Mishima Y, Steitz J A
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812, USA.
EMBO J. 1995 Jun 1;14(11):2679-87. doi: 10.1002/j.1460-2075.1995.tb07266.x.
We have mapped specific RNA-protein contacts between human immunodeficiency virus (HIV) type I reverse transcriptase (RT) and its natural primer, human tRNA(3Lys), using a site-specific crosslinking strategy. Four different tRNA(3Lys) constructs with a single 32P-labeled 4-thiouridine (4-thioU) residue at positions -1, 16, 36 or 41 were synthesized. After incubation with RT followed by irradiation, crosslinks were localized to either the p66 or p51 subunit of RT by digestion with nuclease and SDS gel fractionation. 4-thioU at position -1 or 16 transferred label to the p66 subunit almost exclusively (> 90%), whereas position 36 labeled both p66 and p51 (3:1). Position 41 yielded no detectable crosslinks. The region of p66 contacted by position -1 of tRNA(3Lys) was localized to the 203 C-terminal amino acids of RT by CNBr cleavage, whereas a 127 amino acid-CNBr peptide (residues 230-357) from both p66 and p51 was labeled by position 36. Functionality of the 4-thioU-modified tRNA(3Lys)(-1) crosslinked to RT in the presence of an RNA but not a DNA template was demonstrated by the ability of the tRNA to be extended. These results localize the 5' half of the tRNA on the interface between the two RT subunits, closer to the RNase H domain than to the polymerase active site, in accord with previous suggestions. They argue further that a specific binding site for the 5' end of the primer tRNA(3Lys) may exist within the C-terminal portion of the p66 subunit, which could be important for the initiation of reverse transcription.
我们采用位点特异性交联策略，绘制了人类免疫缺陷病毒1型逆转录酶（RT）与其天然引物人tRNA3Lys之间特定的RNA-蛋白质相互作用图谱。合成了四种不同的tRNA3Lys构建体，它们在-1、16、36或41位带有单个32P标记的4-硫尿苷（4-thioU）残基。与RT孵育后进行照射，通过核酸酶消化和SDS凝胶分级分离，将交联定位到RT的p66或p51亚基上。-1或16位的4-thioU几乎只将标记转移到p66亚基（>90%），而36位则同时标记了p66和p51（3:1）。41位未产生可检测到的交联。通过CNBr切割，tRNA3Lys -1位接触的p66区域定位到RT的203个C末端氨基酸，而来自p66和p51的127个氨基酸的CNBr肽（残基230-357）被36位标记。在RNA而非DNA模板存在的情况下，与RT交联的4-thioU修饰的tRNA3Lys（-1）能够被延伸，证明了其功能性。这些结果将tRNA的5'端定位在两个RT亚基之间的界面上，比聚合酶活性位点更靠近RNase H结构域，这与之前的推测一致。它们进一步表明，引物tRNA3Lys 5'端的特异性结合位点可能存在于p66亚基的C末端部分，这可能对逆转录的起始很重要。
