RNA Polymerase III Transcription Repressed by Rb through Its Interactions with TFIIIB and TFIIIC2

Wen-Ming Chu,Zengxin Wang,Robert G Roeder,Carl W Schmid

doi:10.1074/jbc.272.23.14755

Abstract

The retinoblastoma susceptibility gene product (Rb) generally represses RNA polymerase III (Pol III)-directed transcription. This implies that Rb interacts with essential transcription factors. Mutations in either the A or B subdomains in the Rb pocket interfere with Rb-mediated repression of Pol III-directed transcription, which indicates that both subdomains are directly involved in this activity. Addition of either purified TFIIIB or purified TFIIIC2 partially relieves Rb-mediated repression and restores activity to nuclear extracts that had been depleted of essential factors by binding to Rb. Pull down and coimmunoprecipitation experiments as well as functional assays indicate that Rb interacts with both TFIIIB and TFIIIC2 and that the A subdomain is primarily required for binding TFIIIB and the B subdomain for binding TFIIIC2. While Rb interacts with both factors, the A subdomain is more important than the B subdomain in directing Rb-mediated repression, and TFIIIB is the principal target of that activity.

Highlights

Studies examining mechanisms by which retinoblastoma susceptibility gene product (Rb) suppresses cell growth have focused on its interaction with E2F, a transcription factor that is implicated in the expression of genes required during the S phase of the cell cycle [1]
Extensive mutation of the Rb A subdomain markedly inhibits its interaction with TFIIIB90 (Fig. 5B, lanes 8 and 10), whereas mutation of the B subdomain has little effect (Fig. 5B, lanes 7 and 9). Consistent with this finding, the C706F mutation in the B subdomain has no effect on TFIIIB binding (Fig. 5B, lanes 1 and 2). These results indicate that Rb binds both TFIIIB and TFIIIC2, with the A domain being primarily responsible for TFIIIB binding and the B subdomain primarily being responsible for TFIIIC2 binding
The observation that Rb represses transcription from all known classes of polymerase III (Pol III) promoters suggested either that it targets one or more components (Pol III, TFIIIC2, or specific subunits of TFIIIB) that are commonly required for all Pol III genes

Summary

The abbreviations used are

Polymerase; GST, glutathione Stransferase; PAGE, polyacrylamide gel electrophoresis; TBP, TATAbinding protein. Pression is entirely independent of promoter structure This indicates that Rb must interact with one or more of the common components of the Pol III transcriptional machinery to cause this general repression of Pol III-directed transcription. The participation of TBP in both Pol II- and Pol III-mediated transcription, as well as the similarity of a TFIIIB subunit, TFIII B90, to TFIIB, potentially extends this hypothesis to Pol III-directed transcription [5, 8]. We investigated the possibility that the A and B subdomains in Rb might mediate repression of Pol III transcription through interactions with Pol III accessory factors. TFIIIB, which contains TBP, TFIIIB90, and possibly other subunits, is recruited to the promoter by TFIIIC and facilitates Pol III recruitment

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION