title: A class I ligase ribozyme with reduced Mg2+ dependence: Selection, sequence analysis, and identification of functional tertiary interactions.
abstract: The class I ligase was among the first ribozymes to have been isolated from random sequences and represents the catalytic core of several RNA-directed RNA polymerase ribozymes. The ligase is also notable for its catalytic efficiency and structural complexity. Here, we report an improved version of this ribozyme, arising from selection that targeted the kinetics of the chemical step. Compared with the parent ribozyme, the improved ligase achieves a modest increase in rate enhancement under the selective conditions and shows a sharp reduction in [Mg(2+)] dependence. Analysis of the sequences and kinetics of successful clones suggests which mutations play the greatest part in these improvements. Moreover, backbone and nucleobase interference maps of the parent and improved ligase ribozymes complement the newly solved crystal structure of the improved ligase to identify the functionally significant interactions underlying the catalytic ability and structural complexity of the ligase ribozyme.
authors: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
authors: Sarah C
authors: SC
authors: Bagby
authors: Nicholas H
authors: NH
authors: Bergman
authors: David M
authors: DM
authors: Shechner
authors: Catherine
authors: C
authors: Yen
authors: David P
authors: DP
authors: Bartel
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D014644:Genetic Variation; D007700:Kinetics; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D054730:Protein Interaction Domains and Motifs; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D016337:RNA, Catalytic; D008025:Ligases; D008274:Magnesium