Saturation mutagenesis of 5S rRNA in Saccharomyces cerevisiae
Saturation mutagenesis of 5S rRNA in Saccharomyces cerevisiae
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DOI：
10.1128/mcb.21.24.8264-8275.2001
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发表时间：
2001-12-01
影响因子：
5.3
通讯作者：
Dinman, JD
中科院分区：
文献类型：
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作者：
Smith, MW;Meskauskas, A;Dinman, JD
RRNAs是核糖体催化反应的核心分子，单个rRNAs积极参与不同的核糖体功能。我们之前的研究表明，酵母5S rRNA突变体(称为mof9)可以影响翻译阅读框架的维持，这表明这种普遍存在的生物分子具有意想不到的功能。然而，在当时，编码rRNA的基因高度重复的性质排除了更详细的遗传和分子分析。一种新的遗传系统允许细胞中的所有5S rRNA从一个小的、易于操作的质粒处转录。该系统也适用于其他rRNA的研究，并为详细的结构和功能研究提供了一个理想的遗传平台。饱和突变揭示了细胞存活、翻译准确性和病毒繁殖所需的5S rRNA区域。出乎意料的是，几乎没有发现致命的等位基因，这表明了这种分子的弹性。遗传表型在5S rRNA物理图谱上的叠加揭示了突变体表型簇的存在，表明5S rRNA的特定区域对特定功能是重要的。将这些突变体映射到Haloarcula marismortui大亚基上，发现这些簇发生在5S rRNA和核糖体不同功能中心之间的物理相互作用的重要点上。我们的分析导致我们提出5S rRNA的主要功能之一可能是通过在核糖体的所有不同功能中心之间充当信息的物理转换器来提高翻译保真度。
rRNAs are the central players in the reactions catalyzed by ribosomes, and the individual rRNAs are actively involved in different ribosome functions. Our previous demonstration that yeast 5S rRNA mutants (called mof9) can impact translational reading frame maintenance showed an unexpected function for this ubiquitous biomolecule. At the time, however, the highly repetitive nature of the genes encoding rRNAs precluded more detailed genetic and molecular analyses. A new genetic system allows all 5S rRNAs in the cell to be transcribed from a small, easily manipulated plasmid. The system is also amenable for the study of the other rRNAs, and provides an ideal genetic platform for detailed structural and functional studies. Saturation mutagenesis reveals regions of 5S rRNA that are required for cell viability, translational accuracy, and virus propagation. Unexpectedly, very few lethal alleles were identified, demonstrating the resilience of this molecule. Superimposition of genetic phenotypes on a physical map of 5S rRNA reveals the existence of phenotypic clusters of mutants, suggesting that specific regions of 5S rRNA are important for specific functions. Mapping these mutants onto the Haloarcula marismortui large subunit reveals that these clusters occur at important points of physical interaction between 5S rRNA and the different functional centers of the ribosome. Our analyses lead us to propose that one of the major functions of 5S rRNA may be to enhance translational fidelity by acting as a physical transducer of information between all of the different functional centers of the ribosome.