The non-coding B2 RNA binds to the DNA cleft and active-site region of RNA polymerase II

Abstract

The B2 family of short interspersed elements is transcribed into non-coding RNA by RNA polymerase III. The ~180-nt B2 RNA has been shown to potently repress mRNA transcription by binding tightly to RNA polymerase II (Pol II) and assembling with it into complexes on promoter DNA, where it keeps the polymerase from properly engaging the promoter DNA. Mammalian Pol II is an ~500-kDa complex that contains 12 different protein subunits, providing many possible surfaces for interaction with B2 RNA. We found that the carboxy-terminal domain of the largest Pol II subunit was not required for B2 RNA to bind Pol II and repress transcription in vitro. To identify the surface on Pol II to which the minimal functional region of B2 RNA binds, we coupled multi-step affinity purification, reversible formaldehyde cross-linking, peptide sequencing by mass spectrometry, and analysis of peptide enrichment. The Pol II peptides most highly recovered after cross-linking to B2 RNA mapped to the DNA binding cleft and active-site region of Pol II. These studies determine the location of a defined nucleic acid binding site on a large, native, multi-subunit complex and provide insight into the mechanism of transcriptional repression by B2 RNA.

Keywords: C-terminal domain of RPB1; CTD; EM; EMSA; LC; MS; MS/MS; Pol II; RNA polymerase II; RPB; TATA binding protein; TBP; TFII; XIC; electron microscopy; electrophoretic mobility shift assay; extracted ion chromatogram; liquid chromatography; mass spectrometry; ncRNA; non-coding RNA; protein–RNA cross-linking; subunit of RNA polymerase II; transcription; transcription factor of RNA polymerase II.