all open complexes form at the same position on the DNA, confirming that Improved interaction of Eσ -EG458 with the RNAP is binding to the promoters as expected. Moreover, KAB-TC and KAB-TT promoters the pattern and location of unwinding in the open com- 70 70 70 70 The binding of Eσ and Eσ -EG458 to promoters can plexes formed by both Eσ and Eσ -EG458 are the same be assessed simply using gel mobility shift assays. Labelled at the KAB-TG, KAB-TC and KAB-TT promoters, with DNA fragments carrying the promoters, KAB-TG, KAB- DNA opening confined to positions –11 to 3. The TC or KAB-TT were incubated with either purified core degree of promoter opening by the two forms of RNAP 70 70 enzyme (E), purified Eσ or purified Eσ -EG458 at 37°C holoenzyme, as detected by KMnO , corroborates the for 30 min to allow open complex formation. The resulting β-galactosidase and gel mobility shift assay data. Wild complexes were challenged with heparin (to test the type holoenzyme, Eσ , readily forms open complexes at stability of the RNAP–DNA complexes) before separation KAB-TG, a very small amount of opening is observed at on a native polyacrylamide gel. The assays (Figure 3) KAB-TC, and no opening is found at KAB-TT (compare clearly show that core enzyme (E) is unable to form stable lanes 4, 5 and 6). In contrast, with Eσ -EG458, open complexes with any promoter DNA (lanes 1, 5 and 9) complexes form at all three promoters, confirming that and that the binding of holo-RNAP (Eσ ) to KAB-TG is the EG458 substitution confers a relaxed specificity on greater than to KAB-TC or KAB-TT (lanes 2, 6 and 10). σ and allows RNAP to initiate transcription at promoters 70 70 Comparing the binding of Eσ and Eσ -EG458, at both carrying ‘down’ mutations in the 5-TG-3 motif of KAB-TC and KAB-TT in this assay, Eσ binds relatively extended –10 promoters. poorly, whereas Eσ -EG458 has a greater affinity (com- pare lanes 6 and 10 with lanes 7 and 11). The assay shows The region between conserved regions 2.4 and 3 70 70 that Eσ -EG458 can still bind to the KAB-TG promoter in σ : effects of the HA455 substitution (compare lanes 2 and 3), suggesting that EG458 must be Figure 5 shows an alignment of amino acid sequences 70 70 a relaxed specificity mutant of σ . around E458 in σ with the corresponding sequence in nine bacterial σ factors. E458 is located in the spacer Characterization of open complexes formed by between conserved regions 2.4 and 3. However, it is clear 70 70 Eσ and Eσ -EG458 from the figure that a number of amino acids in this region Since DNA unwinding is an important step in transcription (including E458) are well conserved between different σ 70 70 initiation, we studied the interactions of Eσ and Eσ - factors. For example, the histidine residue at position 455 EG458 in the open complexes by probing with potassium is particularly conserved. We investigated the effect of permanganate (as described by Chan et al., 1990). Open changing this residue in σ to alanine on the activity of complexes were formed by preincubating DNA fragments the three KAB promoters, together with the effects of carrying the KAB-TG, KAB-TC and KAB-TT promoters alanine substitutions at the neighbouring less-conserved 4036 70 E.coli RNA polymerase σ subunit Fig. 5. A PILEUP [Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin] of amino acid sequences of bacterial σ factors between region 2.4 and the start of region 3. The location of E458 and H455 is indicated by open boxes. The position of region 2.4 and the 70 70 start of region 3 is indicated by shaded boxes. The lineup includes: E.coli σ (RP70_ECOLI), Salmonella typhimurium σ (RP70_SALTY), 70 80 A Pseudomonas aeruginosa σ (RP70_PSEAE), Myxococcus xanthus σ (RP80_MYXXA), Bacillus subtilis σ (RPSA_BACSU), Chlamydia 70 HrdB A trachomatis σ (RP70_CHLTR), Streptomyces coelicolor σ (HRDB_STRCO), Anabaena sp. σ (RPSA_ANASP) and Streptomyces aureofaciens σ (RPSA_STRAU). isoleucine 457 and threonine 459. The mutants σ -HA455, 70 70 σ -IA457 and σ -TA459 were constructed in plasmid pKBσ and their ability to drive transcription initiation at the KAB-TG, KAB-TC and KAB-TT promoters was assayed in strain CAG20177 as described above. The results (Figure 6) show that alanine substitutions at either isoleucine 457 or threonine 459 have little or no effect on the ability of RNA polymerase to initiate transcription. In contrast, substitution of histidine 455 results in a 50% reduction in expression from the KAB-TG promoter and smaller effects at the KAB-TC and KAB-TT promoters. We conclude that histidine 455 of σ is also involved in interactions with the 5-TG-3 motif at extended –10 promoters. Discussion It is well documented that the σ subunits of bacterial RNA polymerases are primarily responsible for making sequence-specific contacts with promoter DNA. In pre- vious studies, particular amino acids in regions 2.4 and 4.2 were shown to be responsible for contacting the –10 Fig. 6. The activity of the KAB-TG, KAB-TC and KAB-TT promoters and –35 hexamers respectively. At extended –10 pro- 70 70 with Eσ (shown in dark grey), Eσ -HA455 (shown in black), moters, contacts between region 4.2 of σ and the –35 70 70 Eσ -IA457 (shown in white) and Eσ -TA459 (shown in light grey). region appear to be unimportant for promoter activity, and EcoRI–HindIII fragments carrying the promoters were cloned into the lacZ expression vector, pRW50. The β-galactosidase level was these promoters function because σ makes supplementary measured and is taken to be a direct measure of the activity of the contacts with a 5-TG-3 motif just upstream from the cloned promoter. In the histogram, levels are expressed as a percentage –10 hexamer. To identify the part of σ that interacts of the wild type activity at each promoter. with the 5-TG-3 motif of extended –10 promoters, we developed a genetic screening system for selecting and analysing a mutant form of σ that suppressed ‘down’ motif. This conclusion was corroborated by the gel mobil- mutations in the G–C base pair at position –14 of the ity shift assays shown in Figure 3, and potassium perman- extended –10 region of the KAB-TG promoter. The entire ganate footprints shown in Figure 4. rpoD gene which encodes a product of 613 amino acids Glu458 of σ lies in a region just downstream of was randomly mutated, and yet the screening system twice conserved region 2.4 (amino acids 434–453) which is generated the same σ mutant carrying a single amino known to be involved in recognition of the –10 hexamer acid substitution at position 458. This substitution partially of promoters (Helmann and Chamberlin, 1988; Siegele restores the activity of the KAB-TC and KAB-TT pro- et al., 1989; Daniels et al., 1990). Since Glu458 and other moters, containing a C–G or T–A base pair at position – residues in this region are well conserved between different 14 respectively. The activities of these promoters is σ factors (Figure 5), we suggest that this region, which increased 2.5- to 3-fold in the presence of Eσ -EG458 we propose to call region 2.5, has a function, which is to compared with Eσ , whilst, at the starting KAB-TG recognize 5-TG-3 elements upstream of promoter –10 promoter, activity is reduced to ~85% of that with Eσ . hexamer elements. We conclude that extended –10 regions The mutant σ was stable, retained its core binding affinity are recognized by an extension of region 2 of σ (region and no difference in electrophoretic mobility could be 2.5). One interesting exception, where Glu458 is not 70 32 detected. The simplest conclusion is that Glu458 of σ conserved, is σ , the heat shock σ factor, where this is involved in recognition of the G–C bp of the 5-TG-3 region has a different function and is involved in 4037 K.A.Barne et al. Fig. 7. The amino acid sequence of σ with the four conserved regions highlighted by shading. Regions 1, 2, 3 and 4 are divided into sub-regions. The figure also shows a consensus promoter and indicates that the –10 and –35 promoter elements are recognized by regions 2.4 and 4.2 respectively. Region 2.5 is shown making direct interaction with the 5-TG-3 motif located one base pair upstream of the –10 element. Note that the precise boundaries of different regions are arbitrary: here, we define region 2.5 as the entire sequence between region 2.4 and region 3.1. chaperone-mediated post-translational regulation of heat backbone, accounting for how Eσ -EG458 can drive shock σ levels (Yura, 1996). transcription at the KAB-TC and KAB-TT promoters. It should be noted that a mutant σ subunit carrying Malhotra et al. (1996) recently solved the crystal structure an amino acid change at position 458 had previously been of a σ fragment from amino acid 114 to 448. This isolated by Waldburger et al. (1990), as a suppressor of fragment contains part of conserved region 1.2 and all but mutant ant promoters. The P promoter contains a the C-terminal five residues of conserved region 2. Region ant consensus –35 hexamer and a –10 hexamer that differs 2.4 was found to form an α-helix with the amino acids from consensus at only one position. The σ mutant, rpoD- involved in recognition of the –10 hexamer being solvent EK458, was isolated as a suppressor of a down mutation exposed on one face of the helix. Figure 7 is a model at –12, and also as a suppressor of down mutations at showing the orientation of region 2.4 with respect to the –10 and –9. EK458 causes a small increase in the activity DNA (Malhotra et al., 1996), and also the predicted of 16 P promoters with down mutations at different orientation of region 4.2. The model predicts that amino ant positions in both the –10 and –35 hexamers, and it acids involved in contacting the 5-TG-3 motif would be was suggested that the effects of EK458 on promoter in the region defined by EG458 (which we dub region recognition were position independent. Our result provides 2.5). Finally, extended –10 promoters are ubiquitous in a possible explanation for the observations of Waldburger nature, occurring in most bacteria. Indeed, nearly half of et al. (1990). The P promoter is not an extended –10 the total promoters in some Gram-positive microorganisms ant promoter, and all the promoters used in their study carried can be classified as extended –10 promoters (Helmann, a5-TA-3 at position –15/–14. It is likely that the EK458 1995). We conclude that region 2.5 of σ plays an important substitution is ‘suppressing’ the effect of having 5-TA- role in setting the promoter preferences of many bacterial 3 rather than 5-TG-3 upstream of the –10 hexamer. RNA polymerases. How could region 2.5 of σ make contact with the 5- TG-3 element? Analysis of protein–DNA hydrogen bonds Materials and methods in 28 regulatory protein complexes solved by X-ray crystallography revealed that nine out of ten hydrogen Strains, promoters and plasmids bonds involving glutamic acid involved interactions with The E.coli K12 host strains for this work were: the Δlac, recA strain DH5α (Hanahan, 1983), the rpoD (ts) strain 285c.recA donated by cytosine (Mandel-Gutfreund et al., 1995). This preferred R.Hayward (Harris et al., 1978), the rpoD repressible strain CAG20177 binding of glutamic acid to cytosine suggests that the (donated by M.Lonetto) and the BL21 (λDE3) strain which carries a protein side chain of Glu458 of σ interacts with cytosine copy of the inducible gene for T7 RNAP on its chromosome (Studier rather than the guanine at position –14 of our test pro- and Moffatt, 1986). The promoters KAB-TG, KAB-TC and KAB-TT were cloned as EcoRI–HindIII fragments in either the low copy number moters. Presumably the substitution of other bases, as at lacZ reporter vector pRW50 (Lodge et al., 1992) or the galK fusion the KAB-TC and KAB-TT promoters, leads to electrostatic vector, pAA121 (Kelsall et al., 1985). These promoters, shown in Figure repulsion that is alleviated by the EG458 substitution. 1, are described in detail by Chan and Busby (1989). Derivatives of Note that interactions between Gly458 and other base KAB-TG carrying further substitutions in the –10 or –35 hexamer pairs could occur through interactions involving the protein were made by site-directed mutagenesis and cloned as EcoRI–HindIII 4038 70 E.coli RNA polymerase σ subunit fragments into pRW50. By convention, promoter sequences are numbered given by Akira Ishihama. Plasmid pGEMD-EG458, encoding σ - with the transcription start site as 1, with upstream sequences prefixed EG458, was constructed by digesting the pKBσ derivative encoding with a ‘–’ sign. The plasmid, pKBσ, encoding σ was constructed rpoD-EG458 with BamHI and XhoI and cloning the resulting 444 bp using megaprimer PCR (Barne, 1997: details can be found on http:// fragment carrying rpoD-EG458 into pGEMD. E.coli BL21 (λDE3) cells www.biochemistry.bham.ac.uk/sdm/pkbsigma.html). The construct was were transformed with the different plasmids for overproducing RNAP checked by its ability to complement the E.coli rpoD temperature- subunits: over-expression and purification of the individual subunits and sensitive strain 285c.recA for growth at 42°C. To confirm the construct, reconstitution into functional RNAP was performed as detailed in the complete base sequence of the insert was determined using the Igarashi and Ishihama (1991). Pharmacia T7 Sequencing Kit. All preparations of plasmid DNA, restriction endonuclease treatments and ligations were performed as Electromobility shift assays described by Sambrook et al. (1989). Synthetic oligos were purchased Band shift assays were performed on labelled EcoRI–HindIII promoter from Alta Bioscience at The University of Birmingham. fragments carrying the KAB-TG, KAB-TC and KAB-TT promoters (Figure 1). Typically, labelled fragments (1 nM) were incubated at 37°C In vitro mutagenesis of rpoD and isolation of altered with different preparations of RNAP (100 nM) in transcription buffer specificity mutants of σ (200 mM HEPES pH 8.0, 50 mM MgCl , 500 mM K-glutamate, 10 mM PCR reaction mixes were set up in a final volume of 50 μl with various DTT, 250 μg/ml BSA) for 30 min to allow open complex formation. MgCl concentrations (1–10 mM), primers (1 μM) (upstream primer: Loading buffer (3 μl containing 0.1% bromophenol blue, 0.01% xylene 5-AGGCGTATCACGAGGCCCT-3 and downstream primer: 5-GGG- cyanole FF, 50% glycerol and 160 μg/ml heparin in 1 transcription GAAGCTTTTAATCGTCCAGGAAGCTACGC-3), template DNA buffer) was added immediately prior to loading on a 4.5% polyacrylamide (pKBσ) (50–100 pM), dNTPs (200 mM) (Pharmacia) and Taq DNA gel. After electrophoresis, gels were visualized by autoradiography. polymerase (1 unit) (Boehringer Mannheim). The cycle profile was: 39 cycles of 30 s at 94°C, 30 s at 58°C, 2 min at 72°C; 1 cycle of 30 s at DNA footprinting assays using permanganate 94°C, 30 s at 58°C, 4 min at 72°C. The PCR product carrying randomly DNA footprinting experiments were performed on PstI–HindIII promoter mutagenized rpoD was purified on a 2% agarose gel, digested with fragments (purified from pAA121 derivatives) carrying the KAB-TG, EcoRI and HindIII and recloned into pKBσ. Mutants were isolated KAB-TC and KAB-TT promoters. To examine the lower strand, the by screening against KAB-TC/pRW50 or KAB-TT/pRW50 in DH5α. HindIII ends were labelled with [γ- P]ATP (DuPont NEN) using T4 Transformants were selected on MacConkey indicator plates (Difco polynucleotide kinase (New England Biolabs). Typically, labelled DNA Laboratories) supplemented with lactose, ampicillin (80 μg/ml) and fragments (1 nM) were preincubated at 37°C with different RNAP tetracycline (30 μg/ml). Altered or relaxed specificity mutants of σ preparations (100 nM) in transcription buffer (as above) before addition were identified as red/pink colonies on the indicator plates. Plasmid of freshly prepared KMnO (protocols were as previously used by Chan DNA was recovered from candidates and introduced back into the screen et al., 1990). The reaction was stopped after 4 min by the addition of to check phenotypes. The full rpoD nucleotide sequence of each candidate KMnO stop solution (50 μl containing 3 M ammonium acetate, was determined. 0.1 mM EDTA, 1.5 M β-mercaptoethanol). After phenol/chloroform extraction and ethanol precipitation the samples were cleaved by treat- 70 70 70 Construction of σ -HA455, σ -IA457 and σ -TA459 ment with piperidine. Resulting fragments were electrophoresed on For these mutants, the appropriate base changes were introduced into calibrated 6% sequencing gels (Sequagel, National Diagnostics) that plasmid pKBσ using PCR. The base changes were introduced in an were visualized by autoradiography. initial round of PCR during the synthesis of a megaprimer. These megaprimers were then used in a second PCR reaction to incorporate a Acknowledgements downstream restriction site to aid cloning of the DNA fragments into pKBσ. The primers used were 5-GATGGTCTCAATCATAGCCACCG- We would like to thank Richard Hayward, Mike Lonetto and Akira GAATACGGAT-3,5-CTTGTTGATGGTCTCAGCCATATGCACCG- Ishihama for providing E.coli strains and plasmids. We are most grateful GAAT-3 and 5-GTTGAGCTTGTTGATGGCCTCAATCATATG- to the Wellcome Trust for generous funding of this work with a project CAC-3 for the construction of rpoD-HA455, rpoD-IA457 and rpoD- grant, and to the UK BBSRC for a studentship to K.A.B. TA459 respectively (the DNA modifications are highlighted in bold). These primers were used with an upstream primer (5-GCAACATCGGT- CTGA-3) flanking a unique BamHI restriction site in the template References pKBσ. The PCR products were gel purified and then used as primers in a second round of PCR in conjunction with a ‘downstream’ primer 5- Barne,K. (1997) Studies on promoter recognition by Escherichia coli GCTTCACGCGCGGTCAG-3 that flanks a unique XhoI restriction site. RNA polymerase. PhD thesis, The University of Birmingham, UK. The final PCR products were gel purified, restricted with BamHI and Bown,J., Barne,K., Minchin,S. and Busby,S. (1997) Extended –10 XhoI, cloned into plasmid pKBσ and the entire σ sequence was checked. promoters. Nucleic Acids Mol. Biol., 11, 41–52. Burns,H., Belyaeva,T., Busby,S. and Minchin,S. (1996) Temperature Determination of promoter activity in vivo dependence of open complex formation at two E. coli promoters with β-galactosidase expression in cells carrying different promoter::lacZ extended –10 sequences. Biochem. J., 317, 305–311. fusions cloned in pRW50, and different σ mutants cloned in pKBσ, Chan,B. and Busby,S. (1989) Recognition of nucleotide sequences at was measured as described by Lodge et al. (1992) and was taken to the Escherichia coli galactose operon P1 promoter by RNA reflect the activity of the cloned promoters. Assays were performed in polymerase. Gene, 84, 227–236. the E.coli strain CAG20177, carrying rpoD under the control of the Chan,B., Spassky,A. and Busby,S. (1990) The organization of open repressible trp promoter, which allows the level of transcription initiation complexes between Escherichia coli RNA polymerase and DNA in the absence of host σ to be determined (M.Lonetto, V.Rhodius and fragments carrying promoters either with or without consensus –35 C.Gross, personal communication). Pre-cultures were grown in Lennox region sequences. Biochem. J., 270, 141–148. broth plus ampicillin (80 μg/ml), tetracycline (30 μg/ml) and 3-β- Daniels,D., Zuber,P. and Losick,R. (1990) Two amino acids in an RNA indoleacrylic acid (0.2 mM). Assay cultures were grown in Lennox polymerase σ factor involved in the recognition of adjacent base pairs broth plus ampicillin (80 μg/ml) and tetracycline (30 μg/ml) (in the in the –10 region of a cognate promoter. Proc. Natl Acad. Sci. USA, absence of 3-β-indoleacrylic acid, expression of chromosomal rpoD is 87, 8075–8079. suppressed in strain CAG20177). Each assay was performed independ- Hanahan,D. (1983) Studies on transformation of Escherichia coli with ently at least six times and activities were reproducible to within 10%. plasmids. J. Mol. Biol., 166, 557–580. To exclude the possibility that some effects were due to changes in Harley,C.B. and Reynolds,R.P. (1987) Analysis of E. coli promoter plasmid copy number, plasmids were checked by gel electrophoresis. sequences. Nucleic Acids Res., 15, 2343–2361. Assays were also performed in the E.coli DH5α. Assayed in this strain, Harris,J.D., Heilig,J.S., Martinez,I.I., Calendar,R. and Isaksson,L.A. both the mutant σ and the wild type chromosomal σ were present. These (1978) Temperature-sensitive Escherichia coli mutant producing a assays resulted in the same conclusions as assays in strain CAG20177. temperature-sensitive σ subunit of DNA-dependent RNA polymerase. Proc. Natl Acad. Sci. USA, 75, 6177–6181. Reconstitution of RNA polymerase Hawley,D.K. and McClure,W.R. (1983) Compilation and analysis of Plasmids pGEMAX185, pGEMBC and pGEMD, which enable over- Escherichia coli promoter DNA sequences. Nucleic Acids Res., 11, production of the RNAP α, β and β, and σ subunits respectively were 2237–2255. 4039 K.A.Barne et al. Helmann,J.D. (1995) Compilation and analysis of Bacillus subtilis σ - dependent promoter sequences: Evidence for extended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res. 23, 2351–2360. Helmann,J.D. and Chamberlin,M.J. (1988) Structure and function of bacterial sigma factors. Annu. Rev. Biochem., 57, 839–872. Igarashi,K. and Ishihama,A. (1991) Bipartite functional map of the E. coli RNA polymerase α subunit: Involvement of the C-terminal region in transcription activation by cAMP-CRP. Cell, 65, 1015–1022. Keilty,S. and Rosenberg,M. (1987) Constitutive function of a positively regulated promoter reveals new sequences essential for activity. J. Biol. Chem., 262, 6389–6395. Kelsall,A., Evans,C. and Busby,S. (1985) A plasmid vector that allows fusion of the Escherichia coli galactokinase gene to the translation start point of other genes. FEBS Lett., 180, 155–159. Kumar,A., Malloch,R.A., Fujita,N., Smillie,D.A., Ishihama,A. and Hayward,R.S. (1993) The minus 35-recognition region of Escherichia coli sigma 70 is inessential for initiation of transcription at an ‘Extended Minus 10’ promoter. J. Mol. Biol., 232, 406–418. Lodge,J., Fear,J., Busby,S., Gunasekaran,P. and Kamini,N.R. (1992) Broad host range plasmids carrying the Escherichia coli lactose and galactose operons. FEMS Microbiol. Lett., 95, 271–276. Malhotra,A., Severinova,E. and Darst,S.A. (1996) Crystal-structure of a σ subunit fragment from Escherichia coli RNA polymerase. Cell, 87, 127–136. Mandel-Gutfreund,Y., Schueler,O. and Margalit,H. (1995) Compre- hensive analysis of hydrogen bonds in regulatory protein–DNA complexes: In search of common principles. J. Mol. Biol., 253, 370–382. Minchin,S. and Busby,S. (1993) Location of close contacts between Escherichia coli RNA polymerase and guanine residues at promoters either with or without consensus –35 region sequences. Biochem. J., 289, 771–775. Ponnambalam,S., Webster,C., Bingham,A. and Busby,S. (1986) Transcription initiation at the Escherichia coli galactose operon promoters in the absence of the normal –35 region sequences. J. Biol. Chem., 261, 16043–16048. Sambrook,J., Fritsch,E. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Siegele,D.A., Hu,J.C., Walter,W.A. and Gross,C.A. (1989) Altered promoter recognition by mutant forms of the σ subunit of Escherichia coli RNA polymerase. J. Mol. Biol., 206, 591–603. Studier,F.W. and Moffatt,B.A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol., 189, 113–130. Tatti,K.M., Jones,C.H. and Moran,C.P. (1991) Genetic evidence for interaction of sigma-E with the spoIIID promoter in B. subtilis. J. Bacteriol., 173, 7828–7833. Waldburger,C., Gardella,T., Wong,R. and Susskind,M.M. (1990) Changes in conserved region 2 of Escherichia coli σ affecting promoter recognition. J. Mol. Biol., 215, 267–276. Yura,T. (1996) Regulation and conservation of the heat-shock transcription factor σ . Genes to Cells, 1, 277–284. Zuber,P., Healy,J., Carter,H.L., Cutting,S., Moran,C.P.,Jr and Losick,R. (1989) Mutation changing the specificity of an RNA polymerase sigma factor. J. Mol. Biol., 206, 605–614. Received on January 27, 1997; revised on March 18, 1997

Journal

The EMBO Journal – Springer Journals

Published: Jul 1, 1997

Keywords: promoter recognition; RNA polymerase; sigma subunits; transcription initiation

There are no references for this article.