Formation of a GNRA tetraloop in P5abc can disrupt an interdomain interaction in the Tetrahymena group I ribozyme

Abstract

The secondary structure of a truncated P5abc subdomain (tP5abc, a 56-nucleotide RNA) of the Tetrahymena thermophila group I intron ribozyme changes when its tertiary structure forms. We have now used heteronuclear NMR spectroscopy to determine its conformation in solution. The tP5abc RNA that contains only secondary structure is extended compared with the tertiary folded form; both forms coexist in slow chemical exchange (the interconversion rate constant is slower than 1 s ؊1 ) in the presence of magnesium. Kinetic experiments have shown that tertiary folding of the P5abc subdomain is one of the earliest folding transitions in the group I intron ribozyme, and that it leads to a metastable misfolded intermediate. Previous mutagenesis studies suggest that formation of the extended P5abc structure described here destabilize a misfolded intermediate. This study shows that the P5abc RNA subdomain containing a GNRA tetraloop in P5c (in contrast to the five-nucleotide loop P5c in the tertiary folded ribozyme) can disrupt the base-paired interdomain (P14) interaction between P5c and P2.

Predicting RNA folding thermodynamics with a reduced chain representation model

“…Both our base-pairing probability analysis and the structural prediction algorithm predict the same native structure, and the predicted structure is in good agreement with the NMR experiment (Wu and Tinoco 1998;Zheng et al 2001). We label the three loops in the native structure as L5a, L5b, and L5c, respectively.…”

“…The structural details of the truncated P5abc subdomain (tP5abc) both with and without Mg 2+ have been fully investigated by NMR experiments (Thirumalai 1998;Wu and Tinoco 1998;Zheng et al 2001), which shows the tertiary interactions between the unpaired bases of the secondary structure. Furthermore, nondenaturing gel electrophoresis and NMR spectroscopy show that single point mutations can disrupt the tertiary interaction of the tP5abc subdomain (Silverman et al 1999), a truncated P5abc subdomain.…”

Metal-ion rescue revisited: Biochemical detection of site-bound metal ions important for RNA folding

“…In Mn 2+ , we observed stabilization and accumulation of a folding intermediate corresponding to the P5abc subdomain of P4-P6 (I P5abc ), a structure that contains the two metal-ion binding sites identified above and has previously been shown to fold independently (Murphy and Cech 1993;Wu and Tinoco 1998;Zheng et al 2001). The presence of two different folding pathways subverts simple thermodynamic analysis of the rescue experiments and was a key element limiting the prior data interpretation.…”

“…1C, right). These metal ions bind within the P5abc subdomain, which can undergo divalent metal-ion-dependent folding in isolation (Murphy and Cech 1993;Wu and Tinoco 1998;Zheng et al 2001). Several other metal ions are observed in P4-P6 crystal structures, but are not addressed herein.…”

“…6B, right; Table 3B). The residues that are protected at the lower Mn 2+ concentration (closed squares) correspond to a previously identified folding subdomain, P5abc, which can adopt structure independent of the overall P4-P6 structure (Murphy and Cech 1993;Wu and Tinoco 1998;Zheng et al 2001). Thus, the first protections that appear with increasing Mn 2+ concentration correspond to a previously unseen U-to-I P5abc transition.…”