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.

中文翻译：

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逆转录酶速度和模板灵敏度的直接跟踪：对长 RNA 分子测序和分析的影响

尽管逆转录酶 (RT) 是研究和生物技术的关键试剂，但它们的机械特性尚不清楚。特别是，我们对它们的相对速度和对模板中结构障碍的反应知之甚少。商业逆转录病毒 RT 在混合序列模板的许多位置处停止，导致截短的 cDNA 产物使下游分析复杂化。相比之下，II 组内含子编码的 RT 似乎以高持续合成能力和最少的停止复制长 RNA。然而，它们的速度、一致性和暂停行为尚未得到探索。在这里，我们分析了 RT 速度，因为酶在异质序列和结构中移动，这些序列和结构嵌入在长的非编码 RNA 转录物中。我们观察到模板中的异质性对逆转录病毒 RT 的引物延伸具有高度破坏性。然而，序列组成和模板结构对 II 组内含子 RT 行为的影响可以忽略不计，例如 MarathonRT (MRT)。事实上，MRT 在一次通过中复制长 RNA，并以 25 nt/秒的恒定速度显示同步引物延伸。此外，它通过稳定的 RNA 结构基序而不扰动速度。总之，结果表明一致、稳健的易位行为是 II 组内含子编码 RT 的标志，其中一些以高速运行。并以 25 nt/秒的恒定速度显示同步引物延伸。此外，它通过稳定的 RNA 结构基序而不扰动速度。总之，结果表明一致、稳健的易位行为是 II 组内含子编码 RT 的标志，其中一些以高速运行。并以 25 nt/秒的恒定速度显示同步引物延伸。此外，它通过稳定的 RNA 结构基序而不扰动速度。总之，结果表明一致、稳健的易位行为是 II 组内含子编码 RT 的标志，其中一些以高速运行。