Mougel M, Eyermann F, Westhof E, Romby P, Expert-Bezançon A, Ebel J P, Ehresmann B, Ehresmann C
Laboratoire de Biochimie, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France.
J Mol Biol. 1987 Nov 5;198(1):91-107. doi: 10.1016/0022-2836(87)90460-8.
We have investigated in detail the secondary and tertiary structures of the 16 S rRNA binding site of protein S8 using a variety of chemical and enzymatic probes. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p- toluenesulfonate (at G(N-1) and U(N-3)) and with diethylpyrocarbonate (at A(N-7)). The involvement of phosphates in hydrogen bonds or ion co-ordination was monitored with ethylnitrosourea. RNases T1, U2 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. The RNA region, encompassing nucleotides 582 to 656 was probed within: (1) the complete 16 S rRNA molecule; (2) a 16 S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S8-16 S rRNA complex; (4) the S8-RNA fragment complex; (5) the 30 S subunit. Cleavage or modification sites were detected by primer extension with reverse transcriptase. We present a three-dimensional model derived from mapping experiments and graphic modeling. Nucleotides in area 594-599/639-645 display unusual features: a non-canonical base-pair is formed between U598 and U641; and A595, A640 and A642 are bulging out of the major groove. The resulting helix is slightly unwound. Comparative analysis of probing experiments leads to several conclusions. (1) The synthesized fragment adopts the same conformation as the corresponding region in the complete RNA molecule, thus confirming the existence of independent folding domains in RNAs. (2) A long-range interaction involving cytosine 618 and its 5' phosphate occurs in 16 S rRNA but not in the fragment. (3) The fragment contains the complete information required for S8 binding. (4) The RNA binding site of S8 is centered in the major groove of the slightly unwound helix (594-599/639-645), with the three bulged adenines appearing as specific recognition sites. (5) This same region of the 16 S RNA is not exposed at the surface of the 30 S subunit.
我们使用多种化学和酶促探针，详细研究了蛋白质S8的16 S rRNA结合位点的二级和三级结构。用硫酸二甲酯（作用于A（N - 1）、C（N - 3）和G（N - 7））、N - 环己基 - N' - （2 - （N - 甲基吗啉基） - 乙基） - 碳二亚胺 - 对甲苯磺酸盐（作用于G（N - 1）和U（N - 3））以及焦碳酸二乙酯（作用于A（N - 7））探测碱基。用乙基亚硝基脲监测磷酸参与氢键或离子配位的情况。使用核糖核酸酶T1、U2和核酸酶S1探测未配对的核苷酸，用核糖核酸酶V1监测碱基配对或堆积的核苷酸。对包含核苷酸582至656的RNA区域在以下几种情况下进行了探测：（1）完整的16 S rRNA分子内；（2）通过体外转录获得的对应于核苷酸578至756的16 S rRNA片段；（3）S8 - 16 S rRNA复合物；（4）S8 - RNA片段复合物；（5）30 S亚基。通过逆转录酶引物延伸检测切割或修饰位点。我们展示了一个源自图谱实验和图形建模的三维模型。594 - 599/639 - 645区域的核苷酸呈现出异常特征：U598和U641之间形成了一个非规范碱基对；A595、A640和A642从大沟中凸出。由此形成的螺旋稍有解旋。对探测实验的比较分析得出了几个结论。（1）合成的片段采用与完整RNA分子中相应区域相同的构象，从而证实了RNA中独立折叠结构域的存在。（2）涉及胞嘧啶618及其5'磷酸的长程相互作用在16 S rRNA中存在，但在片段中不存在。（3）该片段包含S8结合所需的完整信息。（4）S8的RNA结合位点位于稍有解旋的螺旋（594 - 599/639 - 645）的大沟中心，三个凸出的腺嘌呤作为特异性识别位点。（5）16 S RNA的这个相同区域在30 S亚基表面未暴露。
