基于核酶技术的人线粒体色氨酸tRNA的高效转录及其活性鉴定

利用T7RNA聚合酶(T7 RNA polymerase,T7 RNAP)的体外转录方法因其简便、高效而在RNA制备中得到广泛应用,但该法由于T7RNAP的启动子跨越转录起始位点而会导致转录产物多出附加序列;如果去掉T7启动子的转录起始位点,则会严重降低T7RNAP的转录活性。本实验很好地消除了以上弊端,将T7RNAP的高效转录系统与核酶高度专一的自剪切技术相结合,成功构建了一种不影响转录效率的体外转录方法,而且转录后核酶能够在设计的特定位点进行自剪切并释放出目的RNA,得到了活性达113.6pmol/μg的人线粒体色氨酸tRNA(HmtRNATrp(UCA))。该方法转录效率高、重复性好,适合RNA的大量精确制备。     

Probing the nucleotide binding sites in T7 RNA polymerase using cibacron blue

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. In this work we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, and its effect on the function of the enzyme. T7 RNAP binds to the dye in a bi-phasic manner. The first phase of the binding is characterized by a high affinity (Kd in the nanomolar range) and reversible inactivation of the enzyme. The second binding site is the common substrate binding site. The association of the dye with T7 RNAP is a good model to understand the physiological significance of a high affinity binding of the initiating nucleotide, GTP, earlier reported from our laboratory. The results will be discussed to understand the role of the high affinity GTP binding.