The path of the nucleic acids through a transcription elongation complex was tracked by mapping cross-links between bacterial RNA polymerase (RNAP) and transcript RNA or template DNA onto the x-ray crystal structure. In the resulting model, the downstream duplex DNA is nestled in a trough formed by the beta' subunit and enclosed on top by the beta subunit. In the RNAP channel, the RNA/DNA hybrid extends from the enzyme active site, along a region of the beta subunit harboring rifampicin resistance mutations, to the beta' subunit "rudder." The single-stranded RNA is then extruded through another channel formed by the beta-subunit flap domain. The model provides insight into the functional properties of the transcription complex.

The 2.6 A crystal structure of a fragment of the sigma 70 promoter specificity subunit of E. coli RNA polymerase is described. Residues involved in core RNA polymerase binding lie on one face of the structure. On the opposite face, aligned along one helix, are exposed residues that interact with the -10 consensus promoter element (the Pribnow box), including four aromatic residues involved in promoter melting. The structure suggests one way in which DNA interactions may be inhibited in the absence of RNA polymerase and provides a framework for the interpretation of a large number of genetic and biochemical analyses.

Summary The amyloid state of protein organization is typically associated with debilitating human neuropathies and seldom observed in physiology. Here, we uncover a systemic program that leverages the amyloidogenic propensity of proteins to regulate cell adaptation to stressors. On stimulus, cells assemble the Amyloid-bodies (A-bodies), nuclear foci containing heterogeneous proteins with amyloid-like biophysical properties. A discrete peptidic sequence, termed the amyloid-converting motif (ACM), is capable of targeting proteins to the A-bodies by interacting with ribosomal intergenic noncoding RNA (rIGSRNA). The pathological β-amyloid peptide, involved in Alzheimer’s disease, displays ACM-like activity and undergoes stimuli-mediated amyloidogenesis in vivo. Upon signal termination, elements of the heat shock chaperone pathway disaggregate the A-bodies. Physiological amyloidogenesis enables cells to store large quantities of proteins and enter a dormant state in response to stressors. We suggest that cells have evolved a post-translational pathway that rapidly and reversibly converts native-fold proteins to an amyloid-like solid phase.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well established, but comparatively little is understood about its 3‐D structure. In this analysis, orientation and distance constraints between elements within the Escherichia coli RNase P RNA‐pre‐tRNA complex were determined by intra‐ and intermolecular crosslinking experiments. A molecular mechanics‐based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models. Seven different structures that satisfy the constraints equally well were generated and compared by superposition to estimate helix positions and orientations. Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pre‐tRNA that is consistent with the crosslinking results and the available phylogenetic comparisons.

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SUMMARY RNase R, an Escherichia coli exoribonuclease important for degradation of structured RNAs, increases 3- to 10-fold under certain stress conditions due to an increased half-life for this usually unstable protein. Components of the trans-translation machinery, tmRNA and its associated protein, SmpB, are essential for RNase R instability. However, it is not understood why exponential phase RNase R is unstable or how it becomes stabilized in stationary phase. We show here that these phenomena are regulated by acetylation catalyzed by YfiQ protein. One residue, Lys544, is acetylated in exponential phase RNase R, but not in the stationary phase protein, resulting in tighter binding of tmRNA-SmpB to the C-terminal region of exponential phase RNase R, and subsequent proteolytic degradation. Removal of the positive charge at Lys544 or a negative charge in the C-terminal region likely disrupts their interaction facilitating tmRNA-SmpB binding. These findings indicate that acetylation can regulate the stability of a bacterial protein.

Sequence differences between members of the mouse olfactory receptor MOR42 subfamily (MOR42-3 and MOR42-1) are likely to be the basis for variation in ligand binding preference among these receptors. We investigated the specificity of MOR42-3 for a variety of dicarboxylic acids. We used site-directed mutagenesis, guided by homology modeling and ligand docking studies, to locate functionally important residues. Receptors were expressed in Xenopus oocytes and assayed using high throughput electrophysiology. The importance of the Val-113 residue, located deep within the receptor, was analyzed in the context of interhelical interactions. We also screened additional residues predicted to be involved in ligand binding site, based on comparison of ortholog/paralog pairs from the mouse and human olfactory receptor genomes (Man, O., Gilad, Y., and Lancet, D. (2004) Protein Sci. 13, 240 -254). A network of 8 residues in transmembrane domains III, V, and VI was identified.These residues form part of the ligand binding pocket of MOR42-3. C12 dicarboxylic acid did not activate the receptor in our functional assay, yet our docking simulations predicted its binding site in MOR42-3. Binding without activation implied that C12 dicarboxylic acid might act as an antagonist. In our functional assay, C12 dicarboxylic acid did indeed act as an antagonist of MOR42-3, in agreement with molecular docking studies. Our results demonstrate a powerful approach based on the synergy between computational predictions and physiological assays.

RNase II is a member of the widely distributed RNR family of exoribonucleases, which are highly processive 3'-->5' hydrolytic enzymes that play an important role in mRNA decay. Here, we report the crystal structure of E. coli RNase II, which reveals an architecture reminiscent of the RNA exosome. Three RNA-binding domains come together to form a clamp-like assembly, which can only accommodate single-stranded RNA. This leads into a narrow, basic channel that ends at the putative catalytic center that is completely enclosed within the body of the protein. The putative path for RNA agrees well with biochemical data indicating that a 3' single strand overhang of 7-10 nt is necessary for binding and hydrolysis by RNase II. The presence of the clamp and the narrow channel provides an explanation for the processivity of RNase II and for why its action is limited to single-stranded RNA.