INTRODUCTION

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Sigma 54 is an obligatory factor in transcription from bacterial promoters that rely on enhancer elements. It associates with the common core RNA polymerase and directs the polymerase to promoters containing appropriate recognition sequences near 12 and 24 (14, 16). The holoenzyme bound to these elements remains inactive until signaled by an activator protein bound to a remote enhancer sequence (3, 17, 20-22). The activation event is the energy-dependent melting of a previously unmelted DNA segment bound by the holoenzyme (1, 20, 21). Once the open promoter complex is formed, the template strand may be read and transcription can proceed.

This reliance on enhancers and on energy for DNA melting is characteristic of eukaryotic RNA polymerase II mechanisms. It is uncharacteristic of typical bacterial transcription mechanisms that use the common sigma 70 family of proteins. Thus, sigma 54 is thought to cause the prokaryotic RNA polymerase to adopt a mechanism that is a hybrid in the sense that it has both eukaryotic and prokaryotic properties.

The amino acid sequence of sigma 54 is not similar to that of any other protein (13, 14), except perhaps for a tiny homologous segment that contributes to RNA polymerase binding (26, 28). The functional domain structure of the protein is complex, but it is thought to contain three primary domains: a C-terminal portion needed for binding DNA, a central portion needed to bind the polymerase, and an N-terminal portion needed for proper regulation of activation (4, 7, 9, 10, 22, 32). When the N-terminal 40 amino acids are deleted, sigma 54 can still bind RNA polymerase and direct it to DNA. However, the bound holoenzyme fails to respond to enhancer protein in that it fails to form a stable open complex that can initiate transcription. If extreme solution conditions that trigger transient DNA melting are used, the holoenzyme with an N-terminally deleted sigma 54 can produce transcript (30), showing that its catalytic activity is intact. This transcript is unusual in that it results from heparin-sensitive transcription. Its production is not enhanced by the addition of activator, implying that the N terminus contains essential activation response determinants.

The N-terminal 40 amino acids have the unusual composition of 40% leucines and glutamines. Site-directed mutagenesis has implicated some of the leucines in protein function (9). Multiple leucine substitutions can alter several properties of the holoenzyme. These include a loss of function, a reduction in ability to melt the DNA, and a reduction in protection of the 12 region of the promoter. The roles of individual leucines in these various processes have not been firmly established.

It is known, however, that a subset of these leucines, including four between amino acids 25 and 31, have a role in keeping unregulated transcription in check. Certain "bypass" mutations in this leucine patch allow some leaky transcription to occur in vivo. These mutants also mimic one of the in vitro properties just described for N-terminally deleted sigma: they allow some transcription to occur in the absence of enhancer protein (25, 31). As with the N-terminal deletion mutants, this in vitro transcription is unusual in being sensitive to heparin and in requiring solution conditions that favor DNA melting.

These leucine patch bypass substitution mutants differ in vitro from the deletion mutant in one important aspect. Deletions destroy the ability to respond to activator, whereas the leucine point substitutions do not. That is, activator can stimulate transcription from the point mutants and can cause the transcription to be heparin resistant (30). Thus, it appears that the N terminus contains additional determinants outside of this leucine patch that are required for the response to activator.

The determinants within the N-terminal region that are needed for the activation response are not known. The aim of this study was to begin to identify these determinants. The initial approach was to create a library of N-terminal mutants and screen it to identify specific amino acids that might be important. Candidate residues were then altered by site-directed mutagenesis and tested for function in vivo. Mutants that showed a defect were then purified and studied further in vitro. The results led to the identification of a small activation response region between amino acids 33 and 37. The properties of mutants in this region assist in understanding how sigma 54 converts the polymerase into an enhancer-responsive enzyme.

	MATERIALS AND METHODS

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Strains, plasmids, and mutagenesis. The plasmid pAS54 was derived from expression plasmid pJF5401 (6, 23). The two differ only in that a proline substitution of unknown origin at position 13 was corrected by replacement with the wild-type leucine. The plasmid carries the Escherichia coli sigma 54 gene and the gene for ampicillin resistance. E. coli YMC109, lacking a wild-type chromosomal copy of the sigma 54 gene and containing a glnAp₂-lacZ fusion on the chromosome, was used as the host. A functional copy of sigma 54 is required to get blue colonies on W-arginine (W-Arg) minimal medium plates (although very small white colonies can grow in the absence of sigma 54).

Functional screen. Transformants from the library in YMC109 cells were grown on LB plates at 30°C. Colonies were then picked and transferred onto W-Arg-X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) plates (500 ml of W-Arg medium contains 5.25 g of K₂HPO₄, 2.25 g of KH₂PO₄, 7.5 g of agar, 10 ml of 20% glucose, 1.0 ml of thiamine [10.0 mg/ml], 0.215 ml of 1 M MgSO₄, 0.5 g of L-arginine, and appropriate antibiotics) and grown overnight at 30°C. The next day the plates were transferred to 43°C and checked every hour to observe the change of color to blue. Colonies with wild-type sigma 54 show a clear blue color after approximately 2 h. After 6 h, all of the colonies begin to turn blue, and subsequently, the screen becomes unreliable.

Protein purification. Sigma 54 was purified as described previously (29-31). In brief, HB101 cells were transformed with expression vector pAS54 and grown in 250 ml of Luria broth medium at 30°C. When the optical density at 600 nm reached 0.8, the culture was shifted to 43°C and the cells were grown for another 3 to 4 h to induce expression. The harvested cells were resuspended in 12 ml of buffer S (10 mM Tris HCl [pH 8.0], 200 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 5% glycerol), and disrupted twice in a French press. The lysate was spun at 1,200 × g for 40 min at 4°C, and the pellet was collected and resuspended in buffer S with 4 M guanidium HCl and 0.1% Nonidet P-40 (nonionic detergent). Sonication was used to help dissolve the pellet, which was then dialyzed, first against buffer S with 1 M guanidium HCl and then against buffer S. After each dialysis the undissolved material was discarded. The dialysate was loaded onto a Q-Sepharose column (1.5 by 15 cm), washed with 10 column volumes of buffer S, and eluted with a 100-ml linear gradient of 0.2 to 0.8 M KCl in buffer S. The protein elutes near fraction 12 to 16, and the fractions with the highest purity were pooled and precipitated with 70% saturated ammonium sulfate (0.436 g/ml at 4°C). The pellet was dissolved in 1 ml of buffer S and dialyzed, first against buffer S and then against buffer S with 40% glycerol. The concentration of sigma 54 protein was determined by A₂₆₀ absorption (1 A₂₆₀ unit = 1.26 mg/ml) and checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis against known protein markers. The proteins purified from inclusion bodies by this one-column purification are >90% pure. The mutant proteins were grown in YMC109 cells (a strain lacking sigma 54) and purified as described above.

In vitro experiments. Standard in vitro transcriptions were done as described previously (30). In brief, 20 µl of reaction mixture contains 1× HEPES buffer (50 mM HEPES [pH 7.9], 50 mM KCl, 10 mM MgCl₂, 0.1 mM EDTA, 1 mM dithiothreitol, 50 µg of bovine serum albumin/ml, and 3.5% (wt/vol) polyethylene glycol), 5 nM pTH8 DNA, 100 nM sigma 54, 36 nM core polymerase (1 U; Epicentre Technologies), 10 mM carbamyl phosphate (Sigma), 4.5 mM ATP, and 100 nM NtrC unless otherwise stated. Reaction mixtures were assembled on ice and preincubated for 20 min at 37°C. Heparin was then added with NTPs (0.5 mM each) and incubated for an additional 10 min at 37°C. The NTPs included 4 µCi of -³²P-labeled UTP (50 µM). The reaction was stopped with urea-saturated formamide dye, loaded directly onto a 6% polyacrylamide-urea gel, and analyzed with a PhosphorImager (Molecular Dynamics). Variations, in which heparin challenge was not used, are described in Results.

	RESULTS

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Screening libraries created by random mutagenesis. The N-terminal region of sigma 54 was targeted for mutagenesis in order to find determinants needed for activation. Error-prone PCR-copying procedures were used to create a library of mutants covering the N-terminal region prior to amino acid 40. The 150-bp PCR product was ligated in frame to the remainder of the wild-type sigma 54 gene contained on a recipient plasmid (Fig. 1). A library was created by transformation into E. coli YMC109. This strain lacks a chromosomal copy of sigma 54. Thus, the sole gene copy is provided from the plasmid containing potential N-terminal mutants. The host cell chromosome contains a fusion between the sigma 54-dependent glnAp₂ promoter and lacZ, allowing color screening for expression (10). The plates contained W-Arg medium, which induces glnAp₂ expression. Thus, functional and nonfunctional forms of sigma 54 may be distinguished by color, with only functional colonies producing a blue color on X-Gal indicator plates.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Outline of the PCR mutagenesis protocol used to create the N-terminal library.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   (a) Summary of changes that are tolerated without loss of function. Sequence changes found in functional forms of sigma 54 are shown in columns beneath the wild-type sequence of the N-terminal region. The total number of tolerated amino acid substitutions and the total number of silent mutations at each position are shown at the bottom. (b) Evaluation of significance of observed pattern of tolerated changes. The fraction of nucleotide changes that led to tolerated amino acid changes was calculated for each codon. This was compared to the theoretical fraction of substitutions expected from random mutagenesis. The ratio of actual fraction/theoretical fraction is shown and is expected to be unity for positions that are not critical for function. A ratio of less than unity indicates that nucleotide changes were introduced but were not tolerated when they led to a changed amino acid. The darkness of the shading of the bars indicates the total number of changes observed, with black bars indicating at least five changes and lighter bars having progressively fewer changes.

Site-directed mutagenesis of candidate amino acids. Next we used site-directed mutagenesis (or existing point mutants) to assess the role of single changes at these four positions. Other mutants were also made for the purposes of comparison. All four candidate amino acids were changed to positively charged amino acids as a common example of an extreme change. The resulting sigmas were assayed for function in vivo with the same screen developed for the library analysis.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Functionality of sigmas made by site-directed mutagenesis. Substitutions are indicated below the wild-type sequence. The results of a plate test for function are shown at the right (+, functional; , nonfunctional). *, mutants from previous work (25); **, mutants from the library.

In vitro transcription. Three sigmas containing point mutations that lead to loss of function in vivo were purified and tested in vitro. The induction and purification was additional confirmation that the in vivo defects were not due to lack of expression. Prior in vitro transcription experiments with point mutants have failed to show significant reductions in function, even when transcription in vivo showed some defect. By contrast the effects of more radical mutations, such as the small N-terminal deletion Sal58 (amino acids 18 to 31 deleted), were easily detectable (30). Sal58 polymerase was used as a negative comparison in these studies.

View larger version (10K): [in this window] [in a new window]   FIG. 4.   In vitro transcription at the glnAp2 promoter with the standard heparin challenge protocol. (a) Results with control sigmas. (b) Results with point mutants in residues deemed important. (c) Summary of results from repeated experiments. (d) Transcription changes as a function of added activator (the arrow indicates 100 nM, above which NtrC can begin to activate by the nonphysiological mechanism from solution).

View larger version (27K): [in this window] [in a new window]   FIG. 5.   Characterization of properties of mutant sigmas. Band shift experiments of mutant holoenzymes with the nifH probe (a) and in vitro transcription without heparin challenge (b) and without activator (c) are shown.

	DISCUSSION

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Prior to this work it was known that the N-terminal 40 residues of sigma 54 were involved in activation, but little was known about the importance of individual amino acids (30). There had been no systematic study of this issue; information was based on the properties of deletions or multiple-substitution mutants. In this work we introduced mutations randomly into this region and screened for function. Analysis of the data led to proposals as to which amino acids were important for function and which were not.

The results showed that most of the amino acids could be changed in multiple ways and still retain function. Analysis indicated that loss of function was likely to be associated with changes in three amino acids within a small patch between residues 33 and 37. Amino acids L33, E36, and L37 appeared to be the most important. This was confirmed directly by creating site-directed mutants in these positions and observing a loss of function. Very similar changes were made in several nearby amino acids, and these retained function. Thus, most amino-terminal residues assayed by substitution did not show defects. It might require specific multiple mutations outside the 33-to-37 region for a loss of function to be observed. Moreover, the type of substitution is probably important, as substitution of serine at position 37 was not deleterious (9) and substitution of glutamine within this region had a modest effect (see Results). It is also possible that the other amino acids within the N terminus might be important for promoter-activator combinations that differed from the one used as the basis for the genetic screen.

Sigma proteins with inactivating changes in each of these positions were purified and studied in vitro. Changes in L33 and L37 caused defects in transcription. A change in E36 did not lead to lower transcription in vitro (discussed further below), as has been observed for some multiple point mutations that were defective in vivo (unpublished data).

L33 and L37 mutants were studied by using more detailed assays to attempt to identify the step at which the transcription defect occurred. Changes at either leucine did not strongly interfere with the binding of polymerase to the promoter or with the formation of a heparin-sensitive transcription complex. However, the formation of a heparin-resistant complex was defective. Prior studies have associated this formation with the use of ATP and activator to form fully stable open complexes (25, 29). Thus, the data indicate that L33 and L37 have specific roles in the enhancer- and energy-dependent melting of the DNA. Recently, we determined that formation of heparin-resistant complexes involves a novel fork-binding activity of sigma 54 (8). Preliminary experiments suggest that mutations in L33 and L37 can strongly inhibit this activity. Thus, the residues may be important in creating or maintaining the fork-binding activity of sigma.

Both leucine 33 and leucine 37 are seen to be very highly conserved when sigma 54 genes from different organisms are compared (2). This suggests that they may play a common role in all organisms. By contrast glutamate 36, which was important in vivo but did not show its defect in vitro, is not conserved. Possibly E36 has a role in steps that are not assayed here, such as reinitiation (27). Alternatively, it may be required for the assembly of transcription complexes in vivo using sets of proteins that are not used in the particular in vitro assay system. It is interesting to note that E36 becomes protease sensitive during initiation (5). Other amino acids near the 33-to-37 patch, such as glutamate 32, are strongly conserved, and their functions remain to be established.

This patch is predominantly leucines and glutamines, and it is within the N-terminal region that has a high (40%) content of these two residues. Some of the residues in this patch have been changed as part of multiple leucine and glutamine mutations in prior studies (9, 10). L33 is part of a heptad hydrophobic repeat that has been previously identified as being important for activation and 12-region recognition, but in vitro studies were not done (9, 10). The 33-to-37 patch is also covered by deletions that were known to eliminate the response to activation (30). The glutamines near and within this patch could be changed without loss of function, consistent with the modest defects associated with multiple glutamine mutations (10). Overall, the identification of L33 and L37 as important residues is consistent with prior studies.

The 33-to-37 region is directly adjacent to a region between amino acids 25 and 31 that is associated with a different critical property of sigma 54. The 25-to-31 region contains leucines that have roles in locking sigma 54 holoenzyme in a closed-complex form prior to activation (25, 31). Mutating leucines in this patch allows some unactivated transcription to occur. However, NtrC and ATP can fully stimulate when leucines between 25 and 31 are mutated, in contrast to the properties of mutations at L33 and L37. The above-mentioned data show that this locking determinant probably extends beyond position 31 to include the small 33-to-37 region identified in the present study. Figure 6 shows the overlap between the locking leucine patch and the activation response determinants uncovered here. The existing data indicate that the entire 25-to-37 region helps keep the polymerase inactive initially. After signal transduction the phosphorylated NtrC would loop (24) to the inactive polymerase and use determinants that include the 33-to-37 subregion to complete the activation process. The 33-to-37 subregion need not contact the activator directly but could be downstream in the response pathway, perhaps, as discussed above, being defective in maintaining an activator-induced DNA fork junction binding activity (8).

View larger version (7K): [in this window] [in a new window]   FIG. 6.   Overlap between inhibitory leucine patch and activation response determinants. The region involved in positive activation (defective) is boxed, and the region required to keep melting in check prior to activation (bypass) is underlined.

Thus, at this stage of investigation it is clear that residues between amino acids 25 and 37 are involved in multiple aspects of activation. The entire region keeps the polymerase from melting the DNA inappropriately. The 33-to-37 subregion is then required in the activation response pathway; the inhibition is first overcome, and eventually the melting reaction is fully completed. One can speculate that NtrC makes contact with sigma (11) to disorganize an inhibitory structure and drive conformational changes (5) that lead eventually to stable melting. The subregion would not be expected to contain all of the required response determinants (19), nor need it control the response to all sigma 54-dependent activators. Other elements within the sigma 54 response pathways are likely to be uncovered.