New Insights into the Assembly Mechanism of an RNA Polymerase III‐Specific Transcription Complex on a Drosophila U6 snRNA Gene Promoter

Abstract

In higher eukaryotes, RNA polymerase III (Pol III) promoters at U6 snRNA genes consist of a TATA box, recognized by TFIIIB, and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). In the fruit fly Drosophila melanogaster, DmSNAPc consists of three subunits DmSNAP190, DmSNAP50, and DmSNAP43; likewise TFIIIB also consists of three subunits, most commonly TBP, Brf1 and Bdp1. At Drosophila tRNA and 5S RNA gene promoters, TBP‐related factor 1 (TRF1) is utilized in place of TBP, but at U6 promoters the canonical TBP is utilized for Pol III transcription (Verma et al. 2013, JBC 288, 27564–27570).Site‐specific protein‐DNA photo‐cross‐linking studies of DmSNAPc and TFIIIB to U6 promoter DNA indicated that Bdp1 is in close proximity to DmSNAP43 and DmSNAP190 on the U6 promoter (Kang et al. 2016, FEBS Lett 590, 1488–1497). This suggested that the interaction between DmSNAPc and TFIIIB may be mediated, at least in part, by Bdp1. We have investigated this further by electrophoretic mobility shift assays (EMSAs). Surprisingly, we found that DmSNAPc, when bound to the U6 PSE, can recruit Bdp1 to the DNA in the absence of TBP and Brf1. Furthermore, EMSAs indicated that the DmSNAPc‐Bdp1 complex, when bound to U6 promoter DNA, can recruit TBP to form a DmSNAPc‐Bdp1‐TBP‐DNA complex of increased stability.In order to understand the protein‐protein interactions taking place, truncation mutations of Bdp1 were used. It was discovered that an area between amino acid 424 and 510 of Bdp1 is required for its recruitment by DmSNAPc. Furthermore, to investigate whether the TATA box is required for Bdp1 and TBP recruitment, the U6 TATA box was mutated to an unrelated sequence. Although mutation of the TATA box interfered with the recruitment of TBP by the DmSNAPc‐Bdp1 complex, the TATA mutation did not prevent the recruitment of Bdp1 by DmSNAPc. Interestingly, the non‐conserved amino‐terminal tail of TBP contributed to the efficiency of TBP recruitment by the DmSNAPc‐Bdp1 complex.A body of previous work from our lab has shown that DmSNAPc binds to U6 (Pol III transcribed) and U1 promoters (Pol II transcribed) in distinct conformations. Interestingly, when we switched the U6 proximal sequence element A (PSEA) to a U1 PSEA by a 5‐nucleotide change, DmSNAPc was unable to recruit Bdp1. This finding suggests that a surface of DmSNAPc that interacts with Bdp1 may be occluded when DmSNAPc binds to a U1 PSEA. It further provides a mechanism for the polymerase specificity seen when comparing the snRNA U1 and U6 genes.Support or Funding InformationThis work was supported by the National Science Foundation and by the California Metabolic Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.