Complete structural model of Escherichia coli RNA polymerase from a hybrid approach.

The Escherichia coli transcription system is the best characterized from a biochemical and genetic point of view and has served as a model system. Nevertheless, a molecular understanding of the details of E. coli transcription and its regulation, and therefore its full exploitation as a model system, has been hampered by the absence of high-resolution structural information on E. coli RNA polymerase (RNAP). We use a combination of approaches, including high-resolution X-ray crystallography, ab initio structural prediction, homology modeling, and single-particle cryo-electron microscopy, to generate complete atomic models of E. coli core RNAP and an E. coli RNAP ternary elongation complex. The detailed and comprehensive structural descriptions can be used to help interpret previous biochemical and genetic data in a new light and provide a structural framework for designing experiments to understand the function of the E. coli lineage-specific insertions and their role in the E. coli transcription program.

Identification of the gate regions in the primary structure of the secretin pIV.

Secretins are a family of large bacterial outer membrane channels that serve as exit ports for folded proteins, filamentous phage and surface structures. Despite the large size of their substrates, secretins do not compromise the barrier function of the outer membrane, implying a gating mechanism. The region in the primary structure that forms the putative gate has not previously been determined for any secretin. To identify residues involved in gating the pIV secretin of filamentous bacteriophage f1, we used random mutagenesis of the gene followed by positive selection for mutants with compromised barrier function ('leaky' mutants). We identified mutations in 34 residues, 30 of which were clustered into two regions located in the centre of the conserved C-terminal secretin family domain: GATE1 (that spanned 39 residues) and GATE2 (that spanned 14 residues). An internal deletion constructed in the GATE2 region resulted in a severely leaky phenotype. Three of the four remaining mutations are located in the region that encodes the N-terminal, periplasmic portion of pIV and could be involved in triggering gate opening. Two missense mutations in the 24-residue region that separates GATE1 and GATE2 were also constructed. These mutant proteins were unstable, defective in multimerization and non-functional.

Interactome and interface protocol (2IP): a novel strategy for high sensitivity topology mapping of protein complexes.

A few well-characterized protein assemblies aside, little is known about the topology and interfaces of multiconstituent protein complexes. Here we report on a novel indirect strategy for low-resolution topology mapping of protein complexes. Following crosslinking, purified protein complexes are subjected to chemical cleavage with cyanogen bromide (CNBr) and the resulting fragments are resolved by 2-D electrophoresis. The side-by-side comparison of a thus generated and a 2-D CNBr fragment map obtained from uncrosslinked material reveals candidate gel spots harboring crosslinked CNBr fragments. In-gel trypsinization and MALDI MS analysis of these informative spots identify the underlying crosslinked CNBr fragments based on unmodified tryptic peptides. Matching the cumulative theoretical molecular mass and predicted pI of these crosslinked CNBr fragments with original gel spot coordinates is required for confident crosslink assignment. The above strategy was successfully validated with the Escherichia coli RNA polymerase (RNAP) core complex and subsequently applied to query the quaternary structure of components of the yeast Skp1-Cdc53/Cullin-F box (SCF) ubiquitin ligase complex. This protocol requires low picomole sample quantities, can be applied to multisubunit protein complexes, and does not rely on specialized data mining software.

Structure and function of the transcription elongation factor GreB bound to bacterial RNA polymerase.

Bacterial GreA and GreB promote transcription elongation by stimulating an endogenous, endonucleolytic transcript cleavage activity of the RNA polymerase. The structure of Escherichia coli core RNA polymerase bound to GreB was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 A, allowing fitting of high-resolution RNA polymerase and GreB structures. In the resulting model, the GreB N-terminal coiled-coil domain extends 45 A through a channel directly to the RNA polymerase active site. The model leads to detailed insights into the mechanism of Gre factor activity that explains a wide range of experimental observations and points to a key role for conserved acidic residues at the tip of the Gre factor coiled coil in modifying the RNA polymerase active site to catalyze the cleavage reaction. Mutational studies confirm that these positions are critical for Gre factor function.

Structure of the filamentous phage pIV multimer by cryo-electron microscopy.

The homo-multimeric pIV protein constitutes a channel required for the assembly and export of filamentous phage across the outer membrane of Escherichia coli. We present a 22 A-resolution three-dimensional reconstruction of detergent-solubilized pIV by cryo-electron microscopy associated with image analysis. The structure reveals a barrel-like complex, 13.5 nm in diameter and 24 nm in length, with D14 point-group symmetry, consisting of a dimer of unit multimers. Side views of each unit multimer exhibit three cylindrical domains named the N-ring, the M-ring and the C-ring. Gold labeling of pIV engineered to contain a single cysteine residue near the N or C terminus unambiguously identified the N-terminal region as the N-ring, and the C-terminal region was inferred to make up the C-ring. A large pore, ranging in inner diameter from 6.0 nm to 8.8 nm, runs through the middle of the multimer, but a central domain, the pore gate, blocks it. Moreover, the pore diameter at the N-ring is smaller than the phage particle. We therefore propose that the pIV multimer undergoes a large conformational change during phage transport, with reorganization of the central domain to open the pore, and widening at the N-ring in order to accommodate the 6.5 nm diameter phage particle.

Stability of rice yellow mottle virus and cellular compartmentalization during the infection process in Oryza sativa (L.).

Rice yellow mottle virus (RYMV) is icosahedral in morphology and known to swell in vitro, but the biological function of swollen particles remains unknown. Anion-exchange chromatography was used to identify three markedly stable forms of RYMV particles from infected plants: (1) an unstable swollen form lacking Ca2+ and dependent upon basic pH; (2) a more stable transitional form lacking Ca2+ but dependent upon acidic pH; and (3) a pH-independent, stable, compact form containing Ca2+. Particle stability increased over the time course of infection in rice plants: transitional and swollen forms were abundant during early infection (2 weeks postinfection), whereas compact forms increased during later stages of infection. Electron microscopy of infected tissue revealed virus particles in vacuoles of xylem parenchyma and mesophyll cells early in the time course of infection and suggested that vacuoles and other vesicles were the major storage compartments for virus particles. We propose a model in which virus maturation is associated with the virus accumulation in vacuoles. In this acidic compartment, virus particles may bind Ca2+ to produce a highly stable, compact form of the virus. The localization of subcellular RYMV isoforms in infected cells and the corresponding biological properties of the virus are discussed.

Conformational flexibility of bacterial RNA polymerase.

The structure of Escherichia coli core RNA polymerase (RNAP) was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 A. Because of the high sequence conservation between the core RNAP subunits, we were able to interpret the E. coli structure in relation to the high-resolution x-ray structure of Thermus aquaticus core RNAP. A very large conformational change of the T. aquaticus RNAP x-ray structure, corresponding to opening of the main DNA/RNA channel by nearly 25 A, was required to fit the E. coli map. This finding reveals, at least partially, the range of conformational flexibility of the RNAP, which is likely to have functional implications for the initiation of transcription, where the DNA template must be loaded into the channel.