Abstract：
:Group I intron RNAs contain a core of highly conserved helices flanked by peripheral domains that stabilize the core structure. In the Tetrahymena group I ribozyme, the P4, P5, and P6 helices of the core pack tightly against a three-helix subdomain called P5abc. Chemical footprinting and the crystal structure of the Tetrahymena intron P4-P6 domain revealed that tertiary interactions between these two parts of the domain create an extensive solvent-inaccessible interface. We have examined the formation and stability of this tertiary interface by providing the P5abc segment in trans to a Tetrahymena ribozyme construct that lacks P5abc (EDeltaP5abc). Equilibrium gel shift experiments show that the affinity of the P5abc and EDeltaP5abc RNAs is exceptionally strong, with a Kd of approximately 100 pM at 10 mM MgCl2 (at 37 degrees C). Chemical and enzymatic footprinting shows that the RNAs are substantially folded prior to assembly of the complex. Solvent accessibility mapping reveals that, in the absence of P5abc, the intron RNA maintains a nativelike fold but its active-site helices are not tightly packed. Upon binding of P5abc, the catalytic core becomes more tightly packed through indirect effects of the tertiary interface formation. This two-component system facilitates quantitative examination of individual tertiary contacts that stabilize the folded intron.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Doherty EA,Herschlag D,Doudna JAdoi
10.1021/bi982113psubject
Has Abstractpub_date
1999-03-09 00:00:00pages
2982-90issue
10eissn
0006-2960issn
1520-4995pii
bi982113pjournal_volume
38pub_type
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