Protein-facilitated folding of group II intron ribozymes.
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 dimethyl sulfate 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 nonspecifically, most binding events do not result in the formation of the compact state, and ATP is required for the protein to dissociate from such nonproductive 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. Dimethyl sulfate 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.
Howard Hughes Medical Institute, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
Olga
O
Fedorova
Amanda
A
Solem
Anna Marie
AM
Pyle
D000255:Adenosine Triphosphate / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007438:Introns / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017510:Protein Folding; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044563:RNA, Ribosomal, Self-Splicing / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D016337:RNA, Catalytic; D012331:RNA, Fungal; D044563:RNA, Ribosomal, Self-Splicing; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C058150:MSS116 protein, S cerevisiae; D053487:DEAD-box RNA Helicases