One of the first steps in ribosome biogenesis is transcription of the ribosomal DNA (rDNA) by RNA polymerase I (Pol I). Processing of the resultant ribosomal RNA (rRNA) begins co-transcriptionally and perturbation of Pol I transcription elongation results in defective rRNA processing. Mechanistic insight regarding the link between transcription elongation and ribosome assembly is lacking due to limited in vivo methods to assay Pol I transcription. Here, we use native elongating transcript sequencing (NET-seq) with a strain of Saccharomyces cerevisiae containing a point mutation in Pol I, rpa190-F1205H, which results in impaired rRNA processing and ribosome assembly. We previously demonstrated that this mutation caused a mild reduction in the transcription elongation rate of Pol I in vitro, however, transcription elongation by the mutant has not been characterized in vivo. Here, our findings demonstrate that the mutant Pol I has an increased pause propensity during processive transcription elongation both in vitro and in vivo. NET-seq reveals that rpa190-F1205H Pol I displays alternative pause site preferences in vivo. Specifically, the mutant is sensitized to A/G residues in the RNA:DNA hybrid and at the last incorporated nucleotide position. Furthermore, both NET-seq and EM analysis of Miller chromatin spreads reveal pileups of rpa190-F1205H Pol I throughout the rDNA, particularly at the 5’ end of the 35S gene. This combination of in vitro and in vivo analyses of the Pol I mutant provides novel insights into Pol I elongation properties and indicates how these properties are crucial for efficient co-transcriptional rRNA processing and ribosome assembly.

核糖体生物发生的第一步是通过 RNA 聚合酶 I (Pol I) 转录核糖体 DNA (rDNA)。由此产生的核糖体 RNA (rRNA) 的加工开始共转录，并且 Pol I 转录延伸的扰动导致 rRNA 加工缺陷。由于测定 Pol I 转录的体内方法有限，缺乏关于转录延伸和核糖体组装之间联系的机制见解。在这里，我们使用原生延伸转录本测序 (NET-seq) 和一株在 Pol I 中含有点突变的酿酒酵母rpa190-F1205H，这会导致 rRNA 加工和核糖体组装受损。我们之前证明，这种突变导致 Pol I在体外的转录延伸率轻度降低，但是，突变体的转录延伸率尚未在体内表征。在这里，我们的研究结果表明，突变体 Pol I 在体外和体内的进行性转录延伸过程中具有增加的停顿倾向。NET-seq 显示rpa190-F1205H Pol I在体内显示替代暂停站点偏好. 具体而言，突变体对 RNA:DNA 杂合体中的 A/G 残基和最后掺入的核苷酸位置敏感。此外，米勒染色质扩散的 NET-seq 和 EM 分析都揭示了rpa190-F1205H Pol I 在整个 rDNA 中的堆积，特别是在 35S 基因的 5' 端。这种对Pol I 突变体的体外和体内分析的结合提供了对 Pol I 延伸特性的新见解，并表明这些特性对于有效的共转录 rRNA 加工和核糖体组装至关重要。