Herschlag D, Khosla M, Tsuchihashi Z, Karpel R L
Department of Biochemistry, Hughes Medical Institute, Stanford University, CA 94305-5307.
EMBO J. 1994 Jun 15;13(12):2913-24. doi: 10.1002/j.1460-2075.1994.tb06586.x.
We have previously shown that a protein derived from the p7 nucleocapsid (NC) protein of HIV type-1 increases kcat/Km and kcat for cleavage of a cognate substrate by a hammerhead ribozyme. Here we show directly that the increase in kcat/Km arises from catalysis of the annealing of the RNA substrate to the ribozyme and the increase in kcat arises from catalysis of dissociation of the RNA products from the ribozyme. A peptide polymer derived from the consensus sequence of the C-terminal domain of the hnRNP A1 protein (A1 CTD) provides similar enhancements. Although these effects apparently arise from non-specific interactions, not all non-specific binding interactions led to these enhancements. NC and A1 CTD exert their effects by accelerating attainment of the thermodynamically most stable species throughout the ribozyme catalytic cycle. In addition, NC protein is shown to resolve a misfolded ribozyme-RNA complex that is otherwise long lived. These in vitro results suggest that non-specific RNA binding proteins such as NC and hnRNP proteins may have a biological role as RNA chaperones that prevent misfolding of RNAs and resolve RNAs that have misfolded, thereby ensuring that RNA is accessible for its biological functions.
我们之前已经表明，一种源自1型人类免疫缺陷病毒（HIV-1）p7核衣壳（NC）蛋白的蛋白质，可提高锤头状核酶切割同源底物的催化常数（kcat）与米氏常数（Km）的比值以及催化常数。在此，我们直接表明，kcat/Km的增加源于RNA底物与核酶退火过程的催化作用，而kcat的增加源于RNA产物从核酶解离过程的催化作用。一种源自异质性核糖核蛋白A1（hnRNP A1）蛋白C端结构域共有序列的肽聚合物也能提供类似的增强作用。尽管这些效应显然源自非特异性相互作用，但并非所有非特异性结合相互作用都会导致这些增强作用。NC和A1 CTD通过在整个核酶催化循环中加速达到热力学上最稳定的物种来发挥其作用。此外，NC蛋白还能解决一种错误折叠的核酶-RNA复合物，否则该复合物会长期存在。这些体外实验结果表明，诸如NC和hnRNP蛋白等非特异性RNA结合蛋白可能具有RNA伴侣的生物学作用，可防止RNA错误折叠并解决已错误折叠的RNA，从而确保RNA能够发挥其生物学功能。
