Molecular structures of unbound and transcribing RNA polymerase III

Abstract

Transcription of genes encoding small structured RNAs such as tRNAs, spliceosomal U6 snRNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. The cryo-EM structures of the S. cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, allow for the first time to build a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82/C34/C31 heterotrimer in close proximity to the stalk. The C53/C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets.

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

“…Electron microscopy analysis of free RNAPII in solution further supported the notion of a conformational flexible clamp, suggesting that Rpb4/7 shifts the equilibrium between the closed and collapsed state of the clamp to the closed state (9). Moreover, the clamp of RNAPIII could recently be imaged in two distinct conformations that differ in the orientation of the stalk and the opening of the DNA cleft (10). These data have led to the theory that the stalk influences the clamp position.…”

Molecular Mechanisms of Transcription Initiation—Structure, Function, and Evolution of TFE/TFIIE-Like Factors and Open Complex Formation

“…Similar to archaeal TFE, TFIIE can stimulate transcription in vitro from negatively supercoiled or partially pre-melted DNA templates [47,60], suggesting that TFE and TFIIE use a conserved mechanism. Likewise, the clamp of RNAPIII adopts open and closed conformations [16]. However, the clamp movement is less pronounced (change of 9 Å) as compared to the archaeal RNAP and RNAPII (change of 17 Å).…”

“…However, the clamp movement is less pronounced (change of 9 Å) as compared to the archaeal RNAP and RNAPII (change of 17 Å). In the RNAPIII system, subunits C82/34 are an integral part of the RNAP and, similar to subunits TFIIEα/β (RNAPII), span the DNA cleft [13,14,16], which is likely to restrict the flexibility of the clamp.…”

“…The archaeal RNAP, RNAPII and -III employ a third conserved factor that stimulates OC formation, called transcription factor E (TFE), TFIIE and the RPC82/34 complex (hRPC62/39 in human), respectively [10]. Recent publications have provided intriguing insights into the structural organisation of these factors [11][12][13][14][15][16][17].…”

RNA polymerase II acts as a selective sensor for DNA lesions and endogenous DNA modifications

“…12,17,26,29 Intriguingly, the similar "above the bridge helix" translocation intermediates are also observed for non-damaged pausing and arrest complexes revealed by the structures of the a-amanitinarrested pol II complex 5,30 and the E. Coli RNAP pausing complex. 31 Another interesting example is revealed by a recent structure of the RNA pol III elongation complex, 32 in which the undamaged template iC1 nucleobase adopts a similar "above the bridge helix" position. Strikingly, this undamaged nucleobase is held at the "above the bridge helix" position through a specific hydrogen-bonding interaction with the pol III residue E506, the counterpart to the pol II Rpb2 Q531 residue that directly senses 5caC/5fC through specific hydrogenbonding interactions.…”