Abstract
SummaryTranscription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5′ enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation 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 use, indicating regulatory significance for transcript folding.
Highlights
Transcription elongation is composed of many successive cycles of nucleotide addition, in which the translocation step is based on Brownian motion without input of external energy
RNAPI Distribution Is Uneven along the Transcription Unit We initially assessed the distribution of RNAPI along the rDNA transcription units using Miller spreads in a wild-type yeast strain (BY4741) growing in YPD medium, containing 2% glucose + 1 M sorbitol at 30C, as described previously (Osheim et al, 2009)
To analyze RNAPI distribution, we selected 60 spreads for which the full-length rDNA could be unambiguously traced, with polymerases positioned at the 50 and 30 ends, and the number of polymerases was around the average number of 50
Summary
Transcription elongation is composed of many successive cycles of nucleotide addition, in which the translocation step is based on Brownian motion without input of external energy. The major driver of transcription elongation is nucleotide addition because pyrophosphate release is essentially irreversible, allowing this step to act as a ratchet (Figure 1A). Dependence on this ‘‘Brownian ratchet’’ rather than an energy-driven processive mechanism makes elongation prone to frequent backtracking and potentially sensitive to inhibition or acceleration by quite modest forces (Dangkulwanich et al, 2013; Guajardo and Sousa, 1997). Deep backtracking is relatively rare compared with the number of nucleotide addition cycles but, in aggregate, is widespread in the cell (Sheridan et al, 2019). Despite functional and structural differences, the basic mechanism of transcription elongation has remained the same throughout evolution
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