Stability and cooperativity of individual tertiary contacts in RNA revealed through chemical denaturation

For proteins, understanding tertiary interactions involved in local versus global unfolding has become increasingly important for understanding the na ture of the native state ensemble, the mechanisms of unfolding, and the sta bility of both the native and intermediate states in folding. In this work we have addressed related questions with respect to RNA structure by combin ing chemical denaturation and hydroxyl radical footprinting methods. We hav e determined unfolding isotherms for each of 26 discrete sites of protectio n located throughout the Tetrahymena thermophila group I ribozyme, The coop erativity of folding, m-value, and the free energy, Delta G degrees(N-U), a ssociated with formation of each tertiary contact was determined by analysi s of the isotherms, The Delta G degrees(N-U) values measured in this study vary from 1.7 +/- 0.2 to 7.6 +/- 1.2 kcal mol(-1), Thus, the stability of t hese discrete tertiary contacts vary by almost 10(4) In addition, an intrad omain contact and three interdomain contacts show high cooperativity (m-val ues of 1.1 +/- 0.2 to 1.7 +/- 0.3 kcal mol(-1) M-1) indicating that these c ontacts exhibit global cooperatively in their folding behavior. This new ap proach to examining RNA stability provides an exciting comparison to our un derstanding of protein structure and folding mechanisms.