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Nucleic Acids Research, 1989, Vol. 17, No. 17 6969-6981
© 1989

MOLECULAR BIOLOGY

A Tetrahymena intron nucleotide connected to the GTP/arginine site

Michael Yarus*, Joseph Levine1,+, Gregg B. Morin1,§ and Thomas R. Cech1

Department of Molecular, Cellular and Development Biology, University of Colorado Boulder, CO 80307-0347 1Department of Chemistry and Biochemistry, University of Colorado Boulder, CO 90309-0215, USA

*To whom correspondence should be addressed

Received May 15, 1989. Revised June 23, 1989. Accepted July 25, 1989.

We have substituted all nucleotides at intron nucleotide 260 (N260) in transcripts related to the self-splicing Tetrahymena rRNA. Substitution slightly affects the binding and reaction of GTP with this group I catalytic center; kcat/Km varies over a three-fold range. The base of N260 therefore communicates with the rG site, but is unlikely to bond directly to GTP. Different nucleotides at this position also alter the binding of L-arginine to the intron, measured by inhibition of the reaction with GTP. Effects of similar small magnitude on interaction of RNA with both GTP and L-arginine support the previous argument from kinetic and structural comparison (Yarus, M. (1988) Science 240, 1751) that placed the two ligands of the RNA in the same binding site. G260 RNA shows the greatest affinity for both D- and L-arginine, but uniquely lacks stereoselectivity for the amino acid. Therefore G260 alters spatial relations within the G site, otherwise conserved in C260, U260, and A260 RNA's. Guanyl urea was used as a probe for the G/guanidino H-bonding part of the rG/arginine site. G260 RNA's dissociation constant for guanyl urea is similar to that of the other RNA's, suggesting that G260 RNA is unaltered at the G/guanidino end of the rG/arginine binding site. To account for all observations, we suggest that the G260 substitution alters the relative location of the RNA backbone near the 5' exon-intron junction, making this location more flexible and closer to the {alpha}-NH3+'s of L- and D-arginine.

Present address: +Somatogenetics, Inc., 350 Interlaken Parkway, Broomfield, CO 80020

§Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510, USA


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