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

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.

转录延伸速率对于RNA加工至关重要，但其序列特异性调控机制尚不明确。本研究通过体内实验，对酿酒酵母中的RNA聚合酶I（RNAPI）进行了分析。通过对Miller染色质扩散的分析，以及利用紫外交联技术对RNAPI进行定位，揭示了显著的5'端偏好和惊人的局部聚合酶占据不均，表明转录速度存在显著差异。新生转录本的两种特征与RNAPI分布相关；即折叠能和G+C含量。体外实验证实，靠近聚合酶的强RNA结构可促进正向迁移并限制反向滑动，而转录泡内的高G+C含量则减缓了延伸速度。我们开发了一个用于RNAPI延伸的数学模型，该模型证实了新生RNA折叠在转录中的重要性。从裂殖酵母（S.pombe）中提取的RNAPI对转录本折叠的敏感性亦然，与酿酒酵母的RNAPII和RNAPIII相同。对于RNAPII而言，未结构化的RNA，有利于减慢延伸速度，与更快的共转录剪接和邻近剪接位点使用相关，表明转录本折叠在调控上具有重要意义。