Dos Santos D V, Vianna A L, Fourrey J L, Favre A
Groupe de Photobiologie Moléculaire, Institut Jacques Monod, CNRS Université Paris VII, France.
Nucleic Acids Res. 1993 Jan 25;21(2):201-7. doi: 10.1093/nar/21.2.201.
Hairpin ribozymes derived from (-)sTRSV RNA exhibit substantial cleavage activity when wobble GU base pairs are introduced in place of the AU pairs normally involved in helices I and II between substrate and ribozyme. This finding prompted us to synthesize by in vitro transcription a new hairpin ribozyme, active against a 14-mer substrate derived from a conserved HIV sequence. Interactions of the canonical and anti-HIV hairpin ribozymes with non cleavable DNA substrate analogues containing the photoaffinity probe deoxy-4-thiouridine (ds4U) at a single site were investigated. Upon near-UV light irradiation (365 nm), all these substrate analogues were covalently attached to ribozyme via single or multiple crosslinks. In contrast, no crosslinks were detected using either a DNA substrate analogue lacking ds4U or a ds4U containing oligomer unrelated to the substrate sequence. As expected, if the dissociation constant is in the range of 5-15 microM, the yield of crosslinked ribozyme increased markedly with increasing the substrate analogue concentration. The ribozyme residues involved in the crosslinks were determined by RNA sequencing. The pattern of crosslinks obtained with the two ribozyme systems provides additional evidence in support of the consensus secondary structure proposed for the hairpin domain. Minor alternative conformations were detected in the case of the (-)sTRSV system.
当引入摆动性GU碱基对取代通常参与底物与核酶之间螺旋I和螺旋II的AU碱基对时，源自（-）sTRSV RNA的发夹状核酶表现出显著的切割活性。这一发现促使我们通过体外转录合成一种新的发夹状核酶，该核酶对源自保守HIV序列的14聚体底物具有活性。研究了标准发夹状核酶和抗HIV发夹状核酶与在单个位点含有光亲和探针脱氧-4-硫尿苷（ds4U）的不可切割DNA底物类似物的相互作用。在近紫外光（365nm）照射下，所有这些底物类似物通过单交联或多交联与核酶共价连接。相比之下，使用缺乏ds4U的DNA底物类似物或与底物序列无关的含ds4U的寡聚物均未检测到交联。正如预期的那样，如果解离常数在5-15 microM范围内，交联核酶的产量会随着底物类似物浓度的增加而显著增加。通过RNA测序确定参与交联的核酶残基。用这两种核酶系统获得的交联模式为支持发夹结构域提出的共有二级结构提供了额外证据。在（-）sTRSV系统中检测到了较小的替代构象。
