Structural insights into RNA polymerase III-mediated transcription termination through trapping poly-deoxythymidine.

Termination of the RNA polymerase III (Pol III)-mediated transcription requires the conversion of an elongation complex (EC) to a pre-termination complex (PTC) on poly-deoxythymidine (dT)-containing non-template strand, a mechanism distinct from Pol I and Pol II. Here, our in vitro transcription elongation assay showed that 5-7 dT-containing DNA template led to transcription termination of Pol III, but not Pol I or Pol II. We assembled human Pol III PTC on a 7 dT-containing DNA template and determined the structure at 3.6 A resolution. The structure reveals that poly-dT are trapped in a narrow exit tunnel formed by RPC2. A hydrophobic gate of the exit tunnel separates the bases of two connected deoxythymidines and may prevent translocation of the non-template strand. The fork loop 2 stabilizes both template and non-template strands around the transcription fork, and may further prevent strand translocation. Our study shows that the Pol III-specific exit tunnel and FL2 allow for efficient translocation of non-poly-dT sequence during transcription elongation but trap poly-dT to promote DNA retention of Pol III, revealing molecular mechanism of poly-dT-dependent transcription termination of Pol III. Termination of eukaryotic RNA polymerase III (Pol III)-mediated transcription occurs when the polymerase reaches a stretch of four or more deoxythymidine nucleotides (poly-dT) on the non-template strand. Here, the authors present the 3.6 A cryo-EM structure of a human Pol III pre-termination complex (PTC) that was assembled on a 7 dT-containing DNA template and discuss the mechanism of poly-dT-dependent transcription termination of Pol III.