Bend and helical twist associated with a symmetric internal loop from 5S ribosomal RNA.
Bend and helical twist associated with a symmetric internal loop from 5S ribosomal RNA.
复制标题
与 5S 核糖体 RNA 的对称内环相关的弯曲和螺旋扭曲。
DOI：
10.1021/bi00199a036
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Draper,DE
中科院分区：
文献类型：
--
作者：
Tang,RS;Draper,DE
MATERIALS AND METHODSPreparation and Electrophoresis of RNA Duplexes. Plasmids containing T7 and T3 RNA polymerase promoters facing each other and having appropriate sequences between them were prepared and transcribed, and the RNA transcripts were annealed and trimmed with Ti RNase to give duplex RNA as described in detail elsewhere (Tang & Draper, 1990, 1994). The internal loops and their sequence contexts are shown in Figure 1. Electrophoresis was carried out in 10 or 12% polyacrylamide gels (0.4 mm thick by 40 cm long) in 75 mM disodium phosphate buffer (adjusted to pH 7.0 with phosphoric acid) with or without 2 mM MgCL. Runs were at 150 V/cm in the cold or at room temperature. The actual temperatures of the gels measured by a surface thermometer were 8 or 30 C, respectively. One strand of each RNA was 32P labeled during transcription, and the RNAs were detected by autoradiography. RNAs having the same number of nucleotides but canonically paired were run as controls in adjacent lanes, and the relative gel mobilities reported are the ratios of the mobilities of loop-containing and control RNAs. For the phasing experiments, plots of relative gel mobility as a function of base pairs between bendsites were fit to an equation describing the end-to-end distance of the RNA, as previously described (Tang & Draper, 1994). Ribosomal Protein L25. L25 protein purifed from E. coli ribosomes was a generous gift from Peter Moore (Yale University). It was dialyzed into 0.1 Mkc1 and 10 mM Tris, pH 7.6, to remove urea, and it showed a single band in SDS gels. For gel shift assays, protein (~ 10/nM) was incubated on ice for 30 min with labeled RNA before electrophoresis, and the gels were run at 30 C in sodium phosphate buffer.
