Synthesis of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukary-otic cell growth. Here we report the 12 A ˚ cryo-electron microscopic structure for the complete 14-subunit yeast Pol I, a homology model for the core enzyme, and the crystal structure of the subcomplex A14/43. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3 0-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2 and, apparently, enables ribosomal RNA proofreading and 3 0-end trimming.

The eukaryotic RNA polymerases Pol I, Pol II, and Pol III are the central multiprotein machines that synthesize ribosomal, messenger, and transfer RNA, respectively. Here we provide a catalog of available structural information for these three enzymes. Most structural data have been accumulated for Pol II and its functional complexes. These studies have provided insights into many aspects of the transcription mechanism, including initiation at promoter DNA, elongation of the mRNA chain, tunability of the polymerase active site, which supports RNA synthesis and cleavage, and the response of Pol II to DNA lesions. Detailed structural studies of Pol I and Pol III were reported recently and showed that the active center region and core enzymes are similar to Pol II and that strong structural differences on the surfaces account for gene class-specific functions.

The eukaryotic RNA polymerases Pol I, II, and III use different promoters to transcribe different classes of genes. Promoter usage relies on initiation factors, including TFIIF and TFIIE, in the case of Pol II. Here, we show that the Pol I-specific subunits A49 and A34.5 form a subcomplex that binds DNA and is related to TFIIF and TFIIE. The N-terminal regions of A49 and A34.5 form a dimerization module that stimulates polymerase-intrinsic RNA cleavage and has a fold that resembles the TFIIF core. The C-terminal region of A49 forms a "tandem winged helix" (tWH) domain that binds DNA with a preference for the upstream promoter nontemplate strand and is predicted in TFIIE. Similar domains are predicted in Pol III-specific subunits. Thus, Pol I/III subunits that have no counterparts in Pol II are evolutionarily related to Pol II initiation factors and may have evolved to mediate promoter specificity and transcription processivity.

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The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-Å three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the threedimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite.

A “breathing” protein: The first structure of the virulence regulator and heat shock protein ClpP from Staphylococcus aureus reveals a previously unobserved compressed state of the ClpP barrel. A conformational switch in the active center “handle region” results in closure of the active sites and opening of equatorial pores. These results confirm proposed modes of processive substrate degradation and product release for the ClpP protease family.

[1] Numerical simulations of transient porous media thermohaline convection including phase separation into a high-density, high-salinity brine phase and a low-density, low-salinity vapor phase at pressures and temperatures well above the critical point of pure H 2 O are presented. Using a novel finite element-finite volume (FEFV) solution technique and a new equation of state for the binary NaCl-H 2 O system, convection of a NaCl-H 2 O fluid in an open top square box of 4 Â 4 km is studied at geologically realistic pressure p, temperature T, and salinity X conditions. In the simulations, the basal temperature and salinity are varied systematically from 200 to 600°C and from 3.2 to 40 or 60 wt % NaCl, for permeabilities of 10 À15 or 10 À14 m 2 and hydrostatic pressure conditions. Resulting flow patterns are diffusive, steady convective, or oscillatory. Singlephase thermohaline convection occurs at temperatures below 400°C. Between 400 and 450°C, phase separation can occur during the buoyant rise of heat and salt if the permeability is high or the salinity low. Above 450°C, the fluid at the basal boundary is a vapor phase coexisting with a brine phase. In this case, convection is dominated by heat and salt transport during the buoyant rise of vapor. Convection sets in almost instantaneously at these conditions. Above 570°C, a nearly pure H 2 O vapor phase coexists with solid salt at the basal boundary. Convection is driven exclusively by the applied temperature gradient. Since fluid properties change by highly nonlinear functions of p, T, and X, parameters such as the Rayleigh number and buoyancy ratio, which are classically used to quantify the different regimes of thermohaline convection, are not meaningful in this context. This implies that parametric studies that make use of the Boussinesq approximation and assume incompressibility are not representative of thermohaline convection in geologic environments. We use the concept of a local Rayleigh number and a fluxibility parameter to provide a better insight into the onset and evolution of thermohaline convective systems.

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