Wu H, Jiang L, Zimmermann R A
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003.
Nucleic Acids Res. 1994 May 11;22(9):1687-95. doi: 10.1093/nar/22.9.1687.
Through specific interactions with rRNA and mRNA, ribosomal protein S8 of Escherichia coli plays a central role in both assembly of the 30S ribosomal subunit and translational regulation of spc operon expression. To better understand S8-RNA association, we have measured the affinity of S8 for a number of variants of its rRNA and mRNA binding sites prepared by in vitro transcription or chemical synthesis. With the aid of site-directed deletions, we demonstrate that an imperfect, 33-nucleotide helical stem encompassing nucleotides 588-603 and 635-651 possesses all of the structural information necessary for specific binding of S8 to the 16S rRNA. This segment consists of two short duplexes that enclose a conserved, asymmetric internal loop which contains features crucial for protein recognition. The S8 binding site in spc operon mRNA is very similar in both primary and secondary structure to that in 16S rRNA except for the presence of two single bulged bases in one of the duplex segments. In addition, the apparent association constant for the S8-mRNA interaction is approximately fivefold less than that for the S8-rRNA interaction. We show that the difference in affinity can be attributed to the effects of the bulged bases. Deletion of the bulged bases from the mRNA site increases its affinity for S8 to a level similar to that of the rRNA, whereas insertion of single-base bulges at equivalent positions within the rRNA site reduces its affinity for S8 to a value typical of the mRNA. Single-base bulges in the proximity of essential recognition features are therefore capable of modulating the strength of protein-RNA interactions.
通过与核糖体RNA（rRNA）和信使RNA（mRNA）的特定相互作用，大肠杆菌的核糖体蛋白S8在30S核糖体亚基的组装以及spc操纵子表达的翻译调控中都起着核心作用。为了更好地理解S8与RNA的结合，我们测量了S8对通过体外转录或化学合成制备的其rRNA和mRNA结合位点的多种变体的亲和力。借助定点缺失，我们证明了一个不完美的、包含核苷酸588 - 603和635 - 651的33个核苷酸的螺旋茎拥有S8与16S rRNA特异性结合所需的所有结构信息。该片段由两个短双链体组成，它们包围着一个保守的、不对称的内部环，该内部环包含对蛋白质识别至关重要的特征。spc操纵子mRNA中的S8结合位点在一级和二级结构上与16S rRNA中的非常相似，只是在其中一个双链片段中存在两个单碱基凸起。此外，S8 - mRNA相互作用的表观缔合常数比S8 - rRNA相互作用的约低五倍。我们表明亲和力的差异可归因于凸起碱基的影响。从mRNA位点删除凸起碱基会使其对S8的亲和力增加到与rRNA相似的水平，而在rRNA位点的等效位置插入单碱基凸起会将其对S8的亲和力降低到mRNA典型的值。因此，在基本识别特征附近的单碱基凸起能够调节蛋白质 - RNA相互作用的强度。
