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Bacterial RNA polymerase holoenzyme consists of a core enzyme (₂') and a dissociable  subunit. Bacteria often contain multiple  factors with individual sequence specificities that direct holoenzyme to different classes of promoters (22). ⁵⁴ is a distinctive bacterial sigma factor that does not share significant sequence homology with other sigma factors. Transcription initiation by ⁵⁴-RNA polymerase holoenzyme (⁵⁴-holoenzyme) occurs through a distinct mechanism that involves an activator or enhancer-binding protein (3). ⁵⁴-Holoenzyme binds to promoters to form stable closed complexes. Isomerization of these closed complexes to transcriptionally active open complexes requires an enhancer-binding protein, which generally binds to sites upstream of the promoter and contacts ⁵⁴-holoenzyme through DNA looping. This isomerization requires nucleoside triphosphate hydrolysis by the enhancer-binding protein.

Deletion analysis of ⁵⁴ has revealed that the protein consists of at least three functional regions (7, 16, 26, 32, 33). The 50 amino-terminal residues of ⁵⁴ constitute region I, which is rich in glutamine and leucine residues and plays a direct role in transcriptional activation (16, 17). Region II of ⁵⁴ from enteric bacteria, which extends from residue 50 to residue 120 and is highly acidic, appears to influence the rate of open complex formation, as well as suppress nonspecific DNA binding by holoenzyme (4, 32). Region III consists of 360 carboxy-terminal residues and contains determinants for binding of both core RNA polymerase and promoter DNA (7, 27, 28, 33). Additional functions for region III are likely, as some substitutions in this region disrupt one or more steps in transcription initiation following initial promoter recognition (11, 18).

When region I of ⁵⁴ is deleted, the resulting holoenzyme assumes a conformation believed to reflect polymerase isomerization (6). This isomerization is inhibited by a peptide containing the region I sequence, indicating that region I can elicit its effects on holoenzyme in trans (6, 14). Holoenzyme containing ⁵⁴ with region I deleted fails to initiate transcription under normal conditions but can initiate transcription independently of enhancer-binding protein from a transiently melted or premelted DNA template (6, 29). These studies suggest that region I inhibits isomerization of the holoenzyme to a form that can stably associate with single-stranded DNA (4). Productive interaction with the enhancer-binding protein apparently relieves this inhibition and permits transcription initiation in a reaction that requires nucleotide hydrolysis by the enhancer-binding protein (14).

We previously isolated several mutant forms of Salmonella enterica serovar Typhimurium ⁵⁴ that were defective in transcription initiation but retained core- and DNA-binding activities (18). Further analysis of these mutant ⁵⁴ proteins, the results of which are presented here, revealed that certain amino acid substitutions in regions I and III allowed the holoenzyme to initiate transcription from a heteroduplex DNA template in the absence of enhancer-binding protein, suggesting that sequences within these regions influence isomerization of the holoenzyme. The remaining ⁵⁴ mutant proteins had amino acid substitutions within region III and represented a second class of mutants that did not allow efficient transcription from the heteroduplex template in the absence of enhancer-binding protein. The strains and plasmids used in this study are described in Table 1.

L179P retains core- and promoter-binding activities. Several mutant forms of ⁵⁴ were isolated previously that failed to function normally at the ⁵⁴-dependent glnA promoter (glnAp2) but retained promoter-binding activities (20). We included here a new ⁵⁴ mutant protein, L179P, that was identified using the same screening procedure following random mutagenesis of a plasmid-borne copy of ntrA (encodes ⁵⁴) with the Epicurian Coli XL1-Red strain (Stratagene, La Jolla, Calif.). We previously used a gel mobility shift assay with labeled oligonucleotides that corresponded to the 9 to 29 region of the Sinorhizobium meliloti nifH promoter to examine interactions between the mutant forms of ⁵⁴ and core RNA polymerase, as well as the binding of the mutant proteins to promoter sequences (20). One oligonucleotide was double stranded over its entire length (double-stranded probe), while in the second oligonucleotide residues 11 to 9 of the template strand were single stranded (fork junction probe). Guo and Gralla first demonstrated fork junction DNA-binding activity of ⁵⁴ using these probes (15), and we subsequently found that the ⁵⁴-holoenzyme bound the fork junction probe significantly better than it did the double-stranded probe (18).

View larger version (66K): [in this window] [in a new window]   FIG. 1.   Gel mobility shift assay with double-stranded and fork junction probes. Binding of mutant forms of 54-holoenzyme to the S. meliloti nifH promoter was analyzed by a gel mobility shift assay as previously described (18). Two different DNA probes were used for the gel mobility shift assays. One probe contained 21 bp of double-stranded DNA that included residues 9 through 29 of the nifH promoter. The second probe had 18 bp of double-stranded DNA, which included residues 12 through 29 of the nifH promoter plus a three-base 5' overhang of the template strand that corresponded to residues 9 through 11. Binding reaction mixtures contained 5 nM DNA probe and 6 µg of sonicated calf thymus DNA/ml along with 300 nM core RNA polymerase (lanes 1 and 2), 600 nM hexahistidine-tagged 54 (lanes 3 and 4), 300 nM core plus 600 nM hexahistidine-tagged 54 (lanes 5 and 6), and 300 nM core plus 600 nM hexahistidine-tagged L179P (lanes 7 and 8). Core RNA polymerase and histidine-tagged 54 proteins were purified as previously described (20, 23). The double-stranded probe was used in odd-numbered lanes, while the fork junction probe was used in even-numbered lanes. The holoenzyme-shifted (h) and 54-shifted () species are indicated. Unbound probes are not shown.

Mutant forms of ⁵⁴ can be cross-linked to DctD. S. enterica serovar Typhimurium ⁵⁴ can be chemically cross-linked to the enhancer-binding protein S. meliloti C₄-dicarboxylic acid transport protein D (DctD), suggesting that ⁵⁴ is a primary target for the enhancer-binding protein (20). Consistent with this hypothesis, mutant forms of DctD that failed to activate transcription and cross-linked poorly to ⁵⁴ have been isolated (31). Further support of this hypothesis comes from the observation that ⁵⁴ bound to heteroduplex DNA in the absence of core RNA polymerase undergoes a conformational change in a reaction that requires enhancer-binding protein and nucleotide hydrolysis (8).

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View this table: [in this window] [in a new window]   TABLE 2.   Abilities of mutant forms of 54 to function with DctD(1-142) at a dctA'-'lacZ reporter gene in vivo

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Certain mutant forms of ⁵⁴ allow activator-independent transcription. We initially used an in vitro transcription assay to examine the abilities of the ⁵⁴ mutant proteins to support transcription from a supercoiled template that carried the S. enterica serovar Typhimurium glnA promoter region. In this transcription assay, heparin was added immediately before the nucleotides to prevent reinitiation but allow transcription from stable open complexes. None of the ⁵⁴ mutant proteins supported transcription under these conditions, except the L46P mutant protein, which had given the highest activity in vivo (Table 2). The level of transcript generated with the L46P mutant protein was ~5% of that generated with wild-type ⁵⁴ (Fig. 2). Addition of heparin to the transcription assay reaction after addition of the nucleotides did not alter the results of the assays with the mutant proteins (data not shown). These data confirmed our earlier in vivo findings that these mutant ⁵⁴ proteins were defective in their function at glnAp2 (18).

View larger version (41K): [in this window] [in a new window]   FIG. 2.   In vitro transcription with mutant forms of 54 from the glnA promoter regulatory region on a supercoiled DNA template. Transcription assays were carried out as described previously (24, 31). Assay reaction mixtures contained 400 nM maltose-binding protein-NtrC, 20 mM carbamoyl phosphate, 100 nM core RNA polymerase, and a 300 nM concentration of either hexahistidine-tagged 54 (lane 1), L37P (lane 2), L46P (lane 3), E32K G189V (lane 4), L124P V148A (lane 5), L179P (lane 6), L199P D231G (lane 7), or L333P (lane 8). The arrow indicates the transcript from the glnA promoter.

View larger version (59K): [in this window] [in a new window]   FIG. 3.   In vitro transcription assays using a heteroduplex DNA template. Transcription assays were carried out using a heteroduplex DNA template that spanned residues 60 to +28 of the S. meliloti nifH promoter and was noncomplementary between residues 10 and 1. The heteroduplex template was generated using two oligonucleotides as previously described (6). Transcription assays were conducted as previously described (6), except that 7.5 µCi of [-32P]CTP (3,000 Ci/mmol) was used to label the transcripts. Reaction mixtures contained 100 nM core RNA polymerase, 300 nM hexahistidine-tagged 54 proteins, and 30 nM heteroduplex DNA template. The 54 proteins assayed were the wild type (lane 1) and the L37P (lane 2), L46P (lane 3), L333P (lane 4), E32K G189A (lane 5), L179P (lane 6), L199P D231G (lane 7), and L124P V148A (lane 8) mutant proteins. The arrow indicates the 28-base transcript.

Alanine scanning mutagenesis of the region around Leu-333. The similarities between the L37P and L333P mutant proteins in the ability to support activator-independent transcription (Fig. 3) and the reduced affinity for fork junction DNA (18) prompted us to examine conserved residues in the vicinity of Leu-333. Leu-333 lies within a well-conserved region of ⁵⁴ that was shown previously to be cross-linked to DNA upon UV irradiation of ⁵⁴-promoter complexes (5). It is also close to a putative helix-turn-helix motif that has been implicated in recognition of the 12 region of the promoter (12, 23). When we compared the sequences of this portion of ⁵⁴ proteins from 25 different bacteria, we noted that it was similar to a motif from single-stranded DNA-binding proteins (Fig. 4A). This motif from single-stranded DNA-binding proteins, (R/K)-N_4-5-(K/R)-N_4-6-(Y/F)-N_5-8-(Y/F/N)-N_6-9-(Y/C)-N₉-(E/D)-N_7-9-(Y/W)-N_4-8-(Y/F/E)-N_11-12-(R/K), is thought to stabilize the binding of single-stranded DNA by stacking interactions between aromatic residues and bases and electrostatic interactions between basic residues and phosphate groups of single-stranded DNA (25).

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Alanine scanning mutagenesis of conserved residues near Leu-333. (A) Amino acid sequence of the 54 protein from S. enterica serovar Typhimurium near Leu-333. The PILEUP program was used to compare sequences around Leu-333 from 54 proteins from 25 bacteria. The sequence shown spans residues 328 to 404. Leu-333 is marked with a dot over the sequence. Boldface letters indicate residues that are identical in at least 22 of the 25 54 protein sequences, and italic letters indicate residues that are similar in at least 22 of the 54 protein sequences. Arrows indicate the residues where alanine substitutions were introduced. The stars indicate residues that align with the single-stranded DNA-binding motif, which is indicated on the bottom line. The sequence that is cross-linked to promoter DNA upon UV irradiation (5) is indicated, as is a putative helix-turn-helix motif that appears to be involved in DNA binding (23). (B) Glutamine synthetase activities observed with 54 mutant proteins. Cells were grown in a modified E medium that lacked sodium ammonium phosphate and was supplemented with acid-hydrolyzed Casamino Acids at 1 mg/liter as described previously (18). Glutamine synthetase activities of strains that express the alanine substitution 54 mutant proteins were carried out using the -glutamyl transferase assay as described previously (18). Glutamine synthetase activities were expressed as micromoles of -glutamyl hydroxymate per minute per milligram of protein, and all assays were done at least twice. Error bars show one standard deviation for each data set. The wild-type and mutant ntrA alleles, which were carried on plasmids, were under the control of the E. coli lac promoter-operator and were overexpressed by including 100 µM isopropyl--thiogalactopyranoside in the growth medium. The no-protein designation indicates the glutamine synthetase activity from S. enterica serovar Typhimurium strain TRH134 without a plasmid-borne ntrA allele. (C) Repression of ant'-'lacZ reporter gene in S. enterica serovar Typhimurium strain TRH107 by 54 proteins. -Galactosidase assays were done in triplicate with strain TRH107, carrying plasmids that encode the 54 proteins indicated, as described previously (1, 18). Error bars show one standard deviation for each data set. The no-protein control shows the activity for strain TRH107 with no plasmid-borne ntrA allele.

Conclusions. Region I of ⁵⁴ functions as an intramolecular inhibitor that prevents isomerization of the closed complex to the open complex, but it also has a role in DNA melting in response to the enhancer-binding protein (6, 13, 29). Two of the mutants that were analyzed here, L37P and L46P, had single amino acid substitutions in region I and allowed activator-independent transcription from the heteroduplex template, indicating that these amino acid substitutions disrupt the inhibitory effect of region I on the holoenzyme. Leu-37 is within a small leucine-rich patch, residues Leu-25 through Leu-37, that was shown previously to be important for mediation of the inhibitory effect of region I on the holoenzyme (10, 26, 30). Substitution of arginine for Leu-37 in E. coli ⁵⁴ was shown previously to allow activator-independent transcription. The L37R mutant protein, however, remained responsive to enhancer-binding protein in vitro, allowing NtrC-mediated activation to about 50% of the level observed with wild-type ⁵⁴ (26). The L37P mutant protein described in this study appeared to be more severely impaired in the ability to respond to enhancer-binding protein, which likely reflects disruption of secondary structure in the protein by the proline substitution. Casaz and coworkers identified two other areas within region I, residues 15 to 17 and 42 to 47, that are important for inhibition of the conformational change in the holoenzyme and responsiveness to the activator (10). Consistent with these previous results, the L46P mutant enzyme was capable of activator-independent transcription from the heteroduplex template and also appeared to be impaired in the ability to respond to enhancer-binding protein, but not as severely as the L37P mutant protein.