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

Journal Article · Mon Jan 01 04:00:00 EST 2007 · Biochemistry

DOI:https://doi.org/10.1021/bi0620149· OSTI ID:930455

Russell, R; Tijerina, P; Chadee, A; Bhaskaran, H

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.

• Research Organization: Brookhaven National Laboratory (BNL) National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: AC02-98CH10886
• OSTI ID: 930455
• Report Number(s): BNL--81207-2008-JA
• Journal Information: Biochemistry, Journal Name: Biochemistry Vol. 46; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CATALYTIC EFFECTS
ENZYMES
HYDROXYL RADICALS
PROBES
PROTEIN STRUCTURE
RNA
TETRAHYMENA
UREA
national synchrotron light source