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首页> 外文期刊>Biochemistry >Kinetic and Thermodynamic Framework for Assembly of the Six-Component bI3 Group I Intron Ribonucleoprotein Catalyst.
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Kinetic and Thermodynamic Framework for Assembly of the Six-Component bI3 Group I Intron Ribonucleoprotein Catalyst.

机译：六组分bI3 I组内含子核糖核蛋白催化剂的动力学和热力学框架。

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摘要

The yeast mitochondrial bI3 group I intron RNA splices in vitro as a six-component ribonucleoprotein complex with the bI3 maturase and Mrs1 proteins. We report a comprehensive framework for assembly of the catalytically active bI3 ribonucleoprotein. (1) In the absence of Mg(2+), two Mrs1 dimers bind independently to the bI3 RNA. The ratio of dissociation to association rate constants, k(off)/k(on), is approximately equal to the observed equilibrium K(1/2) of 0.12 nM. (2) At magnesium ion concentrations optimal for splicing (20 mM), two Mrs1 dimers bind with strong cooperativity to the bI3 RNA. k(off)/k(on) is 15-fold lower than the observed K(1/2) of 11 nM, which reflects formation of an obligate intermediate involving one Mrs1 dimer and the RNA in cooperative assembly of the Mrs1-RNA complex. (3) The bI3 maturase monomer binds to the bI3 RNA at almost the diffusion-controlled limit and dissociates with a half-life of 1 h. k(off)/k(on) is approximately equal to the equilibrium K(D) of 2.8 pM. The bI3 maturase thus represents a rare example of a group I intron protein cofactor whose binding is adequately characterized by a one-step mechanism under conditions that promote splicing. (4) Maturase and Mrs1 proteins each bind the bI3 RNA tightly, but with only modest coupling ( approximately 1 kcal/mol), suggesting that the proteins interact at independent RNA binding sites. Maturase binding functions to slow dissociation of Mrs1; whereas prior Mrs1 binding increases the bI3 maturase k(on) right to the diffusion limit. (5) At effective concentrations plausibly present in yeast mitochondria, a predominant assembly pathway emerges involving rapid, tight binding by the bI3 maturase, followed by slower, cooperative assembly of two Mrs1 dimers. In the absence of other factors, disassembly of all protein subunits will occur in a single apparent step, governed by dissociation of the bI3 maturase.
机译：酵母线粒体bI3组I内含子RNA在体外剪接成六组分核糖核蛋白复合物，并带有bI3成熟酶和Mrs1蛋白。我们报告了催化活性的bI3核糖核蛋白组装的全面框架。 （1）在缺少Mg（2+）的情况下，两个Mrs1二聚体独立结合到bI3 RNA。解离与缔合速率常数的比率k（off）/ k（on）大约等于所观察到的平衡K（1/2）为0.12 nM。 （2）在最适合剪接的镁离子浓度（20 mM）下，两个Mrs1二聚体与bI3 RNA具有很强的协同作用。 k（off）/ k（on）比所观察到的11nM的K（1/2）低15倍，这反映了涉及一个Mrs1二聚体和RNA的专性中间体的形成，Mrs1-RNA复合体协同组装。 （3）bI3成熟酶单体几乎以扩散控制的极限与bI3 RNA结合，并以1 h的半衰期解离。 k（off）/ k（on）大约等于2.8 pM的平衡K（D）。因此，bI3成熟酶代表了I类内含子蛋白辅因子的稀有实例，其结合在促进剪接的条件下通过一步机制被充分表征。 （4）成熟酶和Mrs1蛋白各自紧密结合bI3 RNA，但仅有适度的偶联作用（约1 kcal / mol），表明这些蛋白在独立的RNA结合位点相互作用。成熟酶结合功能可减慢Mrs1的解离;而先前的Mrs1绑定将bI3成熟酶k（on）增加到扩散极限。 （5）在酵母线粒体中可能存在的有效浓度下，主要的组装途径出现，涉及bI3成熟酶的快速紧密结合，随后是两个Mrs1二聚体的缓慢，协同组装。在没有其他因素的情况下，所有蛋白质亚基的分解都将在一个明显的步骤中发生，这取决于bI3成熟酶的解离。

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