Ampt Kirsten A M, van der Werf Ramon M, Nelissen Frank H T, Tessari Marco, Wijmenga Sybren S
Biophysical Chemistry, Institute of Molecules and Materials, Radboud University of Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
Biochemistry. 2009 Nov 10;48(44):10499-508. doi: 10.1021/bi9011385.
Hepatitis B virus (HBV) replication starts with binding of reverse transcriptase (RT) to the apical stem-loop region of epsilon, a conserved element of the RNA pregenome. For duck HBV, an in vitro replication system has provided molecular details of this interaction. Further insights can be obtained from the structure and dynamics of the duck and human apical stem-loops. Previously, we reported these for the human apical stem-loop. Here, we present the same for the duck counterpart. Unlike its human counterpart, the duck apical stem is unstable in its middle/upper part and contains noncanonical base pairs. This dynamics study is the first of an unstable RNA-DNA stem. Similar to the human stem, the duck apical stem comprises two helical segments with a bend angle of ca. 10 degrees , separated by a nonpaired mobile U residue. It is capped by a well-structured conserved UGUU loop with two residues mobile on the pico- to nanosecond time scale, one of which is involved in RT binding. Remarkably, the unstable middle/upper part of the stem does not show enhanced pico- to nanosecond time scale dynamics. Instead, adenine dispersion relaxation studies indicate enhanced millisecond time scale dynamics involving base pair opening. It can then be concluded that base pair opening is essential for epsilon-RT binding, because stabilization of the stem abolishes binding. We hypothesize that binding occurs by conformational capture of bases in the base pair open state. The unstable secondary structure of the apical stem-loop makes duck epsilon-RT binding unusual in light of recent classifications of RNA target interactions that assume stable secondary structures.
乙型肝炎病毒（HBV）的复制始于逆转录酶（RT）与ε的顶端茎环区域结合，ε是RNA前基因组的一个保守元件。对于鸭乙型肝炎病毒，体外复制系统提供了这种相互作用的分子细节。从鸭和人类顶端茎环的结构和动力学中可以获得进一步的见解。此前，我们报道了人类顶端茎环的相关情况。在此，我们展示鸭类对应物的相同情况。与人类对应物不同，鸭顶端茎在其中部/上部不稳定，且包含非经典碱基对。这项动力学研究是对不稳定RNA-DNA茎的首次研究。与人类茎相似，鸭顶端茎由两个螺旋段组成，弯曲角度约为10度，由一个未配对的可移动U残基隔开。它由一个结构良好的保守UGUU环封端，其中两个残基在皮秒到纳秒时间尺度上可移动，其中一个参与RT结合。值得注意的是，茎的不稳定中部/上部在皮秒到纳秒时间尺度上并未表现出增强的动力学。相反，腺嘌呤色散弛豫研究表明在毫秒时间尺度上存在涉及碱基对打开的增强动力学。由此可以得出结论，碱基对打开对于ε-RT结合至关重要，因为茎的稳定会消除结合。我们假设结合是通过在碱基对开放状态下对碱基的构象捕获发生的。鉴于最近对假设稳定二级结构的RNA靶标相互作用的分类，顶端茎环的不稳定二级结构使得鸭ε-RT结合与众不同。
