Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294.
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294
Proc Natl Acad Sci U S A. 2018 Dec 11;115(50):E11633-E11641. doi: 10.1073/pnas.1809421115. Epub 2018 Nov 27.
DNA sequence motifs that affect RNA polymerase transcription elongation are well studied in prokaryotic organisms and contribute directly to regulation of gene expression. Despite significant work on the regulation of eukaryotic transcription, the effect of DNA template sequence on RNA polymerase I (Pol I) transcription elongation remains unknown. In this study, we examined the effects of DNA sequence motifs on Pol I transcription elongation kinetics in vitro and in vivo. Specifically, we characterized how the rho-independent terminator motif from directly affects Pol I activity, demonstrating evolutionary conservation of sequence-specific effects on transcription. The insight gained from this analysis led to the identification of a homologous sequence in the ribosomal DNA of We then used native elongating transcript sequencing (NETSeq) to determine whether Pol I encounters pause-inducing sequences in vivo. We found hundreds of positions within the ribosomal DNA (rDNA) that reproducibly induce pausing in vivo. We also observed significantly lower Pol I occupancy at G residues in the rDNA, independent of other sequence context, indicating differential nucleotide incorporation rates for Pol I in vivo. These data demonstrate that DNA template sequence elements directly influence Pol I transcription elongation. Furthermore, we have developed the necessary experimental and analytical methods to investigate these perturbations in living cells going forward.
在原核生物中，影响 RNA 聚合酶转录延伸的 DNA 序列基序得到了很好的研究，这些基序直接参与了基因表达的调控。尽管在真核转录调控方面做了大量工作，但 DNA 模板序列对 RNA 聚合酶 I（Pol I）转录延伸的影响仍不清楚。在这项研究中，我们研究了 DNA 序列基序对 Pol I 转录延伸动力学的体外和体内影响。具体来说，我们描述了 中 ρ 独立终止子基序如何直接影响 Pol I 活性，证明了序列特异性对转录的影响在进化上是保守的。从该分析中获得的见解导致了在核糖体 DNA 中鉴定出一个同源序列。然后，我们使用天然延伸转录测序（NETSeq）来确定 Pol I 是否在体内遇到诱导暂停的序列。我们在核糖体 DNA（rDNA）内发现了数百个位置，这些位置在体内可重复地诱导暂停。我们还观察到 rDNA 中 G 残基处的 Pol I 占有率显著降低，这与其他序列背景无关，表明 Pol I 在体内的核苷酸掺入率存在差异。这些数据表明，DNA 模板序列元件直接影响 Pol I 转录延伸。此外，我们已经开发了必要的实验和分析方法，以便在未来的活细胞中研究这些扰动。
