Zacharias M, Hagerman P J
Department of Biochemistry, Biophysics, and Genetics, University of Colorado Health Sciences Center, Denver 80262, USA.
Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6052-6. doi: 10.1073/pnas.92.13.6052.
The trans-activation response element (TAR) found near the 5' end of the viral RNA of the human immunodeficiency virus contains a 3-nt bulge that is recognized by the virally encoded trans-activator protein (Tat), an important mediator of transcriptional activation. Insertion of the TAR bulge into double-stranded RNA is known to result in reduced electrophoretic mobility, suggestive of a bulge-induced bend. Furthermore, NMR studies indicate that Arg causes a change in the structure of the TAR bulge, possibly reducing the bulge angle. However, neither of these effects has been quantified, nor have they been compared with the effects of the TAR-Tat interaction. Recently, an approach for the quantification of bulge-induced bends has been described in which hydrodynamic measurements, employing the method of transient electric birefringence, have yielded precise estimates for the angles of a series of RNA bulges, with the angles ranging from 7 degrees to 93 degrees. In the current study, transient electric birefringence measurements indicate that the TAR bulge introduces a bend of 50 degrees +/- 5 degrees in the absence of Mg2+. Addition of Arg leads to essentially complete straightening of the helix (to < 10 degrees) with a transition midpoint in the 1 mM range. This transition demonstrates specificity for the TAR bulge: no comparable transition was observed for U3 or A3 (control) bulges with differing flanking sequences. An essentially identical structural transition is observed for the Tat-derived peptide, although the transition midpoint for the latter is near 1 microM. Finally, low concentrations of Mg2+ alone reduce the bend angle by approximately 50%, consistent with the effects of Mg2+ on other pyrimidine bulges. This last observation is important in view of the fact that most previous structural/binding studies were performed in the absence of Mg2+.
在人类免疫缺陷病毒的病毒RNA 5'端附近发现的反式激活应答元件（TAR）含有一个3个核苷酸的凸起，该凸起可被病毒编码的反式激活蛋白（Tat）识别，Tat是转录激活的重要介导因子。已知将TAR凸起插入双链RNA会导致电泳迁移率降低，这表明存在凸起诱导的弯曲。此外，核磁共振研究表明，精氨酸会导致TAR凸起结构发生变化，可能会减小凸起角度。然而，这些效应均未被量化，也未与TAR-Tat相互作用的效应进行比较。最近，一种用于量化凸起诱导弯曲的方法被描述出来，其中利用瞬态电双折射方法进行的流体动力学测量，对一系列RNA凸起的角度进行了精确估计，角度范围从7度到93度。在当前研究中，瞬态电双折射测量表明，在没有Mg2+的情况下，TAR凸起会引入50度±5度的弯曲。添加精氨酸会导致螺旋基本完全伸直（至<10度），转变中点在1 mM范围内。这种转变显示出对TAR凸起的特异性：对于具有不同侧翼序列的U3或A3（对照）凸起，未观察到类似的转变。对于源自Tat的肽，观察到基本相同的结构转变，尽管后者的转变中点接近1 microM。最后，单独的低浓度Mg2+会使弯曲角度降低约50%，这与Mg2+对其他嘧啶凸起的影响一致。鉴于大多数先前的结构/结合研究是在没有Mg2+的情况下进行的，这一最后的观察结果很重要。
