Baranov Pavel V, Henderson Clark M, Anderson Christine B, Gesteland Raymond F, Atkins John F, Howard Michael T
Department of Human Genetics, University of Utah, 15 N 2030 E, Room 7410, Salt Lake City, UT 84112-5330, USA.
Virology. 2005 Feb 20;332(2):498-510. doi: 10.1016/j.virol.2004.11.038.
Programmed ribosomal frameshifting is an essential mechanism used for the expression of orf1b in coronaviruses. Comparative analysis of the frameshift region reveals a universal shift site U_UUA_AAC, followed by a predicted downstream RNA structure in the form of either a pseudoknot or kissing stem loops. Frameshifting in SARS-CoV has been characterized in cultured mammalian cells using a dual luciferase reporter system and mass spectrometry. Mutagenic analysis of the SARS-CoV shift site and mass spectrometry of an affinity tagged frameshift product confirmed tandem tRNA slippage on the sequence U_UUA_AAC. Analysis of the downstream pseudoknot stimulator of frameshifting in SARS-CoV shows that a proposed RNA secondary structure in loop II and two unpaired nucleotides at the stem I-stem II junction in SARS-CoV are important for frameshift stimulation. These results demonstrate key sequences required for efficient frameshifting, and the utility of mass spectrometry to study ribosomal frameshifting.
程序性核糖体移码是冠状病毒中用于表达orf1b的一种重要机制。对移码区域的比较分析揭示了一个通用的移码位点U_UUA_AAC，随后是一个预测的下游RNA结构，其形式为假结或亲吻茎环。使用双荧光素酶报告系统和质谱法，在培养的哺乳动物细胞中对严重急性呼吸综合征冠状病毒（SARS-CoV）的移码进行了表征。对SARS-CoV移码位点的诱变分析以及对亲和标记的移码产物的质谱分析证实了在序列U_UUA_AAC上的串联tRNA滑动。对SARS-CoV中移码的下游假结刺激物的分析表明，SARS-CoV中II环中一个提议的RNA二级结构以及茎I-茎II交界处的两个未配对核苷酸对移码刺激很重要。这些结果证明了有效移码所需的关键序列，以及质谱法在研究核糖体移码方面的实用性。
