Architecture of a Diels-Alderase ribozyme with a preformed catalytic pocket.
Artificial ribozymes catalyze a variety of chemical reactions. Their structures and reaction mechanisms are largely unknown. We have analyzed a ribozyme catalyzing Diels-Alder cycloaddition reactions by comprehensive mutation analysis and a variety of probing techniques. New tertiary interactions involving base pairs between nucleotides of the 5' terminus and a large internal loop forming a pseudoknot fold were identified. The probing data indicate a preformed tertiary structure that shows no major changes on substrate or product binding. Based on these observations, a molecular architecture featuring a Y-shaped arrangement is proposed. The tertiary structure is formed in a rather unusual way; that is, the opposite sides of the asymmetric internal loop are clamped by the four 5'-terminal nucleotides, forming two adjacent two base-pair helices. It is proposed that the catalytic pocket is formed by a wedge within one of these helices.
Keiper
Sonja
S
Universität Heidelberg, Institut für Pharmazie und Molekulare Biotechnologie, D-69120 Heidelberg, Germany.
Bebenroth
Dirk
D
Seelig
Burckhard
B
Westhof
Eric
E
Jäschke
Andres
A
D000873:Anthracenes / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007854:Lead / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013463:Sulfuric Acid Esters / Q000737:chemistry
D000873:Anthracenes; D008301:Maleimides; D016337:RNA, Catalytic; D013463:Sulfuric Acid Esters; C043592:maleimide; D007854:Lead; D012260:Ribonucleases; C034020:anthracene; C007482:dimethyl sulfate; D004047:Diethyl Pyrocarbonate