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Protein-facilitated folding of group II intron ribozymes.
J Mol Biol. 2010 Feb 4;
Authors: Fedorova O, Solem A, Pyle AM
Multiple studies hypothesize that DEAD-box proteins facilitate folding of the ai5gamma group II intron. However, these conclusions are generally inferred from splicing kinetics, and not from direct monitoring of DEAD-box protein-facilitated folding of the intron. Using native gel electrophoresis and DMS structural probing, we monitored Mss 116-facilitated folding of ai5gamma intron ribozymes and a catalytically active self-splicing RNA containing full length intron and short exons. We found that the protein directly stimulates folding of these RNAs by accelerating formation of the compact near-native state. This process occurs in an ATP-independent manner, although, ATP is required for the protein turnover. As Mss 116 binds RNA non-specifically, most of binding events do not result in the formation of the compact state, and ATP is required for the protein to dissociate from such non-productive complexes and rebind the unfolded RNA. Results obtained from experiments at different concentrations of magnesium ions suggest that Mss 116 stimulates folding of ai5gamma ribozymes by promoting the formation of unstable folding intermediates, which is then followed by a cascade of folding events resulting in the formation of the compact near-native state. DMS probing results suggest that the compact state formed in the presence of the protein is identical to the near-native state formed more slowly in its absence. Our results also indicate that Mss 116 does not stabilize the native state of the ribozyme, but that such stabilization results from binding of attached exons.
PMID: 20138894 [PubMed - as supplied by publisher]