Direct tracking of reverse-transcriptase speed and template sensitivity: implications for sequencing and analysis of long RNA molecules.
Direct tracking of reverse-transcriptase speed and template sensitivity: implications for sequencing and analysis of long RNA molecules.
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DOI：
10.1093/nar/gkac518
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发表时间：
2022-07-08
影响因子：
14.9
通讯作者：
Pyle, Anna Marie
中科院分区：
文献类型：
--
作者：
Guo, Li-Tao;Olson, Sara;Patel, Shivali;Graveley, Brenton R.;Pyle, Anna Marie
虽然逆转录酶（RT）酶是研究和生物技术的关键试剂，但其机械特性尚未得到很好的理解。特别是，我们对它们的相对速度和对模板中结构性障碍的反应知之甚少。商业逆转录病毒RT在混合序列模板的沿着许多位置处停止，导致使下游分析复杂化的截短cDNA产物。相比之下，第二组内含子编码的RT似乎复制长RNA具有高的持续合成能力和最小的停止。然而，他们的速度，一致性和暂停行为还没有被探索。在这里，我们分析RT速度的酶移动通过异质序列和结构，嵌入在一个长的非编码RNA转录。我们观察到，在模板的异质性是高度破坏性的引物延伸逆转录病毒RT。然而，序列组成和模板结构对II组内含子RT的行为，如马拉松RT（MRT）的影响可以忽略不计。事实上，MRT在单次通过中复制长RNA，并以25 nt/sec的恒定速度显示同步的引物延伸。此外，它通过稳定的RNA结构基序没有扰动的速度。两者合计，结果表明，一致的，强大的易位行为是第二组内含子编码的RT的标志，其中一些在高速运行。
Although reverse-transcriptase (RT) enzymes are critical reagents for research and biotechnology, their mechanical properties are not well understood. In particular, we know little about their relative speed and response to structural obstacles in the template. Commercial retroviral RTs stop at many positions along mixed sequence templates, resulting in truncated cDNA products that complicate downstream analysis. By contrast, group II intron-encoded RTs appear to copy long RNAs with high processivity and minimal stops. However, their speed, consistency and pausing behavior have not been explored. Here, we analyze RT velocity as the enzyme moves through heterogeneous sequences and structures that are embedded within a long noncoding RNA transcript. We observe that heterogeneities in the template are highly disruptive to primer extension by retroviral RTs. However, sequence composition and template structure have negligible effects on behavior of group II intron RTs, such as MarathonRT (MRT). Indeed, MRT copies long RNAs in a single pass, and displays synchronized primer extension at a constant speed of 25 nt/sec. In addition, it passes through stable RNA structural motifs without perturbation of velocity. Taken together, the results demonstrate that consistent, robust translocative behavior is a hallmark of group II intron-encoded RTs, some of which operate at high velocity.
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影响因子：
2.9
作者：
Sexton AN;Wang PY;Rutenberg-Schoenberg M;Simon MD
通讯作者：
Simon MD
影响因子：
48
作者：
Sedlazeck FJ;Rescheneder P;Smolka M;Fang H;Nattestad M;von Haeseler A;Schatz MC
通讯作者：
Schatz MC
影响因子：
11.4
作者：
XIONG, Y;EICKBUSH, TH
通讯作者：
EICKBUSH, TH
影响因子：
4.8
作者：
Kelleher, CD;Champoux, JJ
通讯作者：
Champoux, JJ
影响因子：
4.8
作者：
Bibillo, A;Eickbush, TH
通讯作者：
Eickbush, TH