Nishikawa Fumiko, Shirai Miho, Nishikawa Satoshi
Functional Nucleic Acids Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
Eur J Biochem. 2002 Dec;269(23):5792-803. doi: 10.1046/j.1432-1033.2002.03280.x.
Human hepatitis delta (HDV) ribozyme is one of small ribozymes, such as hammerhead and hairpin ribozymes, etc. Its secondary structure shows pseudoknot structure composed of four stems (I to IV) and three single-stranded regions (SSrA, -B and -C). The 3D structure of 3'-cleaved product of genomic HDV ribozyme provided extensive information about tertiary hydrogen bonding interactions between nucleotide bases, phosphate oxygens and 2'OHs including new stem structure P1.1. To analyze the role of these hydrogen bond networks in the catalytic reaction, site-specific atomic-level modifications (such as deoxynucleotides, deoxyribosyl-2-aminopurine, deoxyribosylpurine, 7-deaza-ribonucleotide and inosine) were incorporated in the smallest trans-acting HDV ribozyme (47-mer). Kinetic analysis of these ribozyme variants demonstrated the importance of the two W-C base pairs of P1.1 for cleavage; in addition, the results suggest that all hydrogen bond interactions detected in the crystal structure involving 2'-OH and N7 atoms are present in the active ribozyme structure. In most of the variants, the relative reduction in kobs caused by substitution of the 2'-OH group correlated with the number of hydrogen bonds affected by the substitution. However G74 and C75 may have more than one hydrogen bond involving the 2'-OH in both the trans- and cis-acting HDV ribozyme. Moreover, in variants in which N7 was deleted, kobs was reduced 5- to 15-fold, it may suggest that N7 assists in coordinating Mg2+ ions or water molecules which bind with weak affinity in the active structure.
人丁型肝炎病毒（HDV）核酶是小型核酶之一，如锤头状核酶和发夹状核酶等。其二级结构呈现为由四个茎（I至IV）和三个单链区域（SSrA、-B和-C）组成的假结结构。基因组HDV核酶3'-切割产物的三维结构提供了有关核苷酸碱基、磷酸氧和2'-OH之间三级氢键相互作用的广泛信息，包括新的茎结构P1.1。为了分析这些氢键网络在催化反应中的作用，在最小的反式作用HDV核酶（47个核苷酸）中引入了位点特异性原子水平修饰（如脱氧核苷酸、脱氧核糖基-2-氨基嘌呤、脱氧核糖基嘌呤、7-脱氮核糖核苷酸和次黄苷）。对这些核酶变体的动力学分析表明，P1.1的两个沃森-克里克碱基对对于切割很重要；此外，结果表明，在晶体结构中检测到的所有涉及2'-OH和N7原子的氢键相互作用都存在于活性核酶结构中。在大多数变体中，由2'-OH基团取代引起的kobs相对降低与受取代影响的氢键数量相关。然而，在反式和顺式作用的HDV核酶中，G74和C75可能有不止一个涉及2'-OH的氢键。此外，在缺失N7的变体中，kobs降低了5至15倍，这可能表明N7有助于在活性结构中配位弱结合的Mg2+离子或水分子。
