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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.

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 Å, allowing fitting of high-resolution RNA polymerase and GreB structures. In the resulting model, the GreB N-terminal coiled-coil domain extends 45 Å 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.

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 Å. 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 Å, 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.

Rice yellow mottle virus (RYMV) is a major pathogen that dramatically reduces rice production in many African countries. RYMV belongs to the genus sobemovirus, one group of plant viruses with icosahedral capsids and single-stranded, positive-sense RNA genomes.The structure of RYMV was determined and refined to 2.8 A resolution by X-ray crystallography. The capsid contains 180 copies of the coat protein subunit arranged with T = 3 icosahedral symmetry. Each subunit adopts a jelly-roll beta sandwich fold. The RYMV capsid structure is similar to those of other sobemoviruses. When compared with these viruses, however, the betaA arm of the RYMV C subunit, which is a molecular switch that regulates quasi-equivalent subunit interactions, is swapped with the 2-fold-related betaA arm to a similar, noncovalent bonding environment. This exchange of identical structural elements across a symmetry axis is categorized as 3D domain swapping and produces long-range interactions throughout the icosahedral surface lattice. Biochemical analysis supports the notion that 3D domain swapping increases the stability of RYMV.The quasi-equivalent interactions between the RYMV proteins are regulated by the N-terminal ordered residues of the betaA arm, which functions as a molecular switch. Comparative analysis suggests that this molecular switch can also modulate the stability of the viral capsids.

Transcription in all cellular organisms is orchestrated by the multisubunit DNA-dependent RNA polymerase (RNAP), a multifunctional enzyme that synthesizes RNA and is a major target for the regulation of gene expression. The Escherichia coli RNAP, which is the best characterized, comprises an essential catalytic core of two α-subunits (each 36.5 kDa), one β-subunit (150.6 kDa), and one β′-subunit (155.2 kDa) that is responsible for transcript elongation and termination. The holoenzyme contains an additional regulatory subunit, normally σ70 (70.2 kDa), and is capable of promoter-specific recognition and transcription initiation. To identify the location of subunits within the RNAP, we developed a strategy to target insertions of a protein domain into surface-exposed, nonessential regions that can be visualized by electron microscopy (EM) and image processing. This strategy was readily applicable to either of the two largest subunits, β and β′, which have colinearly arranged regions of strong amino acid sequence similarity from bacteria to humans (1–3). The highly conserved regions are separated by relatively nonconserved spacer regions. In some organisms, the nonconserved regions contain large gaps or insertions compared with E. coli. In the β-subunit, nine conserved regions, labeled A through I, have been identified (ref. 3; Fig. ​Fig.1).1). In addition, the β-subunit of E. coli RNAP contains two regions of poor sequence conservation, centered about residues 300 and 1,000, which are often missing in β-homologs from other organisms (Fig. ​(Fig.1).1). Figure 1 Staphylococcus aureus protein A (SPA) insertion in the β-subunit of E. coli RNAP. The primary sequence of the β-subunit is represented as a black bar. Evolutionarily conserved regions are shaded grey and labeled A–I (3). Evolutionarily ... As expected, mutations affecting the conserved regions frequently cause severe defects in RNAP function (ref. 4 and references therein). β-Residues 1,065 and 1,237, in conserved regions H and I, respectively, participate in the formation of the initiating site of the enzyme (5). Mutations in the β-subunit render the enzyme resistant to the antibiotic inhibitors rifampicin and streptolydigin (refs. 6–9; Fig. ​Fig.11). Alternatively, mutations in poorly conserved regions can have little or no apparent effect on RNAP function. In fact, large deletions in the two poorly conserved regions in β do not affect RNAP assembly and basic function in vitro (10, 11). These regions have thus been termed DRI (situated between conserved regions B and C, extending approximately from residues 170 to 430) and DRII (situated between conserved regions G and H, extending approximately from residues 930 to 1,030). Both of these regions are poorly conserved or absent in β-subunit homologs from other organisms; both can tolerate numerous mutations without fatal consequences in vivo; and both must be structurally autonomous to accommodate large deletions and insertions without disturbing the critical functions of the enzyme. Thus, both DRs are likely to comprise separate and relatively isolated domains on the RNAP structure. Although neither DR of the β-subunit plays an important role in RNAP assembly or basic transcription activity, it is assumed that their presence points to a role in regulatory functions specific to E. coli that have not yet been identified. Indeed, DRI is targeted by the bacteriophage T4-Alc protein, which selectively induces premature termination of E. coli RNAP transcription on E. coli DNA (11), indicating that regulatory factors can impact transcription through these DRs. Herein, we report a structural analysis of a mutant E. coli RNAP harboring an insertion of a protein domain in β-DRII. The mutant RNAP was used to locate directly the inserted domain on the three-dimensional structure of RNAP by using cryo-EM and difference analysis with the previously determined native RNAP structure, thereby locating its site of insertion. The approach used for this study is similar to the peptide-based difference mapping reported by Conway et al. (12), except that a larger protein domain was used to ensure visualization at the lower resolution of our analysis (20-Å compared with 11-Å resolution), and an internal rather than a terminal region of the polypeptide was located.

A combination of structural approaches yields a complete atomic model of the highly biochemically characterized Escherichia coli RNA polymerase, enabling fuller exploitation of E. coli as a model for understanding transcription., 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., Author Summary Transcription, or the synthesis of RNA from DNA, is one of the most important processes in the cell. The central enzyme of transcription is the DNA-dependent RNA polymerase (RNAP), a large, macromolecular assembly consisting of at least five subunits. Historically, much of our fundamental information on the process of transcription has come from genetic and biochemical studies of RNAP from the model bacterium Escherichia coli. More recently, major breakthroughs in our understanding of the mechanism of action of RNAP have come from high resolution crystal structures of various bacterial, archaebacterial, and eukaryotic enzymes. However, all of our high-resolution bacterial RNAP structures are of enzymes from the thermophiles Thermus aquaticus or T. thermophilus, organisms with poorly characterized transcription systems. It has thus far proven impossible to obtain a high-resolution structure of E. coli RNAP, which has made it difficult to relate the large collection of genetic and biochemical data on RNAP function directly to the available structural information. Here, we used a combination of approaches—high-resolution X-ray crystallography of E. coli RNAP fragments, ab initio structure prediction, homology modeling, and single-particle cryo-electron microscopy—to generate complete atomic models of E. coli RNAP. Our detailed and comprehensive structural models provide the heretofore missing structural framework for understanding the function of the highly characterized E. coli RNAP.

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.

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.

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.

Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 A resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca(2+) from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.

The translocation of rice yellow mottle virus (RYMV) within tissues of inoculated and systemically infected Oryza sativa L. leaves was characterized by Western immunoblotting, Northern blotting, and electron microscopy of thin sections. In inoculated leaves, RYMV RNA and coat protein first were detected at 3 and 5 days postinoculation, respectively. By 6 days postinoculation, RYMV had spread systemically to leaves, and virus particles were observed in most cell types, including epidermal, mesophyll, bundle sheath, and vascular parenchyma cells. Most of the virions accumulated in large crystalline patches in xylem parenchyma cells and sieve elements. Colocalization of a cell wall marker for cellulosic β-(1–4)- d -glucans and anti-RYMV antibodies over vessel pit membranes suggests a pathway for virus migration between vessels. We propose that the partial digestion of pit membranes resulting from programmed cell death may permit virus migration through them, concomitant with autolysis. In addition, displacement of the Ca 2+ from pit membranes to virus particles may contribute to the disruption of the pit membranes and facilitate systemic virus transport.

The microprojectile bombardment of immature embryos has proven to be effective in transforming many indica rice varieties. One of the drawbacks of using immature embryos is the requirement of a large number of high quality immature embryos, which itself is a tedious and laborious process. To circumvent these problems, we have developed a procedure, using indica variety TN1 as a model that generates highly homogenous populations of embryogenic subcultured calli by selectively propagating a small number of regeneration-proficient calli derived from seeds. Thousands of embryogenic calli were produced from 50 seeds within 10 weeks. Ten to 20 independent R0 transgenic lines were regenerated per 500 embryogenic calli bombarded. The convenience and reliability offered by this transformation system has made transformation of indica rice a routine procedure.