Domains 2 and 3 Interact to Form Critical Elements of the Group II Intron Active Site Export

@article{citeulike:5916208, abstract = {Group II introns are self-splicing RNA molecules that also behave as mobile genetic elements. The secondary structure of group II intron RNAs is typically described as a series of six domains that project from a central wheel. Most structural and mechanistic analyses of the intron have focused on domains 1 and 5, which contain the residues essential for catalysis, and on domain 6, which contains the branch-point adenosine. Domains 2 and 3 (D2, D3) have been shown to make important contributions to intronic activity; however, information about their function is quite limited. To elucidate the role of D2 and D3 in group II ribozyme catalysis, we built a series of multi-piece ribozyme constructs based on the ai5γ group II intron. These constructs are designed to shed light on the roles of D2 and D3 in some of the major reactions catalyzed by the intron: 5′-exon cleavage, branching, and substrate hydrolysis. Reactions with these constructs demonstrate that D3 stimulates the chemical rate constant of group II intron reactions, and that it behaves as a form of catalytic effector. However, D3 is unable to associate independently with the ribozyme core. Docking of D3 is mediated by a short duplex that is found at the base of D2. In addition to recruiting D3 into the core, the D2 stem directs the folding of the adjacent j 2/3 linker, which is among the most conserved elements in the group II intron active site. In turn, the D2 stem contributes to 5′-splice site docking and ribozyme conformational change. Nucleotide analog interference mapping suggests an interaction between the D2 stem and D3 that builds on the known θ–θ′ interaction and extends it into D3. These results establish that D3 and the base of D2 are key elements of the group II intron core and they suggest a hierarchy for active-site assembly.}, author = {Fedorova, Olga and Mitros, Therese and Pyle, Anna M.}, citeulike-article-id = {5916208}, citeulike-linkout-0 = {http://dx.doi.org/10.1016/S0022-2836(03)00594-1}, citeulike-linkout-1 = {http://linkinghub.elsevier.com/retrieve/pii/S0022283603005941}, day = {04}, doi = {10.1016/S0022-2836(03)00594-1}, issn = {00222836}, journal = {Journal of Molecular Biology}, keywords = {d2, d3, domain, groupii, intron, j23}, month = {July}, number = {2}, pages = {197--209}, posted-at = {2009-10-09 14:50:39}, priority = {2}, title = {Domains 2 and 3 Interact to Form Critical Elements of the Group II Intron Active Site}, url = {http://dx.doi.org/10.1016/S0022-2836(03)00594-1}, volume = {330}, year = {2003} }

TY - JOUR ID - citeulike:5916208 L3 - citeulike-article-id:5916208 N2 - Group II introns are self-splicing RNA molecules that also behave as mobile genetic elements. The secondary structure of group II intron RNAs is typically described as a series of six domains that project from a central wheel. Most structural and mechanistic analyses of the intron have focused on domains 1 and 5, which contain the residues essential for catalysis, and on domain 6, which contains the branch-point adenosine. Domains 2 and 3 (D2, D3) have been shown to make important contributions to intronic activity; however, information about their function is quite limited. To elucidate the role of D2 and D3 in group II ribozyme catalysis, we built a series of multi-piece ribozyme constructs based on the ai5γ group II intron. These constructs are designed to shed light on the roles of D2 and D3 in some of the major reactions catalyzed by the intron: 5′-exon cleavage, branching, and substrate hydrolysis. Reactions with these constructs demonstrate that D3 stimulates the chemical rate constant of group II intron reactions, and that it behaves as a form of catalytic effector. However, D3 is unable to associate independently with the ribozyme core. Docking of D3 is mediated by a short duplex that is found at the base of D2. In addition to recruiting D3 into the core, the D2 stem directs the folding of the adjacent j 2/3 linker, which is among the most conserved elements in the group II intron active site. In turn, the D2 stem contributes to 5′-splice site docking and ribozyme conformational change. Nucleotide analog interference mapping suggests an interaction between the D2 stem and D3 that builds on the known θ–θ′ interaction and extends it into D3. These results establish that D3 and the base of D2 are key elements of the group II intron core and they suggest a hierarchy for active-site assembly. IS - 2 JF - Journal of Molecular Biology SN - 00222836 EP - 209 TI - Domains 2 and 3 Interact to Form Critical Elements of the Group II Intron Active Site VL - 330 SP - 197 KW - d2 KW - d3 KW - domain KW - groupii KW - intron KW - j23 AU - Fedorova, Olga AU - Mitros, Therese AU - Pyle, Anna PY - 2003/07/04/ UR - http://dx.doi.org/10.1016/S0022-2836(03)00594-1 ER -