Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo.
Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo.
复制标题
DOI：
10.1186/s13059-015-0666-5
复制
发表时间：
2015-05-15
期刊：
影响因子：
12.3
通讯作者：
Kashlev M
中科院分区：
文献类型：
--
作者：
Imashimizu M;Takahashi H;Oshima T;McIntosh C;Bubunenko M;Court DL;Kashlev M
转录延伸经常被DNA中的暂停信号中断，并对基因表达产生下游影响。转录错误还会导致长时间的停顿，这可能会通过干扰DNA复制而导致基因组不稳定。与易位阻断和误结合相关的停顿机制已经在体外得到了表征，但在体内还没有。我们用一种新的方法研究了大肠杆菌中RNA聚合酶(RNAP)的暂停模式，将自然延伸转录序列(NET-SEQ)与转录本的RNase足迹(RNet-SEQ)相结合。我们发现RNA-DNA杂交体5‘端的G-DC碱基对干扰了RNAP的易位。5‘G-DC碱基对和RNA的3’端之间的距离在三个核苷酸宽度上波动。因此，G-DC碱基对可以诱导RNAP在移位后、移位前和回溯状态下的暂停。此外，横跨RNAP活性部位的模板DNA链的CpG序列抑制了延伸并诱导G-to-A错误，从而导致RNAP的回溯。GRE因子有效地纠正了错误，并挽救了回溯的复合体。我们还发现，停顿事件在mRNA基因的5‘非翻译区和反义转录中丰富，在rRNA基因中减少。在大肠杆菌中，强大的转录暂停涉及RNAP与RNA-DNA杂交物上游端的G-DC相互作用，这干扰了易位。CpG DNA序列导致转录暂停和G-to-A错误。本文的在线版本(doi：10.1186/s13059-0150666-5)包含补充材料，授权用户可以使用。
Transcription elongation is frequently interrupted by pausing signals in DNA, with downstream effects on gene expression. Transcription errors also induce prolonged pausing, which can lead to a destabilized genome by interfering with DNA replication. Mechanisms of pausing associated with translocation blocks and misincorporation have been characterized in vitro, but not in vivo. We investigate the pausing pattern of RNA polymerase (RNAP) in Escherichia coli by a novel approach, combining native elongating transcript sequencing (NET-seq) with RNase footprinting of the transcripts (RNET-seq). We reveal that the G-dC base pair at the 5′ end of the RNA-DNA hybrid interferes with RNAP translocation. The distance between the 5′ G-dC base pair and the 3′ end of RNA fluctuates over a three-nucleotide width. Thus, the G-dC base pair can induce pausing in post-translocated, pre-translocated, and backtracked states of RNAP. Additionally, a CpG sequence of the template DNA strand spanning the active site of RNAP inhibits elongation and induces G-to-A errors, which leads to backtracking of RNAP. Gre factors efficiently proofread the errors and rescue the backtracked complexes. We also find that pausing events are enriched in the 5′ untranslated region and antisense transcription of mRNA genes and are reduced in rRNA genes. In E. coli, robust transcriptional pausing involves RNAP interaction with G-dC at the upstream end of the RNA-DNA hybrid, which interferes with translocation. CpG DNA sequences induce transcriptional pausing and G-to-A errors. The online version of this article (doi:10.1186/s13059-015-0666-5) contains supplementary material, which is available to authorized users.
登录
查看更多内容
DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Anders S;Pyl PT;Huber W
通讯作者：
Huber W
影响因子：
12.3
作者：
Giannoukos G;Ciulla DM;Huang K;Haas BJ;Izard J;Levin JZ;Livny J;Earl AM;Gevers D;Ward DV;Nusbaum C;Birren BW;Gnirke A
通讯作者：
Gnirke A
影响因子：
46.9
作者：
Cho, Byung-Kwan;Zengler, Karsten;Qiu, Yu;Park, Young Seoub;Knight, Eric M.;Barrett, Christian L.;Gao, Yuan;Palsson, Bernhard O.
通讯作者：
Palsson, Bernhard O.
影响因子：
3.4
作者：
Grokhovsky, Sergei L.;Il'icheva, Irina A.;Nechipurenko, Yury D.
通讯作者：
Nechipurenko, Yury D.
R环的形成是未甲基化的人类CpG岛启动子的独特特征。
影响因子：
16
作者：
Ginno PA;Lott PL;Christensen HC;Korf I;Chédin F
通讯作者：
Chédin F