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Nucleic Acids Research, 1994, Vol. 22, No. 11 2003-2009
© 1994

RNA

Selection of novel forms of a functional domain within the Tetrahymena ribozyme

Kelly P. Williams+, Hiroshi Imahori§, Denise N. Fujimoto and Tan Inoue*

The Salk Institute for Biological Studies 10010 N. Torrey Pines Road, San Diego, CA 92037, USA

*To whom correspondence should be addressed at: Department of Chemistry, Kyoto 606, Japan

Received March 31, 1994. Revised April 26, 1994. Accepted April 26, 1994.

P5abc is an RNA structure within the self-splicing Tetrahymena group I intron that provides an activation function to the remainder of the ribozyme, either when present in cis or when added in trans. This 69-nucleotide activator domain was replaced with randomized sequence of 20 or 40 nt in length, and individuals among these pools with sequences that could functionally replace P5abc were selected. The basis of selection was a reaction in which two separate halves of the ribozyme became joined; selection was completed by reverse transcription and the polymerase chain reaction, using primers with sequence from either side of the ligation junction. Selectant sequences fell into three families that appear unrelated to P5abc; for example they lack the A-rich bulge thought to be a important feature of P5abc. Thus, rather than defining some consensus sequence for activator domains, this result reveals a certain tolerance in the ribozyme in its ability to derive activation function from diverse sequence types. In the context of splicing precursor RNA, the new sequences supported self-splicing, but failed to activate a related reaction, hydrolysis of the 3' splice site, implying that this region of the intron can differentially control two related reactions.

+Present addresses: Institute of Cell Biology, National Research Council, viale Marx 43, 00137 Rome, Italy

§The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567, Japan


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