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Title: Deletion of the P5abc Peripheral Element Accelerates Early and Late Folding Steps of the Tetrahymena Group I Ribozyme

Abstract

The P5abc peripheral element stabilizes the Tetrahymena group I ribozyme and enhances its catalytic activity. Despite its beneficial effects on the native structure, prior studies have shown that early formation of P5abc structure during folding can slow later folding steps. Here we use a P5abc deletion variant (E{sup {Delta}P5abc}) to systematically probe the role of P5abc throughout tertiary folding. Time-resolved hydroxyl radical footprinting shows that E{sup {Delta}P5abc} forms its earliest stable tertiary structure on the millisecond time scale, {approx}5-fold faster than the wild-type ribozyme, and stable structure spreads throughout E{sup {Delta}P5abc} in seconds. Nevertheless, activity measurements show that the earliest detectable formation of native E{sup {Delta}P5abc} ribozyme is much slower ({approx}0.6 min{sup -1}), in a manner similar to that of the wild type. Also similar, only a small fraction of E{sup {Delta}P5abc} attains the native state on this time scale under standard conditions at 25 {sup o}C, whereas the remainder misfolds; footprinting experiments show that the misfolded conformer shares structural features with the long-lived misfolded conformer of the wild-type ribozyme. Thus, P5abc does not have a large overall effect on the rate-limiting step(s) along this pathway. However, once misfolded, E{sup {Delta}P5abc} refolds to the native state 80-fold faster than themore » wild-type ribozyme and is less accelerated by urea, indicating that P5abc stabilizes the misfolded structure relative to the less-ordered transition state for refolding. Together, the results suggest that, under these conditions, even the earliest tertiary folding intermediates of the wild-type ribozyme represent misfolded species and that P5abc is principally a liability during the tertiary folding process.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930455
Report Number(s):
BNL-81207-2008-JA
Journal ID: ISSN 0006-2960; TRN: US200904%%726
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 46
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CATALYTIC EFFECTS; ENZYMES; HYDROXYL RADICALS; PROBES; PROTEIN STRUCTURE; RNA; TETRAHYMENA; UREA; national synchrotron light source

Citation Formats

Russell,R., Tijerina, P., Chadee, A., and Bhaskaran, H. Deletion of the P5abc Peripheral Element Accelerates Early and Late Folding Steps of the Tetrahymena Group I Ribozyme. United States: N. p., 2007. Web. doi:10.1021/bi0620149.
Russell,R., Tijerina, P., Chadee, A., & Bhaskaran, H. Deletion of the P5abc Peripheral Element Accelerates Early and Late Folding Steps of the Tetrahymena Group I Ribozyme. United States. doi:10.1021/bi0620149.
Russell,R., Tijerina, P., Chadee, A., and Bhaskaran, H. Mon . "Deletion of the P5abc Peripheral Element Accelerates Early and Late Folding Steps of the Tetrahymena Group I Ribozyme". United States. doi:10.1021/bi0620149.
@article{osti_930455,
title = {Deletion of the P5abc Peripheral Element Accelerates Early and Late Folding Steps of the Tetrahymena Group I Ribozyme},
author = {Russell,R. and Tijerina, P. and Chadee, A. and Bhaskaran, H.},
abstractNote = {The P5abc peripheral element stabilizes the Tetrahymena group I ribozyme and enhances its catalytic activity. Despite its beneficial effects on the native structure, prior studies have shown that early formation of P5abc structure during folding can slow later folding steps. Here we use a P5abc deletion variant (E{sup {Delta}P5abc}) to systematically probe the role of P5abc throughout tertiary folding. Time-resolved hydroxyl radical footprinting shows that E{sup {Delta}P5abc} forms its earliest stable tertiary structure on the millisecond time scale, {approx}5-fold faster than the wild-type ribozyme, and stable structure spreads throughout E{sup {Delta}P5abc} in seconds. Nevertheless, activity measurements show that the earliest detectable formation of native E{sup {Delta}P5abc} ribozyme is much slower ({approx}0.6 min{sup -1}), in a manner similar to that of the wild type. Also similar, only a small fraction of E{sup {Delta}P5abc} attains the native state on this time scale under standard conditions at 25 {sup o}C, whereas the remainder misfolds; footprinting experiments show that the misfolded conformer shares structural features with the long-lived misfolded conformer of the wild-type ribozyme. Thus, P5abc does not have a large overall effect on the rate-limiting step(s) along this pathway. However, once misfolded, E{sup {Delta}P5abc} refolds to the native state 80-fold faster than the wild-type ribozyme and is less accelerated by urea, indicating that P5abc stabilizes the misfolded structure relative to the less-ordered transition state for refolding. Together, the results suggest that, under these conditions, even the earliest tertiary folding intermediates of the wild-type ribozyme represent misfolded species and that P5abc is principally a liability during the tertiary folding process.},
doi = {10.1021/bi0620149},
journal = {Biochemistry},
number = ,
volume = 46,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}