Evolution of complex RNA polymerases: the complete archaeal RNA polymerase structure.

The archaeal RNA polymerase (RNAP) shares structural similarities with eukaryotic RNAP II but requires a reduced subset of general transcription factors for promoter-dependent initiation. To deepen our knowledge of cellular transcription, we have determined the structure of the 13-subunit DNA-directed RNAP from Sulfolobus shibatae at 3.35 Å resolution. The structure contains the full complement of subunits, including RpoG/Rpb8 and the equivalent of the clamp-head and jaw domains of the eukaryotic Rpb1. Furthermore, we have identified subunit Rpo13, an RNAP component in the order Sulfolobales, which contains a helix-turn-helix motif that interacts with the RpoH/Rpb5 and RpoA'/Rpb1 subunits. Its location and topology suggest a role in the formation of the transcription bubble.

Molecular architecture of Galphao and the structural basis for RGS16-mediated deactivation.

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an alpha subunit with intrinsic GTPase activity and a betagamma heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the alpha subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Galpha subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Galpha subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 A crystal structure of the enigmatic, neuronal G protein Galpha(o) in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 A structure of apo-RGS16, also presented here, reveals plasticity upon Galpha(o) binding, the determinants for GAP activity, and the structurally unique features of Galpha(o) that likely distinguish it physiologically from other members of the larger Galpha(i) family, affording insight to receptor, GAP and effector specificity.

Crystal structure of a transcription factor IIIB core interface ternary complex.

Transcription factor IIIB (TFIIIB), consisting of the TATA-binding protein (TBP), TFIIB-related factor (Brf1) and Bdp1, is a central component in basal and regulated transcription by RNA polymerase III. TFIIIB recruits its polymerase to the promoter and subsequently has an essential role in the formation of the open initiation complex. The amino-terminal half of Brf1 shares a high degree of sequence similarity with the polymerase II general transcription factor TFIIB, but it is the carboxy-terminal half of Brf1 that contributes most of its binding affinity with TBP. The principal anchoring region is located between residues 435 and 545 of yeast Brf1, comprising its homology domain II. The same region also provides the primary interface for assembling Bdp1 into the TFIIIB complex. We report here a 2.95 A resolution crystal structure of the ternary complex containing Brf1 homology domain II, the conserved region of TBP and 19 base pairs of U6 promoter DNA. The structure reveals the core interface for assembly of TFIIIB and demonstrates how the loosely packed Brf1 domain achieves remarkable binding specificity with the convex and lateral surfaces of TBP.

The x-ray crystal structure of the NF-kappa B p50.p65 heterodimer bound to the interferon beta -kappa B site.

We have determined the x-ray crystal structure of the transcription factor NF-kappaB p50.p65 heterodimer complexed to kappaB DNA from the cytokine interferon beta enhancer (IFNbeta-kappaB). To better understand how the binding modes of NF-kappaB on its two best studied DNA targets might contribute to promoter-specific transcription, this structure is compared with the previously determined complex crystal structure containing NF-kappaB bound to the Ig kappa light chain gene enhancer as well as to a second NF-kappaB.Ig kappa light chain gene enhancer complex also reported in this paper. The global binding modes of all NF-kappaB.DNA complex structures are similar, although crystal-packing interactions lead to differences between identical complexes of the same crystallographic asymmetric unit. An extensive network of stacked amino acid side chains that contribute to base-specific DNA contacts is conserved among the three complexes. Consistent with earlier reports, however, the IFNbeta-kappaB DNA is bent significantly less by NF-kappaB than is the Ig kappa light chain gene enhancer. This and other small structural changes may play a role in explaining why NF-kappaB-directed transcription is sensitive to the context of specific promoters. The precise molecular mechanism behind the involvement of the high mobility group protein I(Y) in interferon beta enhanceosome formation remains elusive.

The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state.

Molecular chaperones assist protein folding by facilitating their "forward" folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 A long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.

Role of the gamma-phosphate of ATP in triggering protein folding by GroEL-GroES: function, structure and energetics.

Productive cis folding by the chaperonin GroEL is triggered by the binding of ATP but not ADP, along with cochaperonin GroES, to the same ring as non-native polypeptide, ejecting polypeptide into an encapsulated hydrophilic chamber. We examined the specific contribution of the gamma-phosphate of ATP to this activation process using complexes of ADP and aluminium or beryllium fluoride. These ATP analogues supported productive cis folding of the substrate protein, rhodanese, even when added to already-formed, folding-inactive cis ADP ternary complexes, essentially introducing the gamma-phosphate of ATP in an independent step. Aluminium fluoride was observed to stabilize the association of GroES with GroEL, with a substantial release of free energy (-46 kcal/mol). To understand the basis of such activation and stabilization, a crystal structure of GroEL-GroES-ADP.AlF3 was determined at 2.8 A. A trigonal AlF3 metal complex was observed in the gamma-phosphate position of the nucleotide pocket of the cis ring. Surprisingly, when this structure was compared with that of the previously determined GroEL-GroES-ADP complex, no other differences were observed. We discuss the likely basis of the ability of gamma-phosphate binding to convert preformed GroEL-GroES-ADP-polypeptide complexes into the folding-active state.