Mougel M, Allmang C, Eyermann F, Cachia C, Ehresmann B, Ehresmann C
UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
Eur J Biochem. 1993 Aug 1;215(3):787-92. doi: 10.1111/j.1432-1033.1993.tb18093.x.
Escherichia coli ribosomal protein S8 was previously shown to bind a 16S rRNA fragment (nucleotides 584-756) with the same affinity as the complete 16S rRNA, and to shield an irregular helical region (region C) [Mougel, M., Eyermann, F., Westhof, E., Romby, P., Expert-Bezançon, Ebel, J. P., Ehresmann, B. & Ehresmann, C. (1987). J. Mol. Biol. 198, 91-107]. Region C was postulated to display characteristic features: three bulged adenines (A595, A640 and A642), a non-canonical U598-U641 pair surrounded by two G.C pairs. In order to delineate the minimal RNA binding site, deletions were introduced by site-directed mutagenesis and short RNA fragments were synthesized. Their ability to bind S8 was assayed by filter binding. Our results show that the RNA binding site can be restricted to a short helical stem (588-605/633-651) containing region C. The second part of the work focused on region C and on the role of conserved nucleotides as potential determinants of S8 recognition. Single and double mutations were introduced by site-directed mutagenesis in fragment 584-756, and their effect on S8 binding was measured. It was found that the three bulged positions are essential and that adenines are required at positions 640 and 642. U598 is also crucial and the highly conserved G597.C643 pair cannot be inverted. These conserved nucleotides are either directly involved in the recognition process as direct contacts or required to maintain a specific conformation. The strong evolutionary pressure and the small number of positive mutants stress the high stringency of the recognition process.
先前研究表明，大肠杆菌核糖体蛋白S8与16S rRNA片段（核苷酸584 - 756）结合的亲和力与完整的16S rRNA相同，并能保护一个不规则螺旋区域（区域C）[穆热尔，M.，埃耶曼，F.，韦斯托夫，E.，龙比，P.，埃克斯佩 - 贝赞松，埃贝尔，J. P.，埃雷斯曼，B. & 埃雷斯曼，C.（1987年）。《分子生物学杂志》198，91 - 107]。区域C被推测具有以下特征：三个凸起的腺嘌呤（A595、A640和A642），一个非经典的U598 - U641碱基对，其两侧为两个G.C碱基对。为了确定最小的RNA结合位点，通过定点诱变引入缺失，并合成短RNA片段。通过滤膜结合测定它们与S8结合的能力。我们的结果表明，RNA结合位点可局限于包含区域C的短螺旋茎（588 - 605/633 - 651）。研究工作的第二部分聚焦于区域C以及保守核苷酸作为S8识别潜在决定因素的作用。通过定点诱变在片段584 - 756中引入单突变和双突变，并测定它们对S8结合的影响。结果发现，三个凸起位置至关重要，640和642位需要腺嘌呤。U598也很关键，高度保守的G597.C643碱基对不能颠倒。这些保守核苷酸要么作为直接接触直接参与识别过程，要么是维持特定构象所必需的。强大的进化压力和少量的正向突变强调了识别过程的高度严格性。
