Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis

Maximizing RNA folding rates: A balancing act

“…The well-studied Tetrahymena group I ribozyme can be divided into two principal domains of tertiary interactions and several peripheral helical regions (Fig+ 1; Kim & Cech, 1987;Michel & Westhof, 1990)+ The folded structure of the domain containing paired (P) regions P4, P5, and P6 is stabilized by tertiary interactions between a three-helix junction (P5abc) on one side and helices P4 and P6 on the other (Cate et al+, 1996)+ This domain is independently stable (Murphy & Cech, 1993) and folds at a rate of 1 s Ϫ1 at low ionic strength (Sclavi et al+, 1998)+ In contrast, the region containing P3, P7, and P9, which is much less stable and not able to fold independently (Celander & Cech, 1991;Doherty & Doudna, 1997), requires minutes or hours to form in vitro due to misfolding of nucleotides in P3 (Pan & Woodson, 1998)+ Thus, the formation of active ribozyme is limited by slow reorganization of residues in the P3-P9 domain, which make up half of the catalytic core (Zarrinkar & Williamson, 1994Sclavi et al+, 1998)+ Similarly, the tertiary structure of Bacillus subtilis RNase P ribozyme can be divided into two domains (Pan, 1995;Loria & Pan, 1996)+ The catalytic domain alone folds with a time constant of ;150 ms in vitro (Fang et al+, 1999)+ However, a second domain that is required for specific recognition of pre-tRNA substrates folds much more slowly and increases the folding time for the active wild-type ribozyme to ;2-4 min by populating metastable intermediates Pan & Sosnick, 1997)+…”

“…Relationship between folding times (t) and relative domain stabilities (⌬G 46 /⌬G 39 )+ On the right side of the graph, the minimum kinetic barrier ⌬G I ‡ is proportional to the stability of the P4-P6 domain, ⌬G 46 + This corresponds to the folding kinetics of the wildtype ribozyme+ On the left, the minimum barrier depends on the free energy for folding the P3-P9 domain, ⌬G 39 + The shortest folding times are predicted to occur when these trends converge (⌬G 46 / ⌬G 39 ; 1)+ The complete curve can be approximated by the sum of the terms in equation (3)+ Many group I ribozymes (and other RNAs) contain structural elaborations that reinforce the central fold (Michel & Westhof, 1990;Westhof et al+, 1996)+ These peripheral "stability elements," such as the P5abc threehelix junction, appear to lengthen the folding time of the Tetrahymena ribozyme Russell & Herschlag, 1999)+ This differs from the idea that a stable nugget of tertiary structure, which in this case is outside of the structural core, functions as a scaffold upon which the rest of the structure is built+ Interestingly, the stabilizing function of the P5abc region is replaced by the protein CYT-18 in group I introns from Neurospora mitochondria (Mohr et al+, 1992;Caprara et al+, 1996)+ This hints at the possibility that RNA-protein complexes offer the advantage of a more flexible assembly process, in which the RNA-RNA interactions remain fluid until they are solidified by binding of proteins (e+g+, see Buchmueller et al+, 2000)+…”

Contributions of 2'-hydroxyl groups of the RNA substrate to binding and catalysis by the Tetrahymena ribozyme. An energetic picture of an active site composed of RNA

“…in the -100 sequenced group I introns have three base pairs 5' of the cleavage site (Michel & Westhof, 1990). It will be important to determine whether these tertiary interactions are conserved throughout evolution.…”

Evidence of a tertiary interaction functional in group I 3′‐splicing

“…Once S-cleavage has taken place and concurrently, the S-extremity of the intron is no longer engaged in basepairing, PlO may form thereby inducing exon-exon ligation. This is compatible with the fact that a perturbed intron forced away from this splicing pathway becomes prone to 3'-hydrolysis [3]. Further experimental probes have been designed [1] by showing that PlO may be allowed to form prematurely (that is, prior to habilitation of the 3'-extremity of the 5'-exon as a nucleophilic agent) by selectively destabilizing competing interaction Pl.…”

“…This interaction has been inferred by phylogenetic analysis [3] and has only been confirmed experimentally for the fifth intron of the apocytochrome b gene (YCOBS) by means of site-directed mutagenesis and compensatory basepairing [2]. The interaction involves the S-extremity of the 3'-exon and a portion of the internal guide sequence (ES) (Fig.…”