Three conserved guanosines approach the reaction site in native and minimal hammerhead ribozymes.
Three conserved guanosines approach the reaction site in native and minimal hammerhead ribozymes.
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三个保守的鸟苷接近天然和最小锤头核酶中的反应位点。
DOI：
10.1021/bi052457x
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Burke,JohnM
中科院分区：
文献类型：
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作者：
Lambert,Dominic;Heckman,JoyceE;Burke,JohnM
天然锤头状核酶含有在生理条件下能够实现高催化活性的RNA结构域，其中最小锤头状核酶显示出很小的活性。然而，鲜为人知的是，在本地与最小的核酶折叠的潜在差异。在这里，我们提出了光交联分析的本地和最小的锤头含有光反应性核碱基6-硫代鸟苷，2，6-二氨基嘌呤，4-硫代尿苷，和pyrrolocytidine，在特定的网站内的催化核心。在观察到催化活性的条件下，跨越切割位点的两个底物核碱基接近并堆叠在天然锤头的G8和G12上，这两个保守的核碱基在最小构建体中显示出相似的行为，涉及一般的酸碱催化，并且在晶体结构中距离切割位点>15 μ m。在切割位点位置17处的吡咯胞苷与G12形成有效的交联，并且交联的RNA保留催化活性。多个交联物质指向U形转弯内的结构重排，将残基G5定位在切割位点位置1.1附近。有趣的交联由交联残基远端位置的核苷酸类似物触发;例如，5位的6-硫代鸟苷诱导G12和C17之间的交联，表明这三个核碱基之间存在密切的功能通信。总之，这些结果支持了一个模型，其中的本地锤头折叠到一个活跃的结构类似的最小的核酶，并显着不同的晶体结构。
Native hammerhead ribozymes contain RNA domains that enable high catalytic activity under physiological conditions, where minimal hammerheads show little activity. However, little is known about potential differences in native versus minimal ribozyme folding. Here, we present results of photocross-linking analysis of native and minimal hammerheads containing photoreactive nucleobases 6-thioguanosine, 2,6-diaminopurine, 4-thiouridine, and pyrrolocytidine, introduced at specific sites within the catalytic core. Under conditions where catalytic activity is observed, the two substrate nucleobases spanning the cleavage site approach and stack upon G8 and G12 of the native hammerhead, two conserved nucleobases that show similar behavior in minimal constructs, have been implicated in general acid−base catalysis, and are >15 Å from the cleavage site in the crystal structures. Pyrrolocytidine at cleavage site position 17 forms an efficient crosslink to G12, and the crosslinked RNA retains catalytic activity. Multiple cross-linked species point to a structural rearrangement within the U-turn, positioning residue G5 in the vicinity of cleavage site position 1.1. Intriguing crosslinks were triggered by nucleotide analogues at positions distal to the crosslinked residues; for example, 6-thioguanosine at position 5 induced a crosslink between G12 and C17, suggesting an intimate functional communication among these three nucleobases. Together, these results support a model in which the native hammerhead folds to an active structure similar to that of the minimal ribozyme, and significantly different from the crystallographic structures.