Patel P H, Jacobo-Molina A, Ding J, Tantillo C, Clark A D, Raag R, Nanni R G, Hughes S H, Arnold E
Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854-5638, USA.
Biochemistry. 1995 Apr 25;34(16):5351-63. doi: 10.1021/bi00016a006.
When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemical mechanism of DNA polymerization with the structure of HIV-1 RT complexed with a dsDNA template-primer. The first step of polymerization involves the physical association of a polymerase with its nucleic acid substrate. A comparison of the structures of HIV-1 RT in the presence and absence of DNA indicates that the tip of the p66 thumb moves approximately 30 A upon DNA binding. This conformational change permits numerous interactions between residues of alpha-helices H and I in the thumb subdomain and the DNA. Measurements of DNA binding affinity for nucleic acids with double-stranded DNAs that have an increasing number of bases in the template overhang and molecular modeling suggest that portions of beta 3 and beta 4 within the fingers subdomain bind single-stranded regions of the template. Measurements of nucleotide incorporation efficiency (kcat/Km) show that the binding and incorporation of the next complementary nucleotide are not dependent on the length of the template overhang. Molecular modeling of an incoming nucleotide triphosphate (dTTP), based in part on the position of mercury atoms in a RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta 10 determine the topology of the dNTP-binding site. These results also suggest that nucleotide incorporation is accompanied by a protein conformational change that positions the dNTP for nucleophilic attack. Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer strand could be metal-mediated, and Asp185 may be directly involved in stabilizing the transition state. The translocation step may be characterized by rotational as well as translational motions of HIV-1 RT relative to the DNA double helix. Some of the energy required for translocation could be provided by dNTP hydrolysis and could be coupled with conformational changes within the nucleic acid. A structural comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase identified regions within T7 RNA polymerase which are not present in the other two polymerases that might help this polymerase to remain bound with nucleic acids and contribute to the ability of the T7 RNA polymerase to polymerize processively.
当HIV-1的单链RNA基因组被复制成双链DNA时，病毒酶逆转录酶（RT）催化添加约20,000个核苷酸；然而，核苷酸添加的确切机制尚不清楚。在本研究中，我们试图将DNA聚合的遗传数据和生化机制与结合有dsDNA模板引物的HIV-1 RT结构整合起来。聚合的第一步涉及聚合酶与其核酸底物的物理结合。对存在和不存在DNA时HIV-1 RT结构的比较表明，p66拇指的尖端在DNA结合时移动约30埃。这种构象变化允许拇指亚结构域中α-螺旋H和I的残基与DNA之间发生大量相互作用。对模板突出端具有越来越多碱基的双链DNA与核酸的DNA结合亲和力的测量以及分子建模表明，手指亚结构域内的β3和β4部分结合模板的单链区域。核苷酸掺入效率（kcat/Km）的测量表明，下一个互补核苷酸的结合和掺入不依赖于模板突出端的长度。基于RT/DNA/Hg-UTP/Fab结构中汞原子的位置，对进入的三磷酸核苷酸（dTTP）进行的分子建模表明，二级结构元件αC-β6、αE、β11b和β9-β10的部分决定了dNTP结合位点的拓扑结构。这些结果还表明，核苷酸掺入伴随着蛋白质构象变化，该变化将dNTP定位用于亲核攻击。引物链3'-OH基团的氧原子进行的亲核攻击可能是金属介导的，并且Asp185可能直接参与稳定过渡态。转位步骤的特征可能是HIV-1 RT相对于DNA双螺旋的旋转和平移运动。转位所需的一些能量可以由dNTP水解提供，并且可以与核酸内的构象变化偶联。HIV-1 RT、Klenow片段和T7 RNA聚合酶的结构比较确定了T7 RNA聚合酶中不存在于其他两种聚合酶中的区域，这些区域可能有助于该聚合酶与核酸保持结合，并有助于T7 RNA聚合酶进行连续聚合的能力。
