Structure-function analysis of hRPC62 provides insights into RNA polymerase III transcription initiation

Abstract

The 17-subunit human RNA polymerase III (hPol III) transcribes small, untranslated RNA genes that are involved in the regulation of transcription, splicing and translation. hPol III subunits hRPC62, hRPC39 and hRPC32 form a stable ternary subcomplex required for promoter-specific transcription initiation by hPol III. Here, we report the crystal structure of hRPC62. This subunit folds as a four-tandem extended winged helix (eWH) protein that is structurally related to the transcription factor TFIIEα N terminus. Through biochemical analyses, we mapped the protein-protein interactions of hRPC62, hRPC32 and hRPC39. In addition, we demonstrated that hRPC62 and hRPC39 bind single-stranded and duplex DNA, respectively, in a sequence-independent manner. Overall, we shed light on structural similarities between the hPol III-specific subunit hRPC62 and TFIIEα and propose specific functions for hRPC39 and hRPC62 in transcription initiation by hPol III.

RNA polymerase III subunit architecture and implications for open promoter complex formation

“…The C37/53 dimerization module was positioned into the electron density adjacent to the lobe domain of the C128 subunit on one side of the polymerase cleft, similar to the localization of the TFIIF dimerization module and the A49/34.5 dimerization module on Pol II and Pol I, respectively (23-25). This structural arrangement was supported by site-specific photo-cross-linking and hydroxyl radical probing analyses that provided direct positional mapping for protein interactions (8).The C82/34/31 subcomplex was proposed to occupy a large electron density region between the clamp and the stalk of the polymerase core, on the side of the cleft opposite the lobe (20,24,25). By fitting the crystal structure of hRPC62 into the yeast Pol III cryo-EM envelope, Lefèvre et al proposed a model in which eWH2, eWH3, and the coiled-coil region are positioned on the clamp and eWH1 and eWH4 are exposed to solvent for singlestranded DNA binding (20).…”

“…By fitting the crystal structure of hRPC62 into the yeast Pol III cryo-EM envelope, Lefèvre et al proposed a model in which eWH2, eWH3, and the coiled-coil region are positioned on the clamp and eWH1 and eWH4 are exposed to solvent for singlestranded DNA binding (20). An alternative orientation was proposed by Fernández-Tornero et al that placed eWH4 closer to the stalk of the polymerase core (26).…”

“…Pol I contains the A49/34.5 subcomplex that features a TFIIF-like dimerization module and a tandem WH domain that contains two Tfa2-like WH folds in the C-terminal region of the A49 subunit (18). The crystal structure of the human C82 homolog RPC62 contains four Tfa1-like eWH domains (eWH1-4) that are structurally organized around a Cterminal coiled-coil stalk (20). A Tfa1-related eWH domain is also present in the archaeal transcription factor E (TFE) (22).…”

“…The two Pol III-specific subcomplexes contain structural domains homologous to domains in the Pol II transcription factors TFIIF and TFIIE, including the TFIIF-related dimerization module in the C37/53 subcomplex and several TFIIE-related winged helix (WH) domains in subunits C82 and C34 (14)(15)(16)(17)(18)(19)(20). TFIIE is composed of two subunits, Tfa1 and Tfa2, in yeast, or TFIIEα and TFIIEβ in humans.…”

“…The C82/34/31 subcomplex was proposed to occupy a large electron density region between the clamp and the stalk of the polymerase core, on the side of the cleft opposite the lobe (20,24,25). By fitting the crystal structure of hRPC62 into the yeast Pol III cryo-EM envelope, Lefèvre et al proposed a model in which eWH2, eWH3, and the coiled-coil region are positioned on the clamp and eWH1 and eWH4 are exposed to solvent for singlestranded DNA binding (20).…”