Chadalavada Durga M, Senchak Susan E, Bevilacqua Philip C
Department of Chemistry, The Pennsylvania State University, Pennsylvania, PA 16802, USA.
J Mol Biol. 2002 Apr 5;317(4):559-75. doi: 10.1006/jmbi.2002.5434.
Hepatitis delta virus (HDV) replicates by a double rolling-circle mechanism that requires self-cleavage by closely related genomic and antigenomic versions of a ribozyme. We have previously shown that the uncleaved genomic ribozyme is subject to a variety of alternative (Alt) pairings. Sequence upstream of the ribozyme can regulate self-cleavage activity by formation of an Alt 1 ribozyme-containing structure that severely inhibits self-cleavage, or a P(-1) self-structure that permits rapid self-cleavage. Here, we test three other alternative pairings: Alt P1, Alt 2, and Alt 3. Alt P1 and Alt 3 contain primarily ribozyme-ribozyme interactions, while Alt 2 involves ribozyme-flanking sequence interaction. A number of single and double mutant ribozymes were prepared to increase or decrease the stability of the alternative pairings, and rates of self-cleavage were determined. Results of these experiments were consistent with the existence of the proposed alternative pairings and their ability to inhibit the overall rate of native ribozyme folding. Local misfolds are treated as internal equilibrium constants in a binding polynomial that modulates the intrinsic rate of cleavage. This model of equilibrium effects of misfolds should be general and apply to other ribozymes. All of the alternative pairings sequester a pseudoknot-forming strand. Folding of ribozymes containing Alt P1 and Alt 2 was accelerated by urea as long as the native ribozyme fold was sufficiently stable, while folding of mutants in which both of these alternative pairings had been removed were not stimulated by urea. This behavior suggests that the pseudoknots form by capture of an unfolded or appropriately rearranged alternative pairing by its complementary native strand. Fast-folding mutants were prepared by either weakening alternative pairings or by strengthening native pairings. A kinetic model was developed that accommodates these features and explains the position of the rate-limiting step for the G11C mutant. Implications of these results for structural and dynamic studies of the uncleaved HDV ribozyme are discussed.
丁型肝炎病毒（HDV）通过一种双滚环机制进行复制，该机制需要一种核酶的紧密相关的基因组和反基因组版本进行自我切割。我们之前已经表明，未切割的基因组核酶会经历多种替代（Alt）配对。核酶上游的序列可以通过形成严重抑制自我切割的含Alt 1核酶结构或允许快速自我切割的P（-1）自我结构来调节自我切割活性。在这里，我们测试了其他三种替代配对：Alt P1、Alt 2和Alt 3。Alt P1和Alt 3主要包含核酶-核酶相互作用，而Alt 2涉及核酶侧翼序列相互作用。制备了许多单突变和双突变核酶以增加或降低替代配对的稳定性，并测定了自我切割速率。这些实验结果与所提出的替代配对的存在及其抑制天然核酶折叠总体速率的能力一致。局部错配在结合多项式中被视为内部平衡常数，该多项式调节内在切割速率。这种错配的平衡效应模型应该是通用的，并适用于其他核酶。所有替代配对都隔离了一条形成假结的链。只要天然核酶折叠足够稳定，含有Alt P1和Alt 2的核酶的折叠就会被尿素加速，而去除了这两种替代配对的突变体的折叠则不会被尿素刺激。这种行为表明假结是通过其互补的天然链捕获未折叠或适当重排的替代配对而形成的。通过削弱替代配对或加强天然配对制备了快速折叠突变体。开发了一个动力学模型，该模型适应这些特征并解释了G11C突变体限速步骤的位置。讨论了这些结果对未切割的HDV核酶的结构和动力学研究的意义。
