Cao Hong, Tamilarasu Natarajan, Rana Tariq M
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Bioconjug Chem. 2006 Mar-Apr;17(2):352-8. doi: 10.1021/bc050277u.
The human immunodeficiency virus (HIV-1) encodes a transcriptional activator protein, Tat, which is expressed early in the viral life cycle and is essential for viral gene expression, replication, and pathogenesis. Tat interacts with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. Tat-derived peptides that contain the basic arginine-rich region of Tat are able to form in vitro complexes with TAR RNA, and these peptides provide a well-characterized system for understanding the mechanism of RNA-protein recognition. It is not known how RNA-binding Tat peptides are folded or docked in the Tat-TAR complex, and to what extent structural reorganization occurs upon TAR binding. To address these questions, we developed a fluorescence resonance energy transfer (FRET) system to analyze the interactions between TAR RNA and a Tat protein fragment (aa 38-72) uniquely labeled with donor and acceptor dye molecules, respectively. Using this FRET assay, we determined the binding affinity of Tat (47-58) and Tat (38-72) for TAR RNA under physiological conditions. We also delineated the distance between the N- and C-terminus of Tat (38-72) and the distance between the two termini and the 5' end of TAR when Tat (38-72) is bound to TAR. Our results suggest that the N- and C-termini of Tat (38-72) are close to each other when the peptide is folded and that the peptide does not go through a large structural change upon TAR binding.
人类免疫缺陷病毒1型（HIV-1）编码一种转录激活蛋白Tat，该蛋白在病毒生命周期早期表达，对病毒基因表达、复制及发病机制至关重要。Tat与反式激活应答区域（TAR）RNA相互作用，TAR RNA是一种位于所有HIV mRNA 5′端的59个碱基的茎环结构。含有Tat富含精氨酸碱性区域的Tat衍生肽能够在体外与TAR RNA形成复合物，这些肽为理解RNA-蛋白质识别机制提供了一个特征明确的系统。目前尚不清楚RNA结合型Tat肽在Tat-TAR复合物中是如何折叠或对接的，以及TAR结合后结构重组的程度如何。为了解决这些问题，我们开发了一种荧光共振能量转移（FRET）系统，以分析TAR RNA与分别用供体和受体染料分子独特标记的Tat蛋白片段（氨基酸38-72）之间的相互作用。使用这种FRET检测方法，我们测定了在生理条件下Tat（47-58）和Tat（38-72）与TAR RNA的结合亲和力。我们还描绘了Tat（38-72）的N端和C端之间的距离，以及当Tat（38-72）与TAR结合时两个末端与TAR 5′端之间的距离。我们的结果表明，当该肽折叠时，Tat（38-72）的N端和C端彼此靠近，并且该肽在TAR结合后不会经历大的结构变化。
