New pathways in folding of the Tetrahymena group I RNA enzyme

JOURNAL OF MOLECULAR BIOLOGY

卷:291

Article

Russell, R ; Herschlag, D

关键词: group I intron;    ribozyme;    RNA folding;    Tetrahymena thermophila;   

DOI  :  10.1006/jmbi.1999.3026

来源: Elsevier

【 摘 要 】

Previous studies have shown that the earliest detectable step in folding of the Tetrahymena ribozyme is tertiary structure formation of the peripheral element P5abc. This, along with other results, has suggested that P5abc may serve as a scaffold upon which additional tertiary structure is built. Herein we use the onset of oligonucleotide cleavage activity as a readout for native state formation and investigate the effect of P5abc on the rate of folding to the native structure. Despite the early folding of P5abc, its removal to give the E-Delta P5abc variant decreases the rate of attainment of an active structure less than fivefold (20-100 mM Mg2+, 15-50 degrees C). Furthermore, P5abc added in trans is able to bind the folded E-Delta P5abc ribozyme and promote oligonucleotide cleavage at least tenfold more rapidly than folding of the wild-type ribozyme, indicating that E-Delta P5abc does not have to first unfold before productively binding P5abc to form the true native state. This suggests that a state with the overall tertiary structure formed but with P5abc unfolded represents a viable on-pathway intermediate for the wild-type ribozyme. These results provide strong evidence for the existence of two pathways to the native state: in one pathway P5abc forms tertiary structure first, and in another it forms late. The pathway in which P5abc forms first is favored because P5abc can fold quickly and because its tertiary structure is stable in the absence of additional structured elements, not because P5abc formation is required for subsequent folding steps. In the course of these experiments, we also found that most of the ribozyme population does not reach the native state directly under standard conditions in vitro, but instead forms an inactive structure that is stable for hours. Finally, the fraction that does fold to the native state folds with a single rate constant of 1 min(-1), suggesting that there are no significantly populated fast-track pathways that reach the native state directly by avoiding slow folding steps. (C) 1999 Academic Press.

【 授权许可】

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