Crystal Structure and RNA Binding of the Tex Protein from Pseudomonas aeruginosa (original) (raw)
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Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein
2010
In bacteria, the binding of a single protein, the initiation factor j, to a multi-subunit RNA polymerase core enzyme results in the formation of a holoenzyme, the active form of RNA polymerase essential for transcription initiation. Here we report the crystal structure of a bacterial RNA polymerase holoenzyme from Thermus thermophilus at 2.6 Å resolution. In the structure, two aminoterminal domains of the j subunit form a V-shaped structure near the opening of the upstream DNA-binding channel of the active site cleft. The carboxy-terminal domain of j is near the outlet of the RNA-exit channel, about 57 Å from the N-terminal domains. The extended linker domain forms a hairpin protruding into the active site cleft, then stretching through the RNA-exit channel to connect the N-and C-terminal domains. The holoenzyme structure provides insight into the structural organization of transcription intermediate complexes and into the mechanism of transcription initiation.
Journal of bacteriology, 2014
Bacteria utilize multiple sigma factors that associate with core RNA polymerase (RNAP) to control transcription in response to changes in environmental conditions. In Escherichia coli and Salmonella enterica, Crl positively regulates the σ(S) regulon by binding to σ(S) to promote its association with core RNAP. We recently characterized the determinants in σ(S) responsible for specific binding to Crl. However, little is known about the determinants in Crl required for this interaction. Here, we present the X-ray crystal structure of a Crl homolog from Proteus mirabilis in conjunction with in vivo and in vitro approaches that probe the Crl-σ(S) interaction in E. coli. We show that the P. mirabilis, Vibrio harveyi, and E. coli Crl homologs function similarly in E. coli, indicating that Crl structure and function are likely conserved throughout gammaproteobacteria. We utilize phylogenetic conservation and bacterial two-hybrid analyses to predict residues in Crl important for the intera...
Structure, 2013
In bacteria, the highly conserved RsmA/CsrA family of RNA-binding proteins functions as global posttranscriptional regulators acting on mRNA translation and stability. Through phenotypic complementation of an rsmA mutant in Pseudomonas aeruginosa, we discovered a family member, termed RsmN. Elucidation of the RsmN crystal structure and that of the complex with a hairpin from the sRNA, RsmZ, reveals a uniquely inserted a helix, which redirects the polypeptide chain to form a distinctly different protein fold to the domain-swapped dimeric structure of RsmA homologs. The overall b sheet structure required for RNA recognition is, however, preserved with compensatory sequence and structure differences, allowing the RsmN dimer to target binding motifs in both structured hairpin loops and flexible disordered RNAs. Phylogenetic analysis indicates that, although RsmN appears unique to P. aeruginosa, homologous proteins with the inserted a helix are more widespread and arose as a consequence of a gene duplication event.
Mechanism of Transcriptional Activation by Pseudomonas aeruginosa ExsA
Journal of Bacteriology, 2009
ExsA is a transcriptional activator of the Pseudomonas aeruginosa type III secretion system (T3SS). The T3SS consists of >40 genes organized within 10 transcriptional units, each of which is controlled by the transcriptional activator ExsA. ExsA-dependent promoters contain two adjacent ExsA binding sites that when occupied protect the ؊30 to ؊70 region from DNase I cleavage. The promoters also possess regions bearing strong resemblance to the consensus ؊10 and ؊35 regions of 70 -dependent promoters. The spacing distance between the putative ؊10 and ؊35 regions of ExsA-dependent promoters, however, is increased by 4 to 5 bp compared to that in typical 70 -dependent promoters. In the present study, we demonstrate that ExsA-dependent transcriptional activation requires a 21-or 22-bp spacer length between the ؊10 and ؊35 regions. Despite the atypical spacing in this region, in vitro transcription assays using 70 -saturated RNA polymerase holoenzyme (RNAP-70 ) confirm that ExsA-dependent promoters are indeed 70 dependent. Potassium permanganate footprinting experiments indicate that ExsA facilitates an early step in transcriptional initiation. Although RNAP-70 binds to the promoters with low affinity in the absence of ExsA, the activator stimulates transcription by enhancing recruitment of RNAP-70 to the P exsC and P exsD promoters. Abortive initiation assays confirm that ExsA enhances the equilibrium binding constant for RNAP while having only a modest effect on the isomerization rate constant.
Structural basis of ribosomal RNA transcription regulation
Nature Communications, 2021
Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ 70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β’ lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ 1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ 1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnera...
Crystal Structure of Escherichia coli Rnk, a New RNA Polymerase-Interacting Protein
Journal of Molecular Biology, 2008
Sequence-based searches identified a new family of genes in proteobacteria, named rnk, that shares high sequence similarity with the C-terminal domains of the Gre-factors (GreA, GreB) and the Thermus/Deinococcus anti-Gre-factor Gfh1. We solved the X-ray crystal structure of Escherichia coli Rnk at 1.9 Å-resolution using the anomalous signal from the native protein. The Rnk structure strikingly resembles those of E. coli GreA, GreB, and Thermus Gfh1, all of which are RNAP secondary channel effectors, and all of which have a C-terminal domain belonging to the FKBP fold. Rnk, however, has a much shorter N-terminal coiled-coil. Rnk does not stimulate transcript cleavage in vitro, nor does it reduce the lifetime of the complex formed by RNAP on promoters. We show that Rnk competes with the Gre-factors and DksA (another RNAP secondary channel effector) for binding to RNAP in vitro, and although we found that the concentration of Rnk in vivo was much lower than that of DksA, it was similar to that of GreB, consistent with a potential regulatory role for Rnk as an anti-Gre factor.
Abstract Background: Sigma factors are proteins that regulate transcription in bacteria. Sigma factors can be activated in response to different environmental conditions. The rpoS (RNA polymerase, sigma S) gene encodes sigma-38 (σ38, or RpoS), a 37.8 kDa protein in Pseudomonas aeruginosa (P. aeruginosa) strains. RpoS is a central regulator of the general stress response and operates in both retroactive and proactive manners; not only does it allow the cell to survive environmental challenges; it also prepares the cell for subsequent stresses (cross-protection). Methods: The significance of RpoS for stress resistance and protein expression in stationary-phase P. aeruginosa cells was assessed. The goal of the current study was to characterize RpoS of P. aeruginosa PAO1 using bioinformatics tools. Results: The results showed that RpoS is an unstable protein that belongs to the sigma-70 factor family. Secondary structure analysis predicted that random coil is the predominant structure followed by extended alpha helix. The three-dimensional (3D) structure was modeled using SWISS-MODEL Workspace. Conclusion: Determination of sequence, function, structure, and predicted epitopes of RpoS is important for modeling of inhibitors that will help in the design of new drugs to combat multi-drug-resistant (MDR) strains. Such information may aid in the development of new diagnostic tools, drugs, and vaccines for treatment in endemic regions.
FEBS Open Bio, 2015
RutR is a member of the large family of TetR transcriptional regulators in Escherichia coli. It was originally discovered as the regulator of the rutABCDEFG operon encoding a novel pathway for pyrimidine utilization, but its highest affinity target is the control region of the carAB operon, encoding carbamoylphosphate synthase. Unlike most other TetR-like regulators, RutR exerts both positive and negative effects on promoter activity. Furthermore, RutR exhibits a very narrow ligand binding specificity, unlike the broad effector specificity that characterizes some of the well-studied multidrug resistance regulators of the family. Here we focus on ligand binding and ligand specificity of RutR. We construct single alanine substitution mutants of amino acid residues of the ligand-binding pocket, study their effect on in vitro DNA binding in absence and presence of potential ligands, and analyse their effect on positive regulation of the carP1 promoter and negative autoregulation in vivo. Although RutR structures have been determined previously, they were deposited in the Protein Data Bank without accompanying publications. All of them have uracil bound in the effectorbinding site, representing the inactive form of the regulator. We determined the crystal structure of an unliganded mutant RutR protein and provide a structural basis for the use of uracil as sole effector molecule and the exclusion of the very similar thymine from the ligand-binding pocket.