Abstract：
Members of the 3\'-5\' RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5\'-3\' RNA and DNA polymerases. Discovery of enzymes capable of this reaction raised the possibility of exploiting 3\'-5\' polymerases for post-transcriptional incorporation of nucleotides to the 5\'-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other non-coding RNA. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3\'-5\' polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5\'-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in trans to guide 5\'-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to 8 bases to 5\'-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5\'-end labeling tool.
摘要：
3'-5'RNA聚合酶家族的成员，由tRNAHis鸟苷酸转移酶（Thg1）和Thg1样蛋白（TLPs）组成，在与所有其他已知的RNA和DNA聚合酶相反的方向上催化RNA的模板合成。能够进行该反应的酶的发现提高了利用3'-5'聚合酶将核苷酸转录后掺入核酸的5'末端而无需连接的可能性，而是通过模板化聚合酶添加。迄今为止,这些酶的研究集中在向高度结构化的RNA中添加核苷酸，如tRNA和其他非编码RNA。因此,RNA底物识别和核苷酸偏好的一般原则可能使3'-5'聚合酶的更广泛的应用尚未阐明。这里,我们研究了使用Thg1或TLP将多个核苷酸掺入到短双链体RNA底物的5'末端的可行性，使用反式提供的模板RNA寡核苷酸来指导特定序列的5'端添加。使用优化的测定条件，我们证明了某些TLP具有显着的能力，以适应不同长度的短RNA底物-模板双链体，导致能够以模板依赖性方式将高达8个碱基的所需核苷酸序列掺入到模型RNA底物的5'末端。这项工作进一步推进了我们的目标，以开发这种非典型酶家族作为通用的核酸5'端标记工具。
