Molecular mechanisms of RNA polymerase--the F/E (RPB4/7) complex is required for high processivity in vitro.

Transcription elongation in vitro is affected by the interactions between RNA polymerase (RNAP) subunits and the nucleic acid scaffold of the ternary elongation complex (TEC, RNAP-DNA-RNA). We have investigated the role of the RNAP subunits F/E (homologous to eukaryotic RPB4/7) during transcription elongation and termination using a wholly recombinant archaeal RNAP and synthetic nucleic acid scaffolds. The F/E complex greatly stimulates the processivity of RNAP, it enhances the formation of full length products, reduces pausing, and increases transcription termination facilitated by weak termination signals. Mutant variants of F/E that are defective in RNA binding show that these activities correlate with the nucleic acid binding properties of F/E. However, a second RNA-binding independent component also contributes to the stimulatory activities of F/E. In summary, our results suggest that interactions between RNAP subunits F/E and the RNA transcript are pivotal to the molecular mechanisms of RNAP during transcription elongation and termination.

Spt4/5 stimulates transcription elongation through the RNA polymerase clamp coiled-coil motif.

Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA-RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5--the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.

Differential substrate specificity of group I and group II chaperonins in the archaeon Methanosarcina mazei.

Chaperonins are macromolecular machines that assist in protein folding. The archaeon Methanosarcina mazei has acquired numerous bacterial genes by horizontal gene transfer. As a result, both the bacterial group I chaperonin, GroEL, and the archaeal group II chaperonin, thermosome, coexist. A proteome-wide analysis of chaperonin interactors was performed to determine the differential substrate specificity of GroEL and thermosome. At least 13% of soluble M. mazei proteins interact with chaperonins, with the two systems having partially overlapping substrate sets. Remarkably, chaperonin selectivity is independent of phylogenetic origin and is determined by distinct structural and biochemical features of proteins. GroEL prefers well-conserved proteins with complex alpha/beta domains. In contrast, thermosome substrates comprise a group of faster-evolving proteins and contain a much wider range of different domain folds, including small all-alpha and all-beta modules, and a greater number of large multidomain proteins. Thus, the group II chaperonins may have facilitated the evolution of the highly complex proteomes characteristic of eukaryotic cells.

RNAP subunits F/E (RPB4/7) are stably associated with archaeal RNA polymerase: using fluorescence anisotropy to monitor RNAP assembly in vitro.

Archaeal and eukaryotic RNAPs (DNA-dependent RNA polymerases) are complex multi-subunit enzymes. Two of the subunits, F and E, which together form the F/E complex, have been hypothesized to associate with RNAP in a reversible manner during the transcription cycle. We have characterized the molecular interactions between the F/E complex and the RNAP core. F/E binds to RNAP with submicromolar affinity and is not in a dynamic exchange with unbound F/E.

Molecular mechanisms of archaeal RNA polymerase.

All cellular life depends on multisubunit RNAPs (RNA polymerases) that are evolutionarily related through the three domains of life. Archaeal RNAPs encompass 12 subunits that contribute in different ways to the assembly and stability of the enzyme, nucleic acid binding, catalysis and specific regulatory interactions with transcription factors. The recent development of methods to reconstitute archaeal RNAP from recombinant materials in conjunction with structural information of multisubunit RNAPs present a potent opportunity to investigate the molecular mechanisms of transcription.

Lytic water dynamics reveal evolutionarily conserved mechanisms of ATP hydrolysis by TIP49 AAA+ ATPases.

Eukaryotic TIP49a (Pontin) and TIP49b (Reptin) AAA+ ATPases play essential roles in key cellular processes. How their weak ATPase activity contributes to their important functions remains largely unknown and difficult to analyze because of the divergent properties of TIP49a and TIP49b proteins and of their homo- and hetero-oligomeric assemblies. To circumvent these complexities, we have analyzed the single ancient TIP49 ortholog found in the archaeon Methanopyrus kandleri (mkTIP49). All-atom homology modeling and molecular dynamics simulations validated by biochemical assays reveal highly conserved organizational principles and identify key residues for ATP hydrolysis. An unanticipated crosstalk between Walker B and Sensor I motifs impacts the dynamics of water molecules and highlights a critical role of trans-acting aspartates in the lytic water activation step that is essential for the associative mechanism of ATP hydrolysis.

Coexistence of group I and group II chaperonins in the archaeon Methanosarcina mazei.

Two distantly related classes of cylindrical chaperonin complexes assist in the folding of newly synthesized and stress-denatured proteins in an ATP-dependent manner. Group I chaperonins are thought to be restricted to the cytosol of bacteria and to mitochondria and chloroplasts, whereas the group II chaperonins are found in the archaeal and eukaryotic cytosol. Here we show that members of the archaeal genus Methanosarcina co-express both the complete group I (GroEL/GroES) and group II (thermosome/prefoldin) chaperonin systems in their cytosol. These mesophilic archaea have acquired between 20 and 35% of their genes by lateral gene transfer from bacteria. In Methanosarcina mazei Gö1, both chaperonins are similarly abundant and are moderately induced under heat stress. The M. mazei GroEL/GroES proteins have the structural features of their bacterial counterparts. The thermosome contains three paralogous subunits, alpha, beta, and gamma, which assemble preferentially at a molar ratio of 2:1:1. As shown in vitro, the assembly reaction is dependent on ATP/Mg2+ or ADP/Mg2+ and the regulatory role of the beta subunit. The co-existence of both chaperonin systems in the same cellular compartment suggests the Methanosarcina species as useful model systems in studying the differential substrate specificity of the group I and II chaperonins and in elucidating how newly synthesized proteins are sorted from the ribosome to the proper chaperonin for folding.