Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569, USA, Advanced Analysis Center, Korea Institute of Science and Technology, Seoul, 136-791, Republic of Korea and Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Nucleic Acids Res. 2014 Mar;42(5):3395-408. doi: 10.1093/nar/gkt1276. Epub 2013 Dec 11.
Telomerase is a unique reverse transcriptase that maintains the 3' ends of eukaryotic chromosomes by adding tandem telomeric repeats. The RNA subunit (TR) of vertebrate telomerase provides a template for reverse transcription, contained within the conserved template/pseudoknot domain, and a conserved regions 4 and 5 (CR4/5) domain, all essential for catalytic activity. We report the nuclear magnetic resonance (NMR) solution structure of the full-length CR4/5 domain from the teleost fish medaka (Oryzias latipes). Three helices emanate from a structured internal loop, forming a Y-shaped structure, where helix P6 stacks on P5 and helix P6.1 points away from P6. The relative orientations of the three helices are Mg2+ dependent and dynamic. Although the three-way junction is structured and has unexpected base pairs, telomerase activity assays with nucleotide substitutions and deletions in CR4/5 indicate that none of these are essential for activity. The results suggest that the junction is likely to change conformation in complex with telomerase reverse transcriptase and that it provides a flexible scaffold that allows P6 and P6.1 to correctly fold and interact with telomerase reverse transcriptase.
端粒酶是一种独特的逆转录酶，通过添加串联的端粒重复序列来维持真核染色体的 3' 末端。脊椎动物端粒酶的 RNA 亚基（TR）为逆转录提供模板，包含在保守的模板/假结结构域和保守区域 4 和 5（CR4/5）结构域内，这些都是催化活性所必需的。我们报告了来自硬骨鱼斑马鱼（Oryzias latipes）的全长 CR4/5 结构域的核磁共振（NMR）溶液结构。三个螺旋从一个结构内部环伸出，形成一个 Y 形结构，其中 P6 螺旋堆叠在 P5 上，而 P6.1 螺旋指向远离 P6 的方向。三个螺旋的相对取向依赖于 Mg2+，并且是动态的。尽管三链结是结构的，并且具有意想不到的碱基对，但在 CR4/5 中进行核苷酸取代和缺失的端粒酶活性测定表明，这些都不是活性所必需的。结果表明，该连接点可能在与端粒酶逆转录酶复合物中改变构象，并且它提供了一个灵活的支架，使 P6 和 P6.1 能够正确折叠并与端粒酶逆转录酶相互作用。
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