Feldstein P A, Buzayan J M, van Tol H, deBear J, Gough G R, Gilham P T, Bruening G
Department of Plant Pathology, College of Agricultural and Environmental Sciences, University of California, Davis 95616.
Proc Natl Acad Sci U S A. 1990 Apr;87(7):2623-7. doi: 10.1073/pnas.87.7.2623.
Both polarities of the satellite RNA of tobacco ringspot virus are sources of self-cleaving sequences. RNA of the less abundant, negative polarity, designated sTobRV-(-)RNA, has cleaving activity that was mapped previously to two noncontiguous regions of the polyribonucleotide chain. Endoribonucleolytic oligoribonucleotides (E) corresponding to the larger of the two regions cleaved smaller substrate oligoribonucleotides, at the ApG phosphodiester that is cleaved in sTobRV(-)RNA. An analogue of the substrate, which has a 2'-5' ApG phosphodiester, was not cleaved by E but acted as a competitive inhibitor of the cleavage of substrate. The analogue served as a primer, and E served as template, for reverse transcriptase-catalyzed copying of specific E sequences. The sequences transcribed suggest base pairing between the 5' region of E and a portion of the substrate that is located 3' to, but does not include, the ApG phosphodiester. Results from other experiments indicate this base pairing is a part of the functional cleavage complex. The association of the ends of E and substrate anticipates a second, 4-base-pair association between E and a portion of substrate that is 5' to, but does not include, the ApG phosphodiester. The effects of compensating mutations in E and substrate oligoribonucleotides support the existence of this second association in the active cleavage complex.
烟草环斑病毒卫星RNA的两种极性都是自我切割序列的来源。含量较少的负链RNA，即sTobRV-(-)RNA，具有切割活性，此前已将其定位到多核糖核苷酸链的两个不连续区域。对应于两个区域中较大区域的核糖核酸内切寡核糖核苷酸（E）在sTobRV(-)RNA中被切割的ApG磷酸二酯处切割较小的底物寡核糖核苷酸。具有2'-5' ApG磷酸二酯的底物类似物不能被E切割，但可作为底物切割的竞争性抑制剂。该类似物作为引物，E作为模板，用于逆转录酶催化的特定E序列的复制。转录的序列表明E的5'区域与底物的一部分之间存在碱基配对，该底物部分位于ApG磷酸二酯的3'端但不包括该磷酸二酯。其他实验结果表明这种碱基配对是功能性切割复合物的一部分。E和底物末端的结合预示着E与底物的一部分之间会形成第二个4碱基对的结合，该底物部分位于ApG磷酸二酯的5'端但不包括该磷酸二酯。E和底物寡核糖核苷酸中补偿性突变的影响支持了活性切割复合物中这种第二个结合结构的存在。
