Examination of the catalytic fitness of the hammerhead ribozyme by in vitro selection.
We have designed a self-cleaving ribozyme construct that is rendered inactive during preparative in vitro transcription by allosteric interactions with ATP. This allosteric ribozyme was constructed by joining a hammerhead domain to an ATP-binding RNA aptamer, thereby creating a ribozyme whose catalytic rate can be controlled by ATP. Upon purification by PAGE, the engineered ribozyme undergoes rapid self-cleavage when incubated in the absence of ATP. This strategy of "allosteric delay" was used to prepare intact hammerhead ribozymes that would otherwise self-destruct during transcription. Using a similar strategy, we have prepared a combinatorial pool of RNA in order to assess the catalytic fitness of ribozymes that carry the natural consensus sequence for the hammerhead. Using in vitro selection, this comprehensive RNA pool was screened for sequence variants of the hammerhead ribozyme that also display catalytic activity. We find that sequences that comprise the core of naturally occurring hammerhead dominate the population of selected RNAs, indicating that the natural consensus sequence of this ribozyme is optimal for catalytic function.
Tang
J
J
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.
Breaker
R R
RR
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012641:Selection, Genetic
D012322:RNA Precursors; D016337:RNA, Catalytic; D000255:Adenosine Triphosphate