Almehdi M, Yoo Y S, Schaup H W
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-6503.
Nucleic Acids Res. 1991 Dec 25;19(24):6895-903. doi: 10.1093/nar/19.24.6895.
Ribosomes play an active role in protein biosynthesis. Ribosomal RNA conformation in ribosomal subunits, intramolecular interactions between different rRNA sequences within the confinement of the particles, and intermolecular interactions are presumed necessary to support efficient and accurate protein synthesis. Here we report an analysis of the disposition of 16S rRNA conserved zones centered about positions 525, 1400, and 1500 in 30S subunits. Complementary oligodeoxyribonucleotides in conjunction with nuclease S1 digestion were used to do this. All of the sequences examined in 30S subunits are accessible to DNA probes of 9 to 12 nucleotide residues in length. However, the kinetic characteristics of the respective DNA interactions with 30S particles vary significantly. In addition to the investigation of normal 30S particles, a four base deletion within the 1400 region of 16S rRNA was analyzed. The deletion was made by using synthetic DNAs to target the deletion site for RNase H digestion. The direct in vitro procedure for manipulating rRNA conserves nucleotide modifications. The alteration causes a significant change in the disposition of 16S rRNA in 30S subunits, suggesting a reduction in the freedom of movement of the altered zone in the particle. In a factor-dependent in vitro protein synthesis system primed with MS2 mRNA and altered 30S subunits, there was a 50% decrease in phage coat protein synthesis. The reduction could be due to a decrease in the rate of translation or premature termination of translation. We present evidence here, based on isotopic studies, which supports the latter possibility.
核糖体在蛋白质生物合成中发挥着积极作用。核糖体亚基中的核糖体RNA构象、颗粒范围内不同rRNA序列之间的分子内相互作用以及分子间相互作用被认为是支持高效且准确的蛋白质合成所必需的。在此，我们报告了对30S亚基中以525、1400和1500位置为中心的16S rRNA保守区分布的分析。我们使用互补寡脱氧核糖核苷酸结合核酸酶S1消化来进行此项分析。在30S亚基中检测的所有序列对于长度为9至12个核苷酸残基的DNA探针都是可及的。然而，各个DNA与30S颗粒相互作用的动力学特征差异显著。除了对正常30S颗粒的研究外，还分析了16S rRNA的1400区域内的一个四碱基缺失。该缺失是通过使用合成DNA靶向缺失位点进行RNase H消化产生的。用于操纵rRNA的直接体外方法保留了核苷酸修饰。这种改变导致30S亚基中16S rRNA的分布发生显著变化，表明颗粒中改变区域的移动自由度降低。在以MS2 mRNA引发并含有改变的30S亚基的因子依赖性体外蛋白质合成系统中，噬菌体外壳蛋白的合成减少了50%。这种减少可能是由于翻译速率降低或翻译过早终止。基于同位素研究，我们在此提供了支持后一种可能性的证据。
