He B, Riggs D L, Hanna M M
Department of Botany/Microbiology, University of Oklahoma, Norman 73019, USA.
Nucleic Acids Res. 1995 Apr 11;23(7):1231-8. doi: 10.1093/nar/23.7.1231.
We report a modified synthesis for 5-mercapto-UTP (5-SH-UTP) and its use for analysis of protein-RNA interactions utilizing Escherichia coli and T7 RNA polymerases and yeast RNA polymerases I and III. 5-SH-UTP did not affect transcriptional pausing, Rho-independent termination or recognition of the E. coli transcription complex by NusA. RNA containing 5-SH-UMP did not crosslink to polymerase when irradiation was 302 or 337 nm. Transcription complexes containing RNA substituted with 5-SH-UMP were post-transcriptionally modified to attach a photocross-linking group to thiol-tagged nucleotides in the RNA on the surface of the polymerase of free in solution. The pKa for 5-SH-UTP was determined to be 5.6, so modification of the thiol groups in the RNA with p-azidophenacyl bromide could be carried out at pH 7. Addition of the transcription termination factor Rho, a RNA binding protein, to E. coli transcription complexes resulted in RNA crosslinking to Rho and to the beta and beta' subunits of polymerase. Using 5-SH-UTP, one can distinguish RNA binding domains on the surface of RNA polymerases or other RNA binding proteins from those buried within the protein.
我们报道了一种5-巯基三磷酸尿苷（5-SH-UTP）的改良合成方法及其在利用大肠杆菌和T7 RNA聚合酶以及酵母RNA聚合酶I和III分析蛋白质-RNA相互作用中的应用。5-SH-UTP不影响转录暂停、不依赖Rho的终止或NusA对大肠杆菌转录复合物的识别。当照射波长为302或337 nm时，含有5-SH-尿苷一磷酸（5-SH-UMP）的RNA不会与聚合酶发生交联。含有被5-SH-UMP取代的RNA的转录复合物在转录后被修饰，以便在聚合酶表面或溶液中游离的RNA上的硫醇标记核苷酸上连接一个光交联基团。测定5-SH-UTP的pKa为5.6，因此可以在pH 7下用对叠氮苯甲酰溴对RNA中的硫醇基团进行修饰。向大肠杆菌转录复合物中添加转录终止因子Rho（一种RNA结合蛋白）会导致RNA与Rho以及聚合酶的β和β'亚基发生交联。使用5-SH-UTP，可以区分RNA聚合酶或其他RNA结合蛋白表面上的RNA结合结构域与那些埋藏在蛋白质内部的结构域。
