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.

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.

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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 highresolution 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 DNA͞RNA 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.

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 ␤-(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.

SummarySecretins 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 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.

To identify the location of a domain of the ␤-subunit of Escherichia coli RNA polymerase (RNAP) on the three-dimensional structure, we developed a method to tag a nonessential surface of the multisubunit enzyme with a protein density easily detectable by electron microscopy and image processing. Four repeats of the IgG-binding domain of Staphylococcus aureus protein A were inserted at position 998 of the E. coli RNAP ␤-subunit. The mutant RNAP supported E. coli growth and showed no apparent functional defects in vitro. The structure of the mutant RNAP was determined by cryoelectron microscopy and image processing of frozen-hydrated helical crystals. Comparison of the mutant RNAP structure with the previously determined wild-type RNAP structure by Fourier difference analysis at 20-Å resolution directly revealed the location of the inserted protein domain, thereby locating the region around position 998 of the ␤-subunit within the RNAP three-dimensional structure and refining a model for the subunit locations within the enzyme. T ranscription 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 ␣-subunits (each 36.5 kDa), one ␤-subunit (150.6 kDa), and one ␤Ј-subunit (155.2 kDa) that is responsible for transcript elongation and termination. The holoenzyme contains an additional regulatory subunit, normally 70 (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, ␤ and ␤Ј, 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 ␤-subunit, nine conserved regions, labeled A through I, have been identified (ref. 3; Fig. 1). In addition, the ␤-subunit of E. coli RNAP contains two regions of poor sequence conservation, centered about residues 300 and 1,000, which are often missing in ␤-homologs from other organisms (Fig. 1).As expected, mutations affecting the conserved regions frequently cause severe defects in RNAP function (ref. 4 and references therein). ␤-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 ␤-subunit render the enzyme resistant to the antibiotic inhibitors rifampicin and streptolydigin (refs. 6-9; Fig. 1).Alternatively, mutations in poorly conserved regions ca...