Structural Basis of RNA Polymerase I Transcription Initiation.

Transcription initiation at the ribosomal RNA promoter requires RNA polymerase (Pol) I and the initiation factors Rrn3 and core factor (CF). Here, we combine X-ray crystallography and cryo-electron microscopy (cryo-EM) to obtain a molecular model for basal Pol I initiation. The three-subunit CF binds upstream promoter DNA, docks to the Pol I-Rrn3 complex, and loads DNA into the expanded active center cleft of the polymerase.

Structural basis of transcription initiation by RNA polymerase II.

Transcription of eukaryotic protein-coding genes commences with the assembly of a conserved initiation complex, which consists of RNA polymerase II (Pol II) and the general transcription factors, at promoter DNA. After two decades of research, the structural basis of transcription initiation is emerging. Crystal structures of many components of the initiation complex have been resolved, and structural information on Pol II complexes with general transcription factors has recently been obtained.

Conserved architecture of the core RNA polymerase II initiation complex.

During transcription initiation at promoters of protein-coding genes, RNA polymerase (Pol) II assembles with TBP, TFIIB and TFIIF into a conserved core initiation complex that recruits additional factors. The core complex stabilizes open DNA and initiates RNA synthesis, and it is conserved in the Pol I and Pol III transcription systems. Here, we derive the domain architecture of the yeast core pol II initiation complex during transcription initiation.

RNA polymerase I structure and transcription regulation.

Transcription of ribosomal RNA by RNA polymerase (Pol) I initiates ribosome biogenesis and regulates eukaryotic cell growth. The crystal structure of Pol I from the yeast Saccharomyces cerevisiae at 2.8 Å resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II.

Structure and function of the initially transcribing RNA polymerase II-TFIIB complex.

The general transcription factor (TF) IIB is required for RNA polymerase (Pol) II initiation and extends with its B-reader element into the Pol II active centre cleft. Low-resolution structures of the Pol II-TFIIB complex indicated how TFIIB functions in DNA recruitment, but they lacked nucleic acids and half of the B-reader, leaving other TFIIB functions enigmatic. Here we report crystal structures of the Pol II-TFIIB complex from the yeast Saccharomyces cerevisiae at 3.

Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) from Neisseria meningitidis.

The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from Neisseria meningitidis, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain.

Structural basis of initial RNA polymerase II transcription.

During transcription initiation by RNA polymerase (Pol) II, a transient open promoter complex (OC) is converted to an initially transcribing complex (ITC) containing short RNAs, and to a stable elongation complex (EC). We report structures of a Pol II-DNA complex mimicking part of the OC, and of complexes representing minimal ITCs with 2, 4, 5, 6, and 7 nucleotide (nt) RNAs, with and without a non-hydrolyzable nucleoside triphosphate (NTP) in the insertion site +1. The partial OC structure reveals that Pol II positions the melted template strand opposite the active site.

Architecture of the RNA polymerase-Spt4/5 complex and basis of universal transcription processivity.

Related RNA polymerases (RNAPs) carry out cellular gene transcription in all three kingdoms of life. The universal conservation of the transcription machinery extends to a single RNAP-associated factor, Spt5 (or NusG in bacteria), which renders RNAP processive and may have arisen early to permit evolution of long genes. Spt5 associates with Spt4 to form the Spt4/5 heterodimer.

Crystal structures of penicillin-binding protein 3 from Pseudomonas aeruginosa: comparison of native and antibiotic-bound forms.

We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft.

The structure of a reduced form of OxyR from Neisseria meningitidis.

Background: Survival of the human pathogen, Neisseria meningitidis, requires an effective response to oxidative stress resulting from the release of hydrogen peroxide by cells of the human immune system. In N. meningitidis, expression of catalase, which is responsible for detoxifying hydrogen peroxide, is controlled by OxyR, a redox responsive LysR-type regulator.

Some lessons from the systematic production and structural analysis of soluble (alpha)(beta) T-cell receptors.

T-cell receptors (TCRs) are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response. The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein. Here we report our systematic approach for generating soluble, (alpha)(beta)-TCRs, for X-ray crystallographic studies.

The structure of CrgA from Neisseria meningitidis reveals a new octameric assembly state for LysR transcriptional regulators.

LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes. The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial-host cell contact. Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings.

T cell-mediated autoimmune disease due to low-affinity crossreactivity to common microbial peptides.

Environmental factors account for 75% of the risk of developing multiple sclerosis (MS). Numerous infections have been suspected as environmental disease triggers, but none of them has consistently been incriminated, and it is unclear how so many different infections may play a role. We show that a microbial peptide, common to several major classes of bacteria, can induce MS-like disease in humanized mice by crossreacting with a T cell receptor (TCR) that also recognizes a peptide from myelin basic protein, a candidate MS autoantigen.

The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis.

The structure of the MarR-family transcription factor NMB1585 from Neisseria meningitidis has been solved using data extending to a resolution of 2.1 A. Overall, the dimeric structure resembles those of other MarR proteins, with each subunit comprising a winged helix-turn-helix (wHtH) domain connected to an alpha-helical dimerization domain.

Crystallization and preliminary X-ray analysis of CrgA, a LysR-type transcriptional regulator from pathogenic Neisseria meningitidis MC58.

Although LysR-type regulators (LTTRs) represent the largest family of transcriptional regulators in bacteria, the full-length structure of only one annotated LTTR (CbnR) has been deposited in the PDB. CrgA, a LTTR from pathogenic Neisseria meningitidis MC58, which is up-regulated upon bacterial cell contact with human epithelial cells, has been cloned, purified and crystallized. Crystals of full-length CrgA were obtained after buffer screening with a thermal shift assay and concentration with 0.

The crystal structure of UMP kinase from Bacillus anthracis (BA1797) reveals an allosteric nucleotide-binding site.

Uridine monophosphate (UMP) kinase is a conserved enzyme that catalyzes the ATP-driven conversion of uridylate monophosphate into uridylate diphosphate, an essential metabolic step. In prokaryotes, the enzyme exists as a homohexamer that is regulated by various metabolites. Whereas the enzymatic mechanism of UMP kinase (UK) is well-characterized, the molecular basis of its regulation remains poorly understood.

Structure of the cold-shock domain protein from Neisseria meningitidis reveals a strand-exchanged dimer.

The structure of the cold-shock domain protein from Neisseria meningitidis has been solved to 2.6 A resolution and shown to comprise a dimer formed by the exchange of two beta-strands between protein monomers. The overall fold of the monomer closely resembles those of other bacterial cold-shock proteins.

Semi-automated microseeding of nanolitre crystallization experiments.

A simple semi-automated microseeding procedure for nanolitre crystallization experiments is described. Firstly, a microseed stock solution is made from microcrystals using a Teflon bead. A dilution series of this microseed stock is then prepared and dispensed as 100 nl droplets into 96-well crystallization plates, facilitating the incorporation of seeding into high-throughput crystallization pipelines.

The structure and transcriptional analysis of a global regulator from Neisseria meningitidis.

Neisseria meningitidis, a causative agent of bacterial meningitis, has a relatively small repertoire of transcription factors, including NMB0573 (annotated AsnC), a member of the Lrp-AsnC family of regulators that are widely expressed in both Bacteria and Archaea. In the present study we show that NMB0573 binds to l-leucine and l-methionine and have solved the structure of the protein with and without bound amino acids. This has shown, for the first time that amino acid binding does not induce significant conformational changes in the structure of an AsnC/Lrp regulator although it does appear to stabilize the octameric assembly of the protein.

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications.

This article describes the construction of a set of versatile expression vectors based on the In-Fusion cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His(6)-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein.

Structure of the PII signal transduction protein of Neisseria meningitidis at 1.85 A resolution.

The P(II) signal transduction proteins GlnB and GlnK are implicated in the regulation of nitrogen assimilation in Escherichia coli and other enteric bacteria. P(II)-like proteins are widely distributed in bacteria, archaea and plants. In contrast to other bacteria, Neisseria are limited to a single P(II) protein (NMB 1995), which shows a high level of sequence identity to GlnB and GlnK from Escherichia coli (73 and 62%, respectively).

Crystal structure of nitrogen regulatory protein IIANtr from Neisseria meningitidis.

Background: The NMB0736 gene of Neisseria meningitidis serogroup B strain MC58 encodes the putative nitrogen regulatory protein, IIANtr (abbreviated to NM-IIANtr). The homologous protein present in Escherichia coli is implicated in the control of nitrogen assimilation. As part of a structural proteomics approach to the study of pathogenic Neisseria spp.

The nsp9 replicase protein of SARS-coronavirus, structure and functional insights.

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single beta-barrel with a fold previously unseen in single domain proteins.