Smith M W, Meskauskas A, Wang P, Sergiev P V, Dinman J D
Department of Molecular Genetics and Microbiology, Rutgers University and University of Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854, USA.
Mol Cell Biol. 2001 Dec;21(24):8264-75. doi: 10.1128/MCB.21.24.8264-8275.2001.
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.
核糖体RNA（rRNAs）是核糖体催化反应的核心参与者，各个rRNAs积极参与核糖体的不同功能。我们之前证明酵母5S核糖体RNA突变体（称为mof9）会影响翻译阅读框的维持，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，当时编码rRNAs的基因具有高度重复性，妨碍了更详细的遗传和分子分析。一种新的遗传系统使细胞中的所有5S rRNAs都能从小的、易于操作的质粒中转录而来。该系统也适用于研究其他rRNAs，并为详细的结构和功能研究提供了理想的遗传平台。饱和诱变揭示了5S rRNA中细胞活力、翻译准确性和病毒繁殖所必需的区域。出乎意料的是，只鉴定出极少数致死等位基因，这表明该分子具有很强的适应能力。将遗传表型叠加到5S rRNA的物理图谱上，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很重要。将这些突变体定位到嗜盐栖热袍菌大亚基上，发现这些簇出现在5S rRNA与核糖体不同功能中心之间物理相互作用的重要位点。我们的分析使我们提出，5S rRNA的主要功能之一可能是通过充当核糖体所有不同功能中心之间信息的物理传递体来提高翻译保真度。
