Farrow M A, Aboul-ela F, Owen D, Karpeisky A, Beigelman L, Gait M J
Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge CB2 2QH, U.K.
Biochemistry. 1998 Mar 3;37(9):3096-108. doi: 10.1021/bi972695v.
The Human Immunodeficiency Virus type 1 Tat protein interacts specifically with a U-rich bulge within an RNA stem-loop known as the trans-activation responsive region (TAR) that occurs in all viral transcripts. We have photochemically cross-linked to Tat peptide (37-72), a model TAR duplex substituted at U23 in the bulge by 4-thioU. We have identified the cross-linked amino acid as Arg55 in the basic region of the Tat peptide by use of a combination of proteolytic digestions and MALDI-TOF mass spectrometric analysis. The identification also required use of a synthetic Tat peptide containing a site-specific, uniformly 13C and 15N isotopically labeled arginine. We also describe a new chemical procedure for obtaining site-specific cross-links to Tat via the use of 2'-beta-alanyl U-substituted TAR and the amino-specific reagent dithiobis(succinimidyl propionate). U23-2'-functionalized TAR was shown to cross-link uniquely to Lys51 in the basic region of Tat, whereas other sites in the upper and lower stems of TAR (U35, U38, and U42) showed cross-linking only to the N-terminus of Tat peptide (37-72). U40 cross-linked to both Lys51 and the N-terminus of the peptide. The results help to establish a preliminary model of the binding of Tat peptide to the major groove of TAR RNA.
1型人类免疫缺陷病毒Tat蛋白与RNA茎环结构中富含尿嘧啶的凸起特异性相互作用，该茎环结构被称为反式激活应答区域（TAR），存在于所有病毒转录本中。我们用光化学方法将Tat肽（37 - 72）与一个在凸起处U23被4 - 硫代尿嘧啶取代的TAR双链体模型进行交联。通过蛋白水解消化和基质辅助激光解吸电离飞行时间质谱分析相结合的方法，我们确定交联的氨基酸是Tat肽碱性区域的Arg55。该鉴定还需要使用含有位点特异性、均匀13C和15N同位素标记精氨酸的合成Tat肽。我们还描述了一种新的化学方法，通过使用2'-β-丙氨酰U取代的TAR和氨基特异性试剂二硫代双（琥珀酰亚胺丙酸酯）来获得与Tat的位点特异性交联。结果表明，U23 - 2'-功能化的TAR仅与Tat碱性区域的Lys51交联，而TAR上下茎中的其他位点（U35、U38和U42）仅与Tat肽（37 - 72）的N端交联。U40与肽的Lys51和N端均发生交联。这些结果有助于建立Tat肽与TAR RNA大沟结合的初步模型。
