NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I.
NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I.
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DOI：
10.1073/pnas.1809421115
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发表时间：
2018-12-11
影响因子：
11.1
通讯作者：
Schneider DA
中科院分区：
文献类型：
--
作者：
Clarke AM;Engel KL;Giles KE;Petit CM;Schneider DA
众所周知，核糖体RNA加工直接受到RNA聚合酶I（Pol I）的转录延伸速率的影响。为了理解这些过程是如何协调的，我们必须仔细定义体外和活细胞中的转录延伸特性。在这里，我们表征DNA序列元素，暂停和终止Pol I转录体外。我们还建立了使用天然延伸转录物测序（NETSeq）分析体内Pol I转录延伸特性的方法。我们的NETSeq数据揭示了Pol I的频繁暂停和G残基处Pol I占用的减少，表明酶的核苷酸掺入率不相等。这些发现重新定义了我们对Pol I转录延长及其体内异质性的理解。影响RNA聚合酶转录延伸的DNA序列基序在原核生物中得到了很好的研究，并直接有助于基因表达的调控。尽管在真核生物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）转录延伸的影响仍然未知。在这项研究中，我们研究了DNA序列基序对Pol I转录延长动力学的影响，在体外和体内。具体而言，我们的特点是如何间谍rho独立终止子基序从大肠杆菌直接影响酿酒酵母Pol I活性，证明进化保守的序列特异性转录的影响。从这个分析中获得的见解导致了在S.啤酒。然后，我们使用天然延长转录测序（NETSeq），以确定是否Pol I遇到暂停诱导序列在体内。我们在核糖体DNA（rDNA）中发现了数百个可重复诱导体内停顿的位置。我们还观察到显着较低的Pol I占用G残基的rDNA，独立于其他序列的背景下，表明差异的核苷酸掺入率Pol I在体内。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来研究活细胞中的这些扰动。
It is well known that ribosomal RNA processing is directly impacted by the rate of transcription elongation by RNA polymerase I (Pol I). To understand how these processes are orchestrated, we must carefully define transcription elongation properties in vitro and in living cells. Here, we characterize DNA sequence elements that pause and terminate Pol I transcription in vitro. We also establish methods for analyzing Pol I transcription elongation properties in vivo using native elongating transcript sequencing (NETSeq). Our NETSeq data revealed frequent pausing by Pol I and decreased Pol I occupancy at G residues, suggesting unequal rates of nucleotide incorporation by the enzyme. These findings redefine our understanding of Pol I transcription elongation and its heterogeneity in vivo. 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 spy rho-independent terminator motif from Escherichia coli directly affects Saccharomyces cerevisiae 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 S. cerevisiae. 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.
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DOI：
10.1073/pnas.0605686103
发表时间：
2006-08-22
影响因子：
11.1
作者：
Schneider, D. A.;French, S. L.;Nomura, M.
通讯作者：
Nomura, M.
影响因子：
12.3
作者：
Imashimizu M;Takahashi H;Oshima T;McIntosh C;Bubunenko M;Court DL;Kashlev M
通讯作者：
Kashlev M
影响因子：
11.4
作者：
Bochkareva, Aleksandra;Yuzenkova, Yulia;Tadigotla, Vasisht R.;Zenkin, Nikolay
通讯作者：
Zenkin, Nikolay
影响因子：
16
作者：
Gusarov, I;Nudler, E
通讯作者：
Nudler, E
DOI：
10.1126/science.1251871
发表时间：
2014-05-30
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Larson MH;Mooney RA;Peters JM;Windgassen T;Nayak D;Gross CA;Block SM;Greenleaf WJ;Landick R;Weissman JS
通讯作者：
Weissman JS