Wang M J, Gegenheimer P
Department of Biochemistry, University of Kansas, Lawrence 66045-2106.
Nucleic Acids Res. 1990 Nov 25;18(22):6625-31. doi: 10.1093/nar/18.22.6625.
Inhibition of an RNA processing reaction after treatment with the Ca2(+)-dependent micrococcal nuclease (MN) is often used as a criterion for the presence of a required RNA or ribonucleoprotein component in the system. Following MN digestion, the nuclease is inactivated with EGTA and radiolabeled substrate is added to assay for remaining RNA processing activity. We found previously that inhibition of RNA processing by MN need not involve RNA hydrolysis: EGTA-inactivated MN can suppress RNA processing if the assay is performed in the absence of carrier RNA. We now demonstrate both by native gel electrophoresis and by nitrocellulose filter retention that EGTA-inactivated MN forms a complex with free RNA which can be dissociated by addition of synthetic polynucleotides or heparin. In the absence of Ca2+, nuclease binds to precursor tRNA with an apparent KD congruent to 1.4 x 10(-6) M, comparable to its reported affinity for DNA. In an assay for endonucleolytic tRNA maturation, inactivated MN bound to radiolabeled pre-tRNA physically blocks the sites of endonuclease cleavage and prevents tRNA processing. We call this phenomenon 'substrate masking'. Addition of excess carrier RNA competes with pre-tRNA for MN binding and restores normal processing.
用依赖Ca2+的微球菌核酸酶（MN）处理后，RNA加工反应受到抑制，这常被用作判断系统中是否存在所需RNA或核糖核蛋白成分的标准。MN消化后，用乙二醇双四乙酸（EGTA）使核酸酶失活，并加入放射性标记的底物以检测剩余的RNA加工活性。我们之前发现，MN对RNA加工的抑制不一定涉及RNA水解：如果在没有载体RNA的情况下进行检测，EGTA失活的MN可以抑制RNA加工。我们现在通过非变性凝胶电泳和硝酸纤维素滤膜滞留实验证明，EGTA失活的MN与游离RNA形成复合物，该复合物可通过添加合成多核苷酸或肝素而解离。在没有Ca2+的情况下，核酸酶与前体tRNA结合，其表观解离常数（KD）约为1.4×10^(-6) M，与其对DNA的报道亲和力相当。在一项核酸内切酶介导的tRNA成熟检测中，与放射性标记的前体tRNA结合的失活MN会物理性地阻断核酸内切酶切割位点，从而阻止tRNA加工。我们将这种现象称为“底物掩盖”。添加过量的载体RNA会与前体tRNA竞争MN的结合，从而恢复正常加工。
