Mutational Analysis of an Extracytoplasmic-Function Sigma Factor To Investigate Its Interactions with RNA Polymerase and DNA
http://www.100md.com
《细菌学杂志》
Department of Biochemistry, University of Otago, Dunedin, New Zealand
ABSTRACT
The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function.
INTRODUCTION
Bacterial core RNA polymerase (RNAP; 2') requires a fifth subunit, the sigma factor, in order to initiate specific transcription from double-stranded templates. Core RNAP-sigma factor complexes bind to gene promoters in a sequence-specific manner (reviewed in references 18, 25, and 46). Sigma factor proteins of the 70 family bind at specific DNA sequences that are usually centered at about the –35 and –10 region of the promoter. Multiple sigma factor proteins can be present in a single bacterium, and each has an important role in controlling gene expression, with different sigma factors recognizing different promoter sequences (25, 66). Primary sigma factors, such as 70 in Escherichia coli, are essential proteins that are responsible for the majority of RNA synthesis in exponentially growing cells. Alternative sigma factors are nonessential proteins required under certain circumstances and, except for 54 and related proteins, have various degrees of sequence similarity to 70. The extracytoplasmic-function (ECF) family is a large group of sigma proteins that regulate the production of various extracytoplasmic products in a variety of bacteria (reviewed in references 20 and 40). Although they have conserved sequence features present in 70-type proteins (34), ECF sigma factor proteins are much smaller than most other sigma factors (typically 20 to 25 kDa) and therefore may have significant differences in their interactions with DNA, core RNAP, or other proteins.
Sigma factors have been divided into regions based on sequence alignments (33, 34, 66), with region 2 sequences showing the highest level of similarity (Fig. 1A). Residues in region 2 are required for binding to core RNAP (regions 2.1 and 2.2), recognition of the –10 sequence element (region 2.4), and melting of promoter DNA (region 2.3). The evidence for this first came from mutagenesis studies with 70 and 32 (H) from E. coli and A and E from Bacillus subtilis (1, 27, 52, 53, 56) and is strongly supported by the crystal structure of 70 in holoenzyme complexes (43, 59). ECF proteins have significant similarities with other 70-related proteins in region 2, but there are also significant differences, and it is necessary to introduce a gap in order to align aromatic residues of region 2.3 (34); these residues are associated with open complex formation (13, 28).
A helix-turn-helix (HTH) DNA-binding motif in region 4.2 of these sigma factors contains residues that make sequence-specific contacts with DNA bases in the –35 hexamer sequence (7, 42). The HTH is a very common motif found in many DNA-binding proteins, and several crystal structures that provide information on how it interacts with DNA are available (reviewed in reference 65). The first helix acts to position the second helix, which protrudes from the surface of the protein to make discriminatory sequence-specific contacts with the DNA nucleotides. Mutations made to residues of the 70 HTH and the E HTH alter the promoter sequences recognized by these sigma factors (17, 29, 54, 57), a fact that emphasizes the role of the HTH in promoter recognition.
PvdS is an ECF protein required for the production of pyoverdine, exotoxin, and PrpL protease, three virulence factors secreted by the pathogen Pseudomonas aeruginosa (11, 41, 45, 62). PvdS is a true sigma factor, binding with core RNAP to pyoverdine synthesis gene promoters and triggering transcription of these genes (32, 63). PvdS binds with core RNAP to promoters containing a DNA sequence motif, TAAAT, spaced 16 bases from a second sequence motif, CGT (60, 64). By analogy with 70, regions 4.2 and 2.4 of PvdS are most likely to be responsible for the recognition of these motifs. The activity of PvdS is controlled posttranslationally by an anti-sigma factor, FpvR, that can bind to PvdS, inhibiting its activity (31, 48).
The research described here aimed to identify residues within region 2 and region 4.2 of PvdS that are involved in interactions with core RNAP and with DNA. Each targeted residue was mutated to an alanine, and the mutations were examined for their effect on PvdS function in vivo. Selected proteins were also purified and used in in vitro assays to determine whether they could bind core RNAP or DNA and direct transcription.
MATERIALS AND METHODS
Bacterial strains and constructs. Plasmids were maintained in E. coli strain MC1061 (9). Wild-type and mutant pvdS genes were in the vector pProEX (GIBCO BRL). E. coli was grown in LB broth (49) at 37°C with aeration. Ampicillin and chloramphenicol were added to final concentrations of 100 μg/ml and 30 μg/ml, respectively.
Site-directed mutagenesis. The GeneEditor site-directed mutagenesis system (Promega) was used following the protocol provided by the manufacturer, with pProEX::pvdS (63) as the template. Mutagenic oligonucleotides (synthesized by GIBCO BRL) were designed for each mutation to replace the target residue codon with one encoding alanine. Each mutagenic oligonucleotide was 33 bases in length and had 15 bases identical to wild-type pvdS sequence on either side of the mutated codon. Oligonucleotides were designed to incorporate a change in a restriction site at the site of the mutation. DNA carrying potential mutations was screened by restriction digestion, and mutant genes were sequenced (Centre for Gene Research, University of Otago) to confirm the presence of the intended mutations and the absence of other mutations.
-Galactosidase assays. E. coli MC1061 cells containing the reporter plasmid pMP190::pvdE were transformed with pProEX plasmids containing wild-type or mutant pvdS genes. Plasmid pMP190::pvdE has a PvdS-dependent promoter, pvdE, cloned upstream of a lacZ reporter gene so that lacZ expression and consequent -galactosidase production are dependent on the activity of PvdS (11, 31, 64). Cultures were grown to an optical density at 600 nm of 0.3, and the expression of pvdS was induced by the addition of IPTG (isopropyl--D-thiogalactopyranoside) to a final concentration of 1 mM. Aliquots (100 μl) were removed after 1 h and assayed for -galactosidase by use of the Miller assay (39).
Purification of PvdS. Wild-type and mutant PvdS proteins were purified as described previously (63). Briefly, the pvdS and mutant pvdS genes were present in the pProEX (GIBCO BRL) vector that results in expression of proteins fused to six-histidine tags at their N termini. E. coli MC1061 cells expressing His-tagged PvdS proteins were collected and lysed by sonication, and PvdS was purified under denaturing conditions on a Ni-nitrilotriacetic column (QIAGEN). Purified proteins were renatured by dialysis and stored in TGED buffer (50% glycerol, 50 mM Tris-HCl, pH 7.9, 0.01% [vol/vol] Triton X-100, 0.1 mM EDTA, 50 mM NaCl, and 0.1 mM dithiothreitol). Protein concentrations were determined by the Bradford protein assay method (Bio-Rad).
Quantification of PvdS. Cells expressing wild-type and mutant PvdS were collected and lysed by boiling in phosphate-buffered saline (0.15 M NaCl, 0.012 M NaH2PO4, 0.036 M Na2HPO4 [pH 7.2], and 0.1% sodium dodecyl sulfate [SDS]), and the protein concentrations were determined by using the Bradford assay (6). Two dilutions were made, and equal amounts of protein (approximately 0.42 and 0.18 ng) from different cultures were loaded onto a nitrocellulose membrane using a slot blot apparatus (Schleicher and Schull Minifold-II). PvdS protein was detected using an anti-PvdS monoclonal antibody as described previously (63), except that bound secondary antibody was detected by use of West Pico chemiluminescent substrate (Pierce). Bands were then quantified using Molecular Analyst software (Bio-Rad), and values were expressed as percentages of the value for the wild type.
Core-binding assays. The protocol used was based on the method of Bowers and Dombroski (5). Core RNAP (4 pmol; Epicenter) was incubated with 4 pmol of histidine-tagged PvdS in protein dilution buffer (PDB; 10 mM Tris-Cl [pH 7.5], 1 mM -mercaptoethanol, 0.3 M NaCl, 10 mM MgCl2, 0.1 mM EDTA, 0.05% Tween 20, 250 μg/ml bovine serum albumin, and 5% glycerol) in a final volume of 30 μl for 15 min at 37°C. Each mixture was incubated with antibodies (1 μl) against core RNA polymerase for 1 h on ice, and then core-sigma complexes were purified using protein A-agarose (Sigma). After they were washed three times with PDB to remove unbound sigma factor, samples were heated in 6x SDS loading buffer, and the supernatant was loaded onto a 10% SDS-polyacrylamide gel. Following electrophoresis, proteins were electroblotted onto a membrane and subjected to Western analysis with anti-PvdS antibodies. Bands were quantified with Molecular Analyst (Bio-Rad) software.
Gel shift assays. Gel shift assays were carried out as described previously (63). Briefly, PvdS proteins (8 pmol) were incubated with core RNAP (3.4 pmol) and digoxigenin-labeled promoter probe DNA (pvdE/F) at 37°C for 15 min and subsequently for 5 min at room temperature. Reaction mixtures were loaded onto a 5% native polyacrylamide gel and subjected to electrophoresis at 4°C. DNA probes were transferred to an N+ membrane (Roche Molecular Biochemicals) and detected using anti-digoxigenin antibodies following the manufacturer's protocol (Roche Molecular Biochemicals).
RESULTS
Mutational analysis of regions 2.1 and 2.2 of PvdS. Studies with other sigma factors have indicated that these regions contain residues required for binding to core RNAP. Residues to be mutated were selected on the basis of their alignment with 70 family members and ECF sigma proteins (Fig. 1) and included positions where (i) the same or similar residues are present in primary, alternative, and ECF sigmas (L25, R31, Q44, F47, R49); (ii) the same or similar residues are present in ECF sigmas but not in primary sigmas (E40, D41, D45) and may confer class-specific functions; or (iii) the residue in PvdS is different from those of other sigmas and may confer PvdS-specific functions or may not be critical for function (N21, R22, R36, S52). Residues likely to be important for maintaining the tertiary structure of PvdS (identified by comparison to the 70 crystal structure) were avoided. Targeted residues were individually mutated to alanine, and the mutated genes were expressed in E. coli as His-tagged fusions from the pProEX expression vector. Quantitative Western analysis showed that all the PvdS derivatives were present in cells in amounts comparable to that for wild-type protein (data not shown), indicating that the mutations did not significantly disrupt protein folding and stability.
The plasmid constructs were then transformed into E. coli containing the reporter plasmid pMP190::pvdE. This has a PvdS-dependent promoter, pvdE, upstream of a lacZ reporter gene such that the expression of lacZ in E. coli is dependent upon the activity of PvdS (11, 31, 64). Mutations of eight residues (R22, L25, E40, D41, Q44, D45, F47, and R49) resulted in a level of activity less than 35% of that of the wild type, and mutations N21A and R31A reduced activity to about 60% of that of the wild type (Fig. 1B). L25, Q44, F47, and R49 are conserved throughout sigma proteins, and residues at these positions are implicated in the binding of core RNAP by 70 (42, 43, 52, 59). D41 and to a lesser extent E40 and D45 are highly conserved in the ECF family (Fig. 1A), and residues at the equivalent positions in 70 have been shown to be involved in binding to core RNAP (52). Residue N21 is not conserved and R31 is semiconserved, with similar residues being present in many but not all sigma factors (Fig. 1A), and the data indicate that these residues are important but not critical for PvdS function. Mutations R36A and S52A, where the residues in PvdS differ from those in other sigmas, had little or no effect on protein activity. Mutation R22A had a major effect on the activity of PvdS, even though this is not a conserved residue.
Four mutant proteins, namely, R22A, D41A, F47A, and R49A, were selected for in vitro study. These proteins were purified as His-tagged fusions, and their abilities to bind to core RNAP were determined by use of an immunoaffinity assay (Fig. 2). All four proteins had significant reductions in their abilities to bind to core RNAP relative to that of wild-type PvdS, although they did retain some core-binding activity. These data suggest that these residues are specifically involved in the recognition of core enzyme by PvdS. It is likely that a reduced affinity of PvdS for core RNAP has a more severe effect on activity in vivo due to the presence of other sigma factors that compete for a limiting pool of core RNAP (35) and are absent in the in vitro studies. The relative effects of the different mutations on core binding did not completely correlate with the relative effects of the mutations in the reporter gene assay (Fig. 1), suggesting that the mutations may have other effects in addition to affecting core binding.
Mutational analysis of regions 2.3 and 2.4 of PvdS. In 70, regions 2.3 and 2.4 form part of a continuous -helix (36, 43, 59) and are involved in DNA melting and recognition of the –10 sequence motif, respectively. Five residues (Y66, Q69, N73, D77, and R80) that are likely to lie on the outward face of a corresponding -helix in PvdS and therefore would be positioned to interact with promoter DNA were selected for mutagenesis. Y66 corresponds to a conserved aromatic residue present at this position in all 70-type proteins (Fig. 1) which has been previously implicated in DNA melting in 70/A proteins (28, 47). Residues N73, D77, and R80 are conserved in other ECF factors thought to recognize similar –10 region sequence elements, whereas different residues are present in sigma factors that recognize different promoters, so that these residues are candidates for DNA recognition (Fig. 3A). Each residue was mutated to an alanine, and quantitative Western analysis showed that all the mutants were expressed in amounts similar to that seen for the wild type (data not shown). Mutant proteins were assayed for activity in vivo (Fig. 3B). All mutations except for Q69A essentially abolished PvdS activity, indicating that these residues are essential for PvdS function. The mutant proteins with negligible activity were purified and tested for the ability to bind to core RNAP and DNA. All four bound to core enzyme to the same extent as wild-type PvdS (Fig. 4A), indicating that the mutations had not affected interactions with core RNAP. Mutant proteins were also tested for the ability to bind with core RNAP to promoter DNA in a gel shift assay (Fig. 4B). Y66A, D77A, and R80A proteins did not give rise to detectable DNA-protein complexes, indicating that these mutations affected the interaction with promoter DNA or the stability of the DNA-protein complexes. N73A gave rise to DNA-protein complexes, although in amounts consistently lower than that found in wild-type protein.
Mutational analysis of region 4.2 of PvdS. Alignment of the sequence of PvdS with those of other sigma factors allowed the identification of the HTH DNA-binding motif in region 4.2. This region of PvdS is shown aligned with other HTH regions in Fig. 5A. The predicted HTH of PvdS was determined to have a 90% chance of having an HTH structure by use of the Dodd-Egan matrix method (14). A model for the PvdS HTH is shown in Fig. 5B. In this model, conserved hydrophobic residues in the second helix (residues V15, I19, and L23) face away from the DNA and form the interior of the HTH, whereas more-hydrophilic residues are on the external face of this helix and are positioned to interact with DNA. This model is consistent with HTH motifs for which a three-dimensional structure has been determined (reviewed in reference 65).
Eight residues that had the potential to interact with DNA (Fig. 5B) were altered to alanine residues. Amounts of six of the mutant proteins were similar to, or slightly greater than, that for the wild type (Fig. 6A). The strain expressing M161A contained about 50% less PvdS protein than the wild type, and the strain expressing P155A contained about 30% less. These mutations may have affected protein stability with a consequent degradation of misfolded PvdS.
The effect of each mutation on PvdS function in vivo was assessed with a lacZ reporter assay. All of the mutations reduced the level of PvdS function below that of the wild type (Fig. 5C), indicating that each mutated residue contributed to PvdS activity, although all of the mutated proteins retained significant activity. Mutations in this part of PvdS are likely to affect the binding of PvdS-RNAP to promoters. To test this, two mutant proteins (F160A and R163A) were purified and their activities assessed in vitro in order to analyze the effects of the mutations. In gel shift assays (Fig. 6B), both mutant proteins had a reduced ability, relative to wild-type PvdS, to bind to promoter DNA with core RNAP. Both mutants bound core RNAP to the same extent as wild-type protein (Fig. 6C), indicating that these proteins were folded correctly and had retained the ability to interact with core RNAP. The mutations therefore specifically affected the ability of the PvdS-core RNAP complex to interact with DNA.
DISCUSSION
Alanine-scanning mutagenesis was carried out on 25 amino acid residues in PvdS. The targeted residues were expected to be surface exposed and available to interact with core RNAP or DNA rather than to form part of the hydrophobic core of PvdS. Most of the mutations had little or no effect on the amount of PvdS in the bacteria (Fig. 6A; also data not shown), and proteins with mutations in regions 2.3/2.4 and 4.2 retained the ability to bind core RNA polymerase. In contrast, a mutation, V158R, at a residue predicted to be required for correct folding of PvdS, and also mutations causing the insertion of short (four-residue) peptides in PvdS, resulted in no detectable PvdS protein (M. J. Wilson, T. T. Caradoc-Davies, and I. L. Lamont, unpublished observations), most likely because PvdS that is not correctly folded is rapidly degraded. It is therefore very likely that, with possible exceptions in the cases of P155A and M161A, the effects of the mutations reflect the roles of the corresponding amino acid residues in the activity of PvdS and are not simply due to misfolding of the protein.
Only 3 of the 17 mutations in region 2 had little or no effect on PvdS function (over 85% of wild-type activity in vivo) (Fig. 1B and 3B), and all of these were at nonconserved positions. For non-ECF sigma factors, region 2.1/2.2 is involved in interactions with core enzyme (27, 52, 53), and our in vitro data (Fig. 2) indicate that the same is true for PvdS. The large number of residues where mutation resulted in reduced or no function indicates a complex interaction between the two proteins involving the cooperative participation of a large number of amino acid residues. In this respect, PvdS is similar to 70, for which mutational and structural studies (43, 52, 59) revealed a very extensive sigma factor-core enzyme interface. This is notable because of the much smaller size of PvdS and its low level of overall sequence similarity with 70. As might be expected, mutations at residues that are conserved or highly conserved among sigma factors (L25, R31, Q44, F47, and R49) greatly reduced transcription from a PvdS-dependent promoter. PvdS F47A and R49A had impaired core binding, and residues corresponding to L25 and Q44 are important for core binding in H and 70, so it is very likely that these residues all play a role in core RNAP interactions of PvdS.
E40, D41, and D45 are conserved amino acids within the ECF family, but different residues are present at corresponding positions in primary sigma factors (Fig. 1). Mutations at each of these residues had major effects on the activity of PvdS (Fig. 1A). The D41A protein had reduced affinity for core enzyme in vitro (Fig. 2), and the mutations lie in region 2.2, for which the primary function is core binding, so it is very likely that these residues contribute to core binding for PvdS. Mutations at a residue of 70 that corresponds to E40 of PvdS, L402, affected the affinity of 70 for core enzyme, showing that this position at least is important for core binding in different sigma factors, even though the residues that are present are quite different. It may be that the exact nature of the residues at these positions contributes to determining the relative affinities of different sigma factors for core enzyme; it has been shown that the affinities of different sigmas for core can vary over at least a 16-fold range (35). Residue R22 was very important for PvdS function (Fig. 1B) and core RNAP binding (Fig. 2), even though quite different residues are present at corresponding positions in other sigma factors. The corresponding residue in 70 contributes to core binding, with an L384A mutation reducing the affinity of 70 for core enzyme (52). This indicates that at this position also, residues in different sigmas contribute to core binding, even though the exact natures of the residues can be quite different. Overall, it is clear that an extended region of PvdS spanning at least residues R22 to R49 in the linear sequence is involved in binding core RNA polymerase and that critical residues include ones that are common to different sigma factors and ones that are sigma factor specific and may determine the relative affinities of different sigmas for core RNA polymerase.
There is good evidence that region 2.3 in 70 is involved in open complex formation and that this involves aromatic residues that contribute to open complex formation by forming stacking interactions between nucleotide bases to stabilize DNA that has melted into the single-stranded form (13, 15, 28, 47). Residues in region 2.3 also participate in the binding of holoenzyme to double-stranded DNA (15). ECF sigma factors are quite different from 70 in region 2.3, with relatively few aromatic residues, and it is necessary to introduce a gap in this region in order to align the different sequences (Fig. 1). Only one conserved aromatic residue, Y66, is present in region 2.3 of PvdS (Fig. 1). The Y66A mutant had wild-type affinity for core enzyme (Fig. 4A) but had no activity in vivo and showed no activity in the gel shift assay (Fig. 4B). This indicates that Y66A directly affects DNA binding by the core-PvdS complex, although it is also possible that the mutation affects DNA melting and that this is required in order to obtain DNA-protein complexes in the band shift assay.
Region 2.4 is involved in the recognition of –10 promoter motifs by 70, H, and E with the N-terminal part of this region nearest to the transcription start site (12, 15, 26, 61). The three mutations in this region (at N73, D77, and R80) greatly affected the activity of PvdS in vivo (Fig. 3B) and in band shift assays (Fig. 4B) but did not affect binding of PvdS to core enzyme (Fig. 4A). These data are consistent with mutations in this part of PvdS affecting the recognition of target promoters. The proposed –10 sequence recognition motifs of various ECF sigma factors, along with the region 2.4 sequence, are shown in Fig. 3A. There is a striking correlation between the residues placed to interact with DNA at the conserved positions in region 2.4 and the sequences of the –10 motifs. All of the proteins except AlgU/RpoE have an R residue at the position corresponding to R80 in PvdS, and all of them except AlgU/RpoE recognize a C · G base pair at the first position in the –10 motif. Similarly, the proteins that have a D residue at the position corresponding to D77 in PvdS are those for which a G · C base pair is present at the second base in the –10 sequence, and those with an N at this position have a C · G base pair in the second position of the –10. For four of the five proteins with an N at position 73, there is a T · A base pair in the third –10 position, with the only exception being HrpL. Furthermore, studies with E and H from B. subtilis (12, 56) indicate that the residues corresponding to N73 and D77 in PvdS make discriminating –10 region contacts for these sigma factors. These findings lead to the hypothesis that the three mutated residues in region 2.4 of PvdS are involved in recognition of the –10 promoter sequence element. It should be noted, however, that C, which has an H at the position corresponding to N73, also recognizes a T · A base pair in the third –10 position.
All of the mutations in region 4.2 affected PvdS function, although all of the mutant proteins retained significant activity in vivo (Fig. 5C). This indicates that at least eight residues in this region of PvdS contribute to protein function, most likely through interactions with the –35 region of the promoter, with no single residue of those examined being critical to DNA binding. Residues at positions equivalent to T156 and T159 in the HTH are involved in sequence-specific DNA-base interactions of 70 (17, 29) as well as in other DNA-binding proteins (2-4, 24, 37, 51). The residues corresponding to positions 156, 159, 163, and 164 can all form hydrogen bonds with bases in DNA and make sequence-specific DNA contacts in many other DNA-binding proteins (37), so they are good candidates for involvement in promoter recognition. Histidine can carry a positive charge and is present in many DNA-binding proteins, making contacts with the DNA phosphate backbone and stabilizing the DNA binding (37), and this may be the role of H168.
The likely roles of P155, M161, and F160 are less clear, as these amino acids do not commonly make sequence-specific contacts with DNA. Mutations P155A and M161A resulted in reduced amounts of PvdS, and this may be at least part of the reason for the lower activities of these proteins in vivo. The F160A mutation had the most detrimental effect on PvdS activity of all the mutations in region 4.2 (Fig. 5C). Phenylalanine is a hydrophobic amino acid, and it is possible that it forms part of the hydrophobic face of the helix, interacting with other parts of PvdS. However, the F160A mutation did not affect amounts of PvdS (Fig. 6A) and purified protein was still able to bind to core RNAP (Fig. 6C), indicating that the F160A mutation did not affect protein stability or folding and therefore is most likely to be at the protein surface, as predicted by analogy with other HTH protein domains (Fig. 5B). The phenylalanine side chain cannot form H bonds that could contribute to sequence-specific interactions between proteins and DNA and is not often found in HTH motifs. The crystal structure of the Arc repressor showed that a phenylalanine residue contacts the DNA backbone, and this residue is important for DNA binding and contributes to binding specificity (50). It may be that F160 makes contacts with the sugar-phosphate backbone at pyoverdine promoters and that these contacts are important in enabling PvdS-RNA polymerase to bind to DNA.
Overall, these results suggest that at least three residues in region 2.4 and several residues in region 4.2 of PvdS collectively contribute to promoter recognition. While this work was in preparation, a study using similar approaches with the non-ECF sigma factor 32 from E. coli was published (30). Four mutations in region 4.2 of 32 reduced 32-dependent gene expression to less than 30% of that of the wild type. This was in contrast to results for PvdS, in which only one mutation had such a marked effect (Fig. 6). Conversely, mutations in regions 2.3 and 2.4 of 32 did not have as marked an effect on gene expression as mutations in these regions of PvdS (Fig. 2). These differences may reflect differences in the molecular recognitions of promoter DNA by the different sigma factors or differences in the initiations of transcription following promoter recognition. Determination of the structure of PvdS in complex with DNA and with core enzyme will be required to obtain a detailed picture of the interactions between PvdS and these binding partners. In addition, the activity of PvdS is posttranslationally regulated by an anti-sigma factor, FpvR (31). Anti-sigma factors bind to their cognate sigmas to prevent their interaction with RNA polymerase core enzyme (19, 23), and FpvR has recently been shown to interact with PvdS in vivo (48). The mutants generated in this study, in conjunction with studies on protein structure, will be very useful in exploring FpvR-PvdS interactions.
ACKNOWLEDGMENTS
We thank Michael Chamberlin and Malay Ray for supplying anti-core RNAP antibodies and Michael Vasil for anti-PvdS antibodies.
This project was funded in part by a grant from the New Zealand Lottery Health Board.
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ABSTRACT
The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function.
INTRODUCTION
Bacterial core RNA polymerase (RNAP; 2') requires a fifth subunit, the sigma factor, in order to initiate specific transcription from double-stranded templates. Core RNAP-sigma factor complexes bind to gene promoters in a sequence-specific manner (reviewed in references 18, 25, and 46). Sigma factor proteins of the 70 family bind at specific DNA sequences that are usually centered at about the –35 and –10 region of the promoter. Multiple sigma factor proteins can be present in a single bacterium, and each has an important role in controlling gene expression, with different sigma factors recognizing different promoter sequences (25, 66). Primary sigma factors, such as 70 in Escherichia coli, are essential proteins that are responsible for the majority of RNA synthesis in exponentially growing cells. Alternative sigma factors are nonessential proteins required under certain circumstances and, except for 54 and related proteins, have various degrees of sequence similarity to 70. The extracytoplasmic-function (ECF) family is a large group of sigma proteins that regulate the production of various extracytoplasmic products in a variety of bacteria (reviewed in references 20 and 40). Although they have conserved sequence features present in 70-type proteins (34), ECF sigma factor proteins are much smaller than most other sigma factors (typically 20 to 25 kDa) and therefore may have significant differences in their interactions with DNA, core RNAP, or other proteins.
Sigma factors have been divided into regions based on sequence alignments (33, 34, 66), with region 2 sequences showing the highest level of similarity (Fig. 1A). Residues in region 2 are required for binding to core RNAP (regions 2.1 and 2.2), recognition of the –10 sequence element (region 2.4), and melting of promoter DNA (region 2.3). The evidence for this first came from mutagenesis studies with 70 and 32 (H) from E. coli and A and E from Bacillus subtilis (1, 27, 52, 53, 56) and is strongly supported by the crystal structure of 70 in holoenzyme complexes (43, 59). ECF proteins have significant similarities with other 70-related proteins in region 2, but there are also significant differences, and it is necessary to introduce a gap in order to align aromatic residues of region 2.3 (34); these residues are associated with open complex formation (13, 28).
A helix-turn-helix (HTH) DNA-binding motif in region 4.2 of these sigma factors contains residues that make sequence-specific contacts with DNA bases in the –35 hexamer sequence (7, 42). The HTH is a very common motif found in many DNA-binding proteins, and several crystal structures that provide information on how it interacts with DNA are available (reviewed in reference 65). The first helix acts to position the second helix, which protrudes from the surface of the protein to make discriminatory sequence-specific contacts with the DNA nucleotides. Mutations made to residues of the 70 HTH and the E HTH alter the promoter sequences recognized by these sigma factors (17, 29, 54, 57), a fact that emphasizes the role of the HTH in promoter recognition.
PvdS is an ECF protein required for the production of pyoverdine, exotoxin, and PrpL protease, three virulence factors secreted by the pathogen Pseudomonas aeruginosa (11, 41, 45, 62). PvdS is a true sigma factor, binding with core RNAP to pyoverdine synthesis gene promoters and triggering transcription of these genes (32, 63). PvdS binds with core RNAP to promoters containing a DNA sequence motif, TAAAT, spaced 16 bases from a second sequence motif, CGT (60, 64). By analogy with 70, regions 4.2 and 2.4 of PvdS are most likely to be responsible for the recognition of these motifs. The activity of PvdS is controlled posttranslationally by an anti-sigma factor, FpvR, that can bind to PvdS, inhibiting its activity (31, 48).
The research described here aimed to identify residues within region 2 and region 4.2 of PvdS that are involved in interactions with core RNAP and with DNA. Each targeted residue was mutated to an alanine, and the mutations were examined for their effect on PvdS function in vivo. Selected proteins were also purified and used in in vitro assays to determine whether they could bind core RNAP or DNA and direct transcription.
MATERIALS AND METHODS
Bacterial strains and constructs. Plasmids were maintained in E. coli strain MC1061 (9). Wild-type and mutant pvdS genes were in the vector pProEX (GIBCO BRL). E. coli was grown in LB broth (49) at 37°C with aeration. Ampicillin and chloramphenicol were added to final concentrations of 100 μg/ml and 30 μg/ml, respectively.
Site-directed mutagenesis. The GeneEditor site-directed mutagenesis system (Promega) was used following the protocol provided by the manufacturer, with pProEX::pvdS (63) as the template. Mutagenic oligonucleotides (synthesized by GIBCO BRL) were designed for each mutation to replace the target residue codon with one encoding alanine. Each mutagenic oligonucleotide was 33 bases in length and had 15 bases identical to wild-type pvdS sequence on either side of the mutated codon. Oligonucleotides were designed to incorporate a change in a restriction site at the site of the mutation. DNA carrying potential mutations was screened by restriction digestion, and mutant genes were sequenced (Centre for Gene Research, University of Otago) to confirm the presence of the intended mutations and the absence of other mutations.
-Galactosidase assays. E. coli MC1061 cells containing the reporter plasmid pMP190::pvdE were transformed with pProEX plasmids containing wild-type or mutant pvdS genes. Plasmid pMP190::pvdE has a PvdS-dependent promoter, pvdE, cloned upstream of a lacZ reporter gene so that lacZ expression and consequent -galactosidase production are dependent on the activity of PvdS (11, 31, 64). Cultures were grown to an optical density at 600 nm of 0.3, and the expression of pvdS was induced by the addition of IPTG (isopropyl--D-thiogalactopyranoside) to a final concentration of 1 mM. Aliquots (100 μl) were removed after 1 h and assayed for -galactosidase by use of the Miller assay (39).
Purification of PvdS. Wild-type and mutant PvdS proteins were purified as described previously (63). Briefly, the pvdS and mutant pvdS genes were present in the pProEX (GIBCO BRL) vector that results in expression of proteins fused to six-histidine tags at their N termini. E. coli MC1061 cells expressing His-tagged PvdS proteins were collected and lysed by sonication, and PvdS was purified under denaturing conditions on a Ni-nitrilotriacetic column (QIAGEN). Purified proteins were renatured by dialysis and stored in TGED buffer (50% glycerol, 50 mM Tris-HCl, pH 7.9, 0.01% [vol/vol] Triton X-100, 0.1 mM EDTA, 50 mM NaCl, and 0.1 mM dithiothreitol). Protein concentrations were determined by the Bradford protein assay method (Bio-Rad).
Quantification of PvdS. Cells expressing wild-type and mutant PvdS were collected and lysed by boiling in phosphate-buffered saline (0.15 M NaCl, 0.012 M NaH2PO4, 0.036 M Na2HPO4 [pH 7.2], and 0.1% sodium dodecyl sulfate [SDS]), and the protein concentrations were determined by using the Bradford assay (6). Two dilutions were made, and equal amounts of protein (approximately 0.42 and 0.18 ng) from different cultures were loaded onto a nitrocellulose membrane using a slot blot apparatus (Schleicher and Schull Minifold-II). PvdS protein was detected using an anti-PvdS monoclonal antibody as described previously (63), except that bound secondary antibody was detected by use of West Pico chemiluminescent substrate (Pierce). Bands were then quantified using Molecular Analyst software (Bio-Rad), and values were expressed as percentages of the value for the wild type.
Core-binding assays. The protocol used was based on the method of Bowers and Dombroski (5). Core RNAP (4 pmol; Epicenter) was incubated with 4 pmol of histidine-tagged PvdS in protein dilution buffer (PDB; 10 mM Tris-Cl [pH 7.5], 1 mM -mercaptoethanol, 0.3 M NaCl, 10 mM MgCl2, 0.1 mM EDTA, 0.05% Tween 20, 250 μg/ml bovine serum albumin, and 5% glycerol) in a final volume of 30 μl for 15 min at 37°C. Each mixture was incubated with antibodies (1 μl) against core RNA polymerase for 1 h on ice, and then core-sigma complexes were purified using protein A-agarose (Sigma). After they were washed three times with PDB to remove unbound sigma factor, samples were heated in 6x SDS loading buffer, and the supernatant was loaded onto a 10% SDS-polyacrylamide gel. Following electrophoresis, proteins were electroblotted onto a membrane and subjected to Western analysis with anti-PvdS antibodies. Bands were quantified with Molecular Analyst (Bio-Rad) software.
Gel shift assays. Gel shift assays were carried out as described previously (63). Briefly, PvdS proteins (8 pmol) were incubated with core RNAP (3.4 pmol) and digoxigenin-labeled promoter probe DNA (pvdE/F) at 37°C for 15 min and subsequently for 5 min at room temperature. Reaction mixtures were loaded onto a 5% native polyacrylamide gel and subjected to electrophoresis at 4°C. DNA probes were transferred to an N+ membrane (Roche Molecular Biochemicals) and detected using anti-digoxigenin antibodies following the manufacturer's protocol (Roche Molecular Biochemicals).
RESULTS
Mutational analysis of regions 2.1 and 2.2 of PvdS. Studies with other sigma factors have indicated that these regions contain residues required for binding to core RNAP. Residues to be mutated were selected on the basis of their alignment with 70 family members and ECF sigma proteins (Fig. 1) and included positions where (i) the same or similar residues are present in primary, alternative, and ECF sigmas (L25, R31, Q44, F47, R49); (ii) the same or similar residues are present in ECF sigmas but not in primary sigmas (E40, D41, D45) and may confer class-specific functions; or (iii) the residue in PvdS is different from those of other sigmas and may confer PvdS-specific functions or may not be critical for function (N21, R22, R36, S52). Residues likely to be important for maintaining the tertiary structure of PvdS (identified by comparison to the 70 crystal structure) were avoided. Targeted residues were individually mutated to alanine, and the mutated genes were expressed in E. coli as His-tagged fusions from the pProEX expression vector. Quantitative Western analysis showed that all the PvdS derivatives were present in cells in amounts comparable to that for wild-type protein (data not shown), indicating that the mutations did not significantly disrupt protein folding and stability.
The plasmid constructs were then transformed into E. coli containing the reporter plasmid pMP190::pvdE. This has a PvdS-dependent promoter, pvdE, upstream of a lacZ reporter gene such that the expression of lacZ in E. coli is dependent upon the activity of PvdS (11, 31, 64). Mutations of eight residues (R22, L25, E40, D41, Q44, D45, F47, and R49) resulted in a level of activity less than 35% of that of the wild type, and mutations N21A and R31A reduced activity to about 60% of that of the wild type (Fig. 1B). L25, Q44, F47, and R49 are conserved throughout sigma proteins, and residues at these positions are implicated in the binding of core RNAP by 70 (42, 43, 52, 59). D41 and to a lesser extent E40 and D45 are highly conserved in the ECF family (Fig. 1A), and residues at the equivalent positions in 70 have been shown to be involved in binding to core RNAP (52). Residue N21 is not conserved and R31 is semiconserved, with similar residues being present in many but not all sigma factors (Fig. 1A), and the data indicate that these residues are important but not critical for PvdS function. Mutations R36A and S52A, where the residues in PvdS differ from those in other sigmas, had little or no effect on protein activity. Mutation R22A had a major effect on the activity of PvdS, even though this is not a conserved residue.
Four mutant proteins, namely, R22A, D41A, F47A, and R49A, were selected for in vitro study. These proteins were purified as His-tagged fusions, and their abilities to bind to core RNAP were determined by use of an immunoaffinity assay (Fig. 2). All four proteins had significant reductions in their abilities to bind to core RNAP relative to that of wild-type PvdS, although they did retain some core-binding activity. These data suggest that these residues are specifically involved in the recognition of core enzyme by PvdS. It is likely that a reduced affinity of PvdS for core RNAP has a more severe effect on activity in vivo due to the presence of other sigma factors that compete for a limiting pool of core RNAP (35) and are absent in the in vitro studies. The relative effects of the different mutations on core binding did not completely correlate with the relative effects of the mutations in the reporter gene assay (Fig. 1), suggesting that the mutations may have other effects in addition to affecting core binding.
Mutational analysis of regions 2.3 and 2.4 of PvdS. In 70, regions 2.3 and 2.4 form part of a continuous -helix (36, 43, 59) and are involved in DNA melting and recognition of the –10 sequence motif, respectively. Five residues (Y66, Q69, N73, D77, and R80) that are likely to lie on the outward face of a corresponding -helix in PvdS and therefore would be positioned to interact with promoter DNA were selected for mutagenesis. Y66 corresponds to a conserved aromatic residue present at this position in all 70-type proteins (Fig. 1) which has been previously implicated in DNA melting in 70/A proteins (28, 47). Residues N73, D77, and R80 are conserved in other ECF factors thought to recognize similar –10 region sequence elements, whereas different residues are present in sigma factors that recognize different promoters, so that these residues are candidates for DNA recognition (Fig. 3A). Each residue was mutated to an alanine, and quantitative Western analysis showed that all the mutants were expressed in amounts similar to that seen for the wild type (data not shown). Mutant proteins were assayed for activity in vivo (Fig. 3B). All mutations except for Q69A essentially abolished PvdS activity, indicating that these residues are essential for PvdS function. The mutant proteins with negligible activity were purified and tested for the ability to bind to core RNAP and DNA. All four bound to core enzyme to the same extent as wild-type PvdS (Fig. 4A), indicating that the mutations had not affected interactions with core RNAP. Mutant proteins were also tested for the ability to bind with core RNAP to promoter DNA in a gel shift assay (Fig. 4B). Y66A, D77A, and R80A proteins did not give rise to detectable DNA-protein complexes, indicating that these mutations affected the interaction with promoter DNA or the stability of the DNA-protein complexes. N73A gave rise to DNA-protein complexes, although in amounts consistently lower than that found in wild-type protein.
Mutational analysis of region 4.2 of PvdS. Alignment of the sequence of PvdS with those of other sigma factors allowed the identification of the HTH DNA-binding motif in region 4.2. This region of PvdS is shown aligned with other HTH regions in Fig. 5A. The predicted HTH of PvdS was determined to have a 90% chance of having an HTH structure by use of the Dodd-Egan matrix method (14). A model for the PvdS HTH is shown in Fig. 5B. In this model, conserved hydrophobic residues in the second helix (residues V15, I19, and L23) face away from the DNA and form the interior of the HTH, whereas more-hydrophilic residues are on the external face of this helix and are positioned to interact with DNA. This model is consistent with HTH motifs for which a three-dimensional structure has been determined (reviewed in reference 65).
Eight residues that had the potential to interact with DNA (Fig. 5B) were altered to alanine residues. Amounts of six of the mutant proteins were similar to, or slightly greater than, that for the wild type (Fig. 6A). The strain expressing M161A contained about 50% less PvdS protein than the wild type, and the strain expressing P155A contained about 30% less. These mutations may have affected protein stability with a consequent degradation of misfolded PvdS.
The effect of each mutation on PvdS function in vivo was assessed with a lacZ reporter assay. All of the mutations reduced the level of PvdS function below that of the wild type (Fig. 5C), indicating that each mutated residue contributed to PvdS activity, although all of the mutated proteins retained significant activity. Mutations in this part of PvdS are likely to affect the binding of PvdS-RNAP to promoters. To test this, two mutant proteins (F160A and R163A) were purified and their activities assessed in vitro in order to analyze the effects of the mutations. In gel shift assays (Fig. 6B), both mutant proteins had a reduced ability, relative to wild-type PvdS, to bind to promoter DNA with core RNAP. Both mutants bound core RNAP to the same extent as wild-type protein (Fig. 6C), indicating that these proteins were folded correctly and had retained the ability to interact with core RNAP. The mutations therefore specifically affected the ability of the PvdS-core RNAP complex to interact with DNA.
DISCUSSION
Alanine-scanning mutagenesis was carried out on 25 amino acid residues in PvdS. The targeted residues were expected to be surface exposed and available to interact with core RNAP or DNA rather than to form part of the hydrophobic core of PvdS. Most of the mutations had little or no effect on the amount of PvdS in the bacteria (Fig. 6A; also data not shown), and proteins with mutations in regions 2.3/2.4 and 4.2 retained the ability to bind core RNA polymerase. In contrast, a mutation, V158R, at a residue predicted to be required for correct folding of PvdS, and also mutations causing the insertion of short (four-residue) peptides in PvdS, resulted in no detectable PvdS protein (M. J. Wilson, T. T. Caradoc-Davies, and I. L. Lamont, unpublished observations), most likely because PvdS that is not correctly folded is rapidly degraded. It is therefore very likely that, with possible exceptions in the cases of P155A and M161A, the effects of the mutations reflect the roles of the corresponding amino acid residues in the activity of PvdS and are not simply due to misfolding of the protein.
Only 3 of the 17 mutations in region 2 had little or no effect on PvdS function (over 85% of wild-type activity in vivo) (Fig. 1B and 3B), and all of these were at nonconserved positions. For non-ECF sigma factors, region 2.1/2.2 is involved in interactions with core enzyme (27, 52, 53), and our in vitro data (Fig. 2) indicate that the same is true for PvdS. The large number of residues where mutation resulted in reduced or no function indicates a complex interaction between the two proteins involving the cooperative participation of a large number of amino acid residues. In this respect, PvdS is similar to 70, for which mutational and structural studies (43, 52, 59) revealed a very extensive sigma factor-core enzyme interface. This is notable because of the much smaller size of PvdS and its low level of overall sequence similarity with 70. As might be expected, mutations at residues that are conserved or highly conserved among sigma factors (L25, R31, Q44, F47, and R49) greatly reduced transcription from a PvdS-dependent promoter. PvdS F47A and R49A had impaired core binding, and residues corresponding to L25 and Q44 are important for core binding in H and 70, so it is very likely that these residues all play a role in core RNAP interactions of PvdS.
E40, D41, and D45 are conserved amino acids within the ECF family, but different residues are present at corresponding positions in primary sigma factors (Fig. 1). Mutations at each of these residues had major effects on the activity of PvdS (Fig. 1A). The D41A protein had reduced affinity for core enzyme in vitro (Fig. 2), and the mutations lie in region 2.2, for which the primary function is core binding, so it is very likely that these residues contribute to core binding for PvdS. Mutations at a residue of 70 that corresponds to E40 of PvdS, L402, affected the affinity of 70 for core enzyme, showing that this position at least is important for core binding in different sigma factors, even though the residues that are present are quite different. It may be that the exact nature of the residues at these positions contributes to determining the relative affinities of different sigma factors for core enzyme; it has been shown that the affinities of different sigmas for core can vary over at least a 16-fold range (35). Residue R22 was very important for PvdS function (Fig. 1B) and core RNAP binding (Fig. 2), even though quite different residues are present at corresponding positions in other sigma factors. The corresponding residue in 70 contributes to core binding, with an L384A mutation reducing the affinity of 70 for core enzyme (52). This indicates that at this position also, residues in different sigmas contribute to core binding, even though the exact natures of the residues can be quite different. Overall, it is clear that an extended region of PvdS spanning at least residues R22 to R49 in the linear sequence is involved in binding core RNA polymerase and that critical residues include ones that are common to different sigma factors and ones that are sigma factor specific and may determine the relative affinities of different sigmas for core RNA polymerase.
There is good evidence that region 2.3 in 70 is involved in open complex formation and that this involves aromatic residues that contribute to open complex formation by forming stacking interactions between nucleotide bases to stabilize DNA that has melted into the single-stranded form (13, 15, 28, 47). Residues in region 2.3 also participate in the binding of holoenzyme to double-stranded DNA (15). ECF sigma factors are quite different from 70 in region 2.3, with relatively few aromatic residues, and it is necessary to introduce a gap in this region in order to align the different sequences (Fig. 1). Only one conserved aromatic residue, Y66, is present in region 2.3 of PvdS (Fig. 1). The Y66A mutant had wild-type affinity for core enzyme (Fig. 4A) but had no activity in vivo and showed no activity in the gel shift assay (Fig. 4B). This indicates that Y66A directly affects DNA binding by the core-PvdS complex, although it is also possible that the mutation affects DNA melting and that this is required in order to obtain DNA-protein complexes in the band shift assay.
Region 2.4 is involved in the recognition of –10 promoter motifs by 70, H, and E with the N-terminal part of this region nearest to the transcription start site (12, 15, 26, 61). The three mutations in this region (at N73, D77, and R80) greatly affected the activity of PvdS in vivo (Fig. 3B) and in band shift assays (Fig. 4B) but did not affect binding of PvdS to core enzyme (Fig. 4A). These data are consistent with mutations in this part of PvdS affecting the recognition of target promoters. The proposed –10 sequence recognition motifs of various ECF sigma factors, along with the region 2.4 sequence, are shown in Fig. 3A. There is a striking correlation between the residues placed to interact with DNA at the conserved positions in region 2.4 and the sequences of the –10 motifs. All of the proteins except AlgU/RpoE have an R residue at the position corresponding to R80 in PvdS, and all of them except AlgU/RpoE recognize a C · G base pair at the first position in the –10 motif. Similarly, the proteins that have a D residue at the position corresponding to D77 in PvdS are those for which a G · C base pair is present at the second base in the –10 sequence, and those with an N at this position have a C · G base pair in the second position of the –10. For four of the five proteins with an N at position 73, there is a T · A base pair in the third –10 position, with the only exception being HrpL. Furthermore, studies with E and H from B. subtilis (12, 56) indicate that the residues corresponding to N73 and D77 in PvdS make discriminating –10 region contacts for these sigma factors. These findings lead to the hypothesis that the three mutated residues in region 2.4 of PvdS are involved in recognition of the –10 promoter sequence element. It should be noted, however, that C, which has an H at the position corresponding to N73, also recognizes a T · A base pair in the third –10 position.
All of the mutations in region 4.2 affected PvdS function, although all of the mutant proteins retained significant activity in vivo (Fig. 5C). This indicates that at least eight residues in this region of PvdS contribute to protein function, most likely through interactions with the –35 region of the promoter, with no single residue of those examined being critical to DNA binding. Residues at positions equivalent to T156 and T159 in the HTH are involved in sequence-specific DNA-base interactions of 70 (17, 29) as well as in other DNA-binding proteins (2-4, 24, 37, 51). The residues corresponding to positions 156, 159, 163, and 164 can all form hydrogen bonds with bases in DNA and make sequence-specific DNA contacts in many other DNA-binding proteins (37), so they are good candidates for involvement in promoter recognition. Histidine can carry a positive charge and is present in many DNA-binding proteins, making contacts with the DNA phosphate backbone and stabilizing the DNA binding (37), and this may be the role of H168.
The likely roles of P155, M161, and F160 are less clear, as these amino acids do not commonly make sequence-specific contacts with DNA. Mutations P155A and M161A resulted in reduced amounts of PvdS, and this may be at least part of the reason for the lower activities of these proteins in vivo. The F160A mutation had the most detrimental effect on PvdS activity of all the mutations in region 4.2 (Fig. 5C). Phenylalanine is a hydrophobic amino acid, and it is possible that it forms part of the hydrophobic face of the helix, interacting with other parts of PvdS. However, the F160A mutation did not affect amounts of PvdS (Fig. 6A) and purified protein was still able to bind to core RNAP (Fig. 6C), indicating that the F160A mutation did not affect protein stability or folding and therefore is most likely to be at the protein surface, as predicted by analogy with other HTH protein domains (Fig. 5B). The phenylalanine side chain cannot form H bonds that could contribute to sequence-specific interactions between proteins and DNA and is not often found in HTH motifs. The crystal structure of the Arc repressor showed that a phenylalanine residue contacts the DNA backbone, and this residue is important for DNA binding and contributes to binding specificity (50). It may be that F160 makes contacts with the sugar-phosphate backbone at pyoverdine promoters and that these contacts are important in enabling PvdS-RNA polymerase to bind to DNA.
Overall, these results suggest that at least three residues in region 2.4 and several residues in region 4.2 of PvdS collectively contribute to promoter recognition. While this work was in preparation, a study using similar approaches with the non-ECF sigma factor 32 from E. coli was published (30). Four mutations in region 4.2 of 32 reduced 32-dependent gene expression to less than 30% of that of the wild type. This was in contrast to results for PvdS, in which only one mutation had such a marked effect (Fig. 6). Conversely, mutations in regions 2.3 and 2.4 of 32 did not have as marked an effect on gene expression as mutations in these regions of PvdS (Fig. 2). These differences may reflect differences in the molecular recognitions of promoter DNA by the different sigma factors or differences in the initiations of transcription following promoter recognition. Determination of the structure of PvdS in complex with DNA and with core enzyme will be required to obtain a detailed picture of the interactions between PvdS and these binding partners. In addition, the activity of PvdS is posttranslationally regulated by an anti-sigma factor, FpvR (31). Anti-sigma factors bind to their cognate sigmas to prevent their interaction with RNA polymerase core enzyme (19, 23), and FpvR has recently been shown to interact with PvdS in vivo (48). The mutants generated in this study, in conjunction with studies on protein structure, will be very useful in exploring FpvR-PvdS interactions.
ACKNOWLEDGMENTS
We thank Michael Chamberlin and Malay Ray for supplying anti-core RNAP antibodies and Michael Vasil for anti-PvdS antibodies.
This project was funded in part by a grant from the New Zealand Lottery Health Board.
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