Yeh L C, Lee J C
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284.
J Mol Biol. 1995 Feb 17;246(2):295-307. doi: 10.1006/jmbi.1994.0085.
Previous studies suggest that the C-terminal region of ribosomal protein L1 from Saccharomyces cerevisiae is important for its interaction with the 5 S rRNA molecule. Within this region are several highly conserved basic amino acids including Lys276, Lys279, Lys289, Arg282, Arg285. To examine potential contributions of these amino acids to RNA-protein interaction and ribosomal assembly, effects of substitutions of these residues by methionine either individually or in combinations were examined. A methionine substitution of any one of the lysine residues did not significantly affect RNA binding in vitro. The mutant RNPs were as stable as the wild-type RNP. Yeast transformants expressing these mutant proteins grew at the same rate as the wild-type. However, mutant proteins containing substitutions of any two of these basic amino acids bound RNA weakly. The resultant RNPs were significantly less stable than the wild-type. Whereas cells expressing mutant L1 with a single substitution at 289 was not lethal, cells expressing mutant L1 with any double substitutions involving Lys289 as one of the substituted amino acids were lethal. These data suggest that Lys289 plays a key role in the binding of ribosomal protein L1 to 5 S rRNA. The other basic residues, particularly Arg282, and Arg285, in this region also contribute to RNA binding. These residues are predicted to locate on the same side of an alpha helix. We would like to propose a structural model for the yeast RNP that involves multiple contact sites located on one side of the helix in the C terminus of the protein and the 5 S rRNA. These basic amino acids also participate, directly or indirectly, in the interaction of the RNP complex with other components of the 60 S ribosomal subunit.
先前的研究表明，酿酒酵母核糖体蛋白L1的C末端区域对于其与5S rRNA分子的相互作用很重要。该区域内有几个高度保守的碱性氨基酸，包括赖氨酸276、赖氨酸279、赖氨酸289、精氨酸282、精氨酸285。为了研究这些氨基酸对RNA-蛋白质相互作用和核糖体组装的潜在贡献，研究了这些残基单独或组合被甲硫氨酸取代的影响。任何一个赖氨酸残基被甲硫氨酸取代都不会显著影响体外RNA结合。突变的核糖核蛋白（RNP）与野生型RNP一样稳定。表达这些突变蛋白的酵母转化体生长速度与野生型相同。然而，含有这些碱性氨基酸中任意两个被取代的突变蛋白与RNA的结合较弱。产生的RNP比野生型的稳定性显著降低。虽然表达在289位点有单个取代的突变L1的细胞没有致死性，但表达涉及赖氨酸289作为被取代氨基酸之一的任何双取代突变L1的细胞是致死的。这些数据表明赖氨酸289在核糖体蛋白L1与5S rRNA的结合中起关键作用。该区域的其他碱性残基，特别是精氨酸282和精氨酸285，也有助于RNA结合。预计这些残基位于α螺旋的同一侧。我们想提出一个酵母RNP的结构模型，该模型涉及位于蛋白质C末端螺旋一侧和5S rRNA上的多个接触位点。这些碱性氨基酸还直接或间接参与RNP复合物与60S核糖体亚基其他成分的相互作用。
