The Amino Terminus of Salmonella enterica Serovar Typhimurium ς ⁵⁴ Is Required for Interactions with an Enhancer-Binding Protein and Binding to Fork Junction DNA

Abstract

(20,28).Interactions between 54 -holoenzyme and enhancer-binding proteins are transient, and little is known about the nature of these interactions. The C 4 -dicarboxylic acid transport protein D (DctD) is an enhancer-binding protein from Sinorhizobium meliloti that can be cross-linked to 54 and the ␤ subunit of RNA polymerase (16). Some mutant forms of DctD that fail to activate transcription also fail to cross-link to these subunits (27), suggesting that DctD contacts these subunits of 54 -holoenzyme to catalyze open-complex formation. To identify the region of 54 that interacts with DctD, we generated a series of truncated 54 proteins and assessed their abilities to cross-link with DctD.Deletion of 179 amino acid residues from the amino terminus of 54 disrupts cross-linking to DctD. The cross-linking reagent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) can cross-link Sinorhizobium meliloti DctD and Salmonella enterica serovar Typhimurium 54 (16). Sulfo-SMCC is a heterobifunctional cross-linking reagent that has a maleimide group and an N-hydroxysuccinimide ester group, which react preferentially with sulfhydryl groups and primary amines, respectively, and are linked by a relatively long and flexible spacer arm. Cys-307 of S. enterica serovar Typhimurium 54 is critical for cross-linking to DctD (16), presumably because it is surface exposed and reacts readily with the maleimide group of sulfo-SMCC. For the cross-linking experiments in this study, we used DctD (⌬1-142) , which is a truncated, constitutively active form of DctD (17). As in previous studies, these cross-linking assays were done with purified 54 proteins and DctD (⌬1-142) in the absence of core RNA polymerase and DNA (16, 27).We initially generated three amino-terminally truncated 54 proteins that had deletions of residues 2 to 56 (⌬2-56), 2 to 105 (⌬2-105), and 2 to 179 (⌬2-179) (Fig. 1). All deletions were generated by using PCR to introduce a unique NdeI site at the desired position of rpoN, the gene encoding 54 . The NdeI site was used to clone the truncated rpoN alleles into the expression vector pCyt3 (provided by Elliot Altman, University of Georgia), which placed the rpoN alleles under the control of the Escherichia coli lac promoter/operator. The rpoN alleles were also cloned into the expression vector pET-28a(ϩ) (Novagen), which resulted in attachment of a hexahistidine tag at the amino terminus of each truncated 54 protein. Hexahistidine-tagged 54 proteins were overexpressed in E. coli BL21 (DE3) [F Ϫ ompT (lon) hsdS B gal DE3::lacI lacUV5-gene 1 (T7 polymerase)] by growing the cells in Luria-Bertani broth supplemented with 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG). Cells were harvested, resuspended in 50 mM Tris-acetate (pH 8.2)-200 mM KCl-1 mM EDTA-1 mM dithiothreitol, and lysed in a French pressure cell at 12,000 lb/in 2 . Like the full-length hexahistidine-tagged 54 protein (15), the truncated hexahistidine-tagged 54 proteins were in the insoluble fraction following centrifugation of the cell ...

Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the σ54 (RpoN) regulon of Salmonella Typhimurium LT2

“…0.6 ml of the resulting extracts were mixed with 2 μl of rabbit anti-serum against S . Typhimurium σ 54 [ 73 ] and incubated with gentle shaking overnight at 4°C. The next day, 50 μl of protein A-Sepharose bead slurry was added to each sample, incubated 1 hr at room temperature and collected by centrifugation.…”

Mechanochemical ATPases and transcriptional activation

“…Each of the DctD C3 mutants (in both the ESELF and the GAFTGA motifs) failed to activate transcription (Wang et al , 1997). Use of σ 54 with Region I deleted showed that σ 54 Region I was dispensable for binding DctD in the cross‐linking assay (Kelly and Hoover, 2000). In direct contrast to these results, the nucleotide‐dependent activator‐σ 54 binding assay using the transition state analogue ADP.AlF x showed that σ 54 Region I was essential for stable binding to PspFΔHTH and the central domain of NifA (Chaney et al , 2001).…”

The Bacterial Enhancer-Dependent ς54(ςN) Transcription Factor

“…Nonetheless, holoenzyme binds tighter than does 54 alone. Two other regions can affect the affinity of binding: the N terminus plays complex multiple roles in binding to both duplex and melted DNA (10,17,41,52,59,60,98,117), and the segment between the C terminus and the major core-binding determinant influences binding affinity (12,50). These regions do not necessarily make direct DNA contact.…”

“…This is accomplished using the ATPase activity of the activator, which loops from the enhancer site (96). The interactions between activator and the 54 -RNA polymerase holoenzyme appear to be transient, and the only direct physical evidence has been by a cross-linking assay using the DctD activator (60,69).…”