Many macromolecular folding reactions, such as the Mg2+-dependent folding of the Tetrahymena thermophila group I intron, occur on timescales ranging from milliseconds to minutes. Kinetic progress curves describing the millisecond folding of P4-P6 and other domains within the L-21 ribozyme have been obtained using a new ?OH ?footprinting? technique, stopped-flow synchrotron x-ray ?footprinting?. The folding of P4-P6 is a highly concerted reaction; the regions of ?OH protection within the interior of the folded domain appear at rates of ? 1.0 sec-1. ?OH protections within the P5c sub-domain appear at rates of ~ 2.0 sec-1, suggesting that folding of this subdomain is the initial step in the folding pathway. The rates of ?OH protection of the ?triple helix? junction between P4-P6 and the P3-P7 domain and those protections in P4-P6 ascribed to interaction with the P9 domain at rates of ~ 0.3 sec-1. These results suggest that these tertiary interactions guide the folding of the catalytic core against an extensively folded P4-P6 domain. To further dissect the folding mechanism of the Tetrahymena ribozyme, which we have solved in detail for a single set of conditions, the magnesium and temperature dependence of the folding mechanism will be explored in order to correlate the changes in energetics and structure that occur along the folding pathway.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001633-18
Application #
6345076
Study Section
Project Start
2000-09-01
Project End
2001-08-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
18
Fiscal Year
2000
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
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