Nascent transcript folding plays a major role in determining RNA polymerase elongation rates

Abstract

Transcription elongation rates are important for RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S.cerevisiae. Analysis of Miller chromatin spreads and mapping RNAPI using UV crosslinking, revealed a marked 5 bias and strikingly uneven local polymerase occupancy, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution; folding energy and G+C-content. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high G+C within the transcription bubble slows elongation. We developed a mathematical model for RNAPI elongation, which confirmed the importance of nascent RNA folding in transcription. RNAPI from S.pombe was similarly sensitive to transcript folding, as were S.cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage indicating regulatory significance for transcript folding. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/969709v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@f3f9fcorg.highwire.dtl.DTLVardef@1a133c8org.highwire.dtl.DTLVardef@4eae0dorg.highwire.dtl.DTLVardef@18502b8_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSStructures in the nascent RNA correlate with rapid elongation by RNAPI in vivo Stable RNA structures limit RNAPI backtracking in vitro GC content in the transcription bubble tunes transcription elongation rate Nascent transcript folding modulates dynamics of all three RNAPs in vivo