Total chemical synthesis of a ribozyme derived from a group I intron.
We describe the complete chemical synthesis of a ribozyme that catalyzes template-directed oligonucleotide ligation. The specific activity of the synthetic ribozyme is nearly identical to that of the same enzyme generated by in vitro transcription with T7 RNA polymerase. The ribozyme is derived from a group I intron and consists of three RNA fragments of 36, 43, and 59 nt that self-assemble to form a catalytically active complex. We have site-specifically substituted ribonucleotide analogs into this enzyme and have identified two 2'-hydroxyl groups that are required for full catalytic activity. In contrast, neither the 2'-hydroxyl nor the exocyclic amino group of the conserved guanosine in the guanosine binding site is necessary for catalysis. By allowing the ribozyme to be modified as easily as its substrates, this synthetic ribozyme system should be useful for testing specific hypotheses concerning ribozyme-substrate interactions and tertiary interactions within the ribozyme.
Whoriskey
S K
SK
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114, USA.
Usman
N
N
Szostak
J W
JW
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D012321:DNA-Directed RNA Polymerases; D007202:Indicators and Reagents; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis*; D016337:RNA, Catalytic / Q000138:chemical synthesis* / Q000378:metabolism; D013698:Templates, Genetic; D014764:Viral Proteins
D007202:Indicators and Reagents; D009843:Oligoribonucleotides; D016337:RNA, Catalytic; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases