Dubreuil Y L, Expert-Bezançon A, Favre A
Institut Jacques Monod, CNRS Université Paris 7, France.
Nucleic Acids Res. 1991 Jul 11;19(13):3653-60. doi: 10.1093/nar/19.13.3653.
The structure in solution of an RNA fragment (218 nucleotides long) containing part of E. coli 16S rRNA domain 2 has been studied using the intrinsic photoaffinity probe 4-thiouridine (s4U). In vitro transcription with T7 polymerase, in the presence of s4U triphosphate yielded complete RNA molecules. An affinity electrophoresis system based on Phenylmercuric substituted polyacrylamide (APM) gels allows separation of the RNA chains as a function of their s4U content. Distribution of s4U within chains follows a binomial law indicating that (i) substitution is close to random, (ii) efficiency of s4U incorporation is 0.22 times that of U. The monothiolated RNA fraction isolated from APM gel was irradiated at 366 nm under native conditions and the intramolecularly crosslinked molecules, (34%), were separated on denaturing polyacrylamide gel according to loop size. The positions of the two partners of bridges were identified by mean of reverse transcription and RNA sequencing. 17 of the 41 possible s4U positions lead to detectable bridges. These crosslinks formed efficiently at the border of bihelical regions or when structural mobility is allowed. The pattern of crosslinks is in agreement with the previously proposed secondary structure but indicates that it is much more flexible than expected.
利用固有光亲和探针4-硫尿苷（s4U）对包含大肠杆菌16S rRNA结构域2一部分的RNA片段（218个核苷酸长）在溶液中的结构进行了研究。在三磷酸s4U存在的情况下，用T7聚合酶进行体外转录产生了完整的RNA分子。基于苯基汞取代聚丙烯酰胺（APM）凝胶的亲和电泳系统能够根据RNA链的s4U含量对其进行分离。s4U在链内的分布遵循二项式规律，这表明：（i）取代接近随机；（ii）s4U掺入效率是U的0.22倍。从APM凝胶中分离出的单硫醇化RNA组分在天然条件下于366 nm处照射，然后根据环大小在变性聚丙烯酰胺凝胶上分离分子内交联的分子（34%）。通过逆转录和RNA测序确定桥接的两个配对体的位置。41个可能的s4U位置中有17个导致可检测到的桥接。这些交联在双螺旋区域的边界处或允许结构移动时有效形成。交联模式与先前提出的二级结构一致，但表明其比预期的要灵活得多。
