We obtained an 11 subunit model of RNA polymerase (Pol) III by combining a homology model of the nine subunit core enzyme with a new X-ray structure of the subcomplex C17/25. Compared to Pol II, Pol III shows a conserved active center for RNA synthesis but a structurally different upstream face for specific initiation complex assembly during promoter selection. The Pol III upstream face includes a HRDC domain in subunit C17 that is translated by 35 A and rotated by 150 degrees compared to its Pol II counterpart. The HRDC domain is essential in vivo, folds independently in vitro, and, unlike other HRDC domains, shows no indication of nucleic acid binding. Thus, the HRDC domain is a functional module that could account for the role of C17 in Pol III promoter-specific initiation. During elongation, C17/25 may bind Pol III transcripts emerging from the adjacent exit pore, because the subcomplex binds to tRNA in vitro.

The ABC ATPase RNase-L inhibitor (RLI) emerges as a key enzyme in ribosome biogenesis, formation of translation preinitiation complexes, and assembly of HIV capsids. To help reveal the structural mechanism of RLI, we determined the Mg2+-ADP bound crystal structure of the twin cassette ATPase of P. furiosus RLI at 1.9 A resolution and analyzed functional motifs in yeast in vivo. RLI shows similarities but also differences to known ABC enzyme structures. Twin nucleotide binding domains (NBD1 and NBD2) are arranged to form two composite active sites in their interface cleft, indicating they undergo the ATP-driven clamp-like motion of the NBDs of ABC transporters. An unusual "hinge" domain along the NBD1:NBD2 interface provides a frame for association and possibly ATP-driven conformational changes of the NBDs. Our results establish a first structural basis for ABC domain heterodimers and suggest that RLI may act as mechanochemical enzyme in ribosome and HIV capsid biogenesis.

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism. Sm-based regulation is diverse and can range in scope from eukaryotic mRNA splicing to bacterial quorum sensing, with at least one step in these processes being mediated by an RNA-associated molecular assembly built on Sm proteins. Despite the availability of several 3D-structures of Sm-like archaeal proteins (SmAPs), their function has remained elusive. The aim of this study was to shed light on the function of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso). Using co-purification followed by RNASeq different classes of non-coding RNAs and mRNAs were identified that co-purified either with both paralogues or solely with Sso-SmAP1 or Sso-SmAP2. The large number of associated intron-containing tRNAs and tRNA/rRNA modifying RNAs may suggest a role of the two Sso-SmAPs in tRNA/rRNA processing. Moreover, the 3D structure of Sso-SmAP2 was elucidated. Like Sso-SmAP1, Sso-SmAP2 forms homoheptamers. The binding of both proteins to distinct RNA substrates is discussed in terms of surface conservation, structural differences in the RNA binding sites and differences in the electrostatic surface potential of the two Sso-SmAP proteins. Taken together, this study may hint to common and different functions of both Sso-SmAPs in Sso RNA metabolism.