Department of Chemistry, University of Houston, 3585 Cullen Blvd, Houston, TX 77204, USA.
Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA.
Chembiochem. 2021 May 14;22(10):1775-1778. doi: 10.1002/cbic.202000863. Epub 2021 Feb 25.
Ribosomal frameshifting is an important pathway used by many viruses for protein synthesis that involves mRNA translocation of various numbers of nucleotides. Resolving the mRNA positions with subnucleotide precision will provide critical mechanistic information that is difficult to obtain with current techniques. We report a method of high-resolution DNA rulers with subnucleotide precision and the discovery of new frameshifting intermediate states on mRNA containing a GA G motif. Two intermediate states were observed with the aid of fusidic acid, one at the "0" reading frame and the other near the "-1" reading frame, in contrast to the "-2" and "-1" frameshifting products found in the absence of the antibiotic. We termed the new near-"-1" intermediate the Post(-1*) state because it was shifted by approximately half a nucleotide compared to the normal "-1" reading frame at the 5'-end. This indicates a ribosome conformation that is different from the conventional model of three reading frames. Our work reveals uniquely precise mRNA motions and subtle conformational changes that will complement structural and fluorescence studies.
核糖体移码是许多病毒用于蛋白质合成的重要途径，涉及到各种数量的核苷酸的 mRNA 易位。以亚核苷酸精度解析 mRNA 的位置将提供难以用现有技术获得的关键机制信息。我们报告了一种具有亚核苷酸精度的高分辨率 DNA 标尺的方法，并在含有 GA G 基序的 mRNA 上发现了新的移码中间状态。在 fusidic acid 的辅助下观察到了两种中间状态，一种在“0”阅读框，另一种在接近“-1”阅读框，与在没有抗生素的情况下发现的“-2”和“-1”移码产物形成对比。我们将新的近“-1”中间状态命名为 Post(-1*)状态，因为与 5' 端正常的“-1”阅读框相比，它移位了大约半个核苷酸。这表明核糖体构象不同于传统的三个阅读框模型。我们的工作揭示了独特的精确 mRNA 运动和微妙的构象变化，将补充结构和荧光研究。
