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

Haifeng Hou,Zishuo Yu,Yanhui Xu,Yan Li,Mo Wang,Aijun Liu,Ke Chen,Dan Zhao

doi:10.1038/s41467-021-26402-9

Abstract

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 Å 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.

Highlights

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 polydeoxythymidine-containing non-template strand, a mechanism distinct from Pol I and Pol II
The recombinant human Pol III complex was transiently expressed in human embryonic kidney Expi293F cells and purified to homogeneity as previously reported[21] (Supplementary Fig. 1a)
Pol III could read-through TS1T1 and TS1T3 (Supplementary Fig. 1b, lanes 3 and 5) but showed largely reduced read-through and obvious termination on TS1T5 and TS1T7 (Supplementary Fig. 1b, lanes 7 and 9), RNA products around 43 nt in length indicated with red box)

Summary

Introduction

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 polydeoxythymidine (dT)-containing non-template strand, a mechanism distinct from Pol I and Pol II. 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 polydT-dependent transcription termination of Pol III. The average lengths of the poly-dT in canonical Pol III termination sites vary across species, with an average of 5–7 dTs in Schizosaccharomyces pombe (S. pombe), 6–9 dT in Saccharomyces cerevisiae (S. cerevisiae), and 4–5 dT in vertebrates This unique termination mechanism is beneficial to the functions of

Methods

Results

Conclusion