Transfer RNA methyltransferase activity in Paramecium aurelia

334 Biochimica et Biophysica Acta, 425 (1976) 334--341

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98544 T R A N S F E R RNA M E T H Y L T R A N S F E R A S E ACTIVITY IN P A R A M E C I U M A U R E L I A

RICHARD A. MAKI, JERRY L. BROWN and DONALD J. CUMMINGS Department of Biochemistry and Microbiology, University of Colorado Medical Center, Denver, Colo. 80220 (U.S.A.) (Received September 29th, 1975)

Summary The t R N A methyltransferases from P a r a m e c i u m aurelia were investigated. The effects of varying the Mg :+ and NH4 ÷ concentrations, pH, and temperature on the methylation of Escherichia coli B tRNA using extracts from P. aurelia were determined. Optimum t R N A methyltransferase activity was observed at pH 7.8 and 37°C. The Mg 2÷ o p t i m u m occurred at 0.66 mM in the absence of NH4 ÷ while the NH4 ÷ o p t i m u m occurred at 100 mM in the absence of Mg 2÷. Analysis of the bases methylated in (E. coli B) tRNA by extracts of P. aurelia showed the presence of 1-methyladenine, 1-methylguanine, N2-methylguanine, N2~V2-dimethylguanine and methylated pyrimidine nucleotides. In comparison, an analysis of the in vivo methylation of tRNA from P. aurelia showed the presence of 1-methyladenine, 6-methyladenine, 6,6-dimethyladenine, 1-methylguanine, NZ-methylguanine, N2,N2-dimethylguanine, 7-methylguanine, and methylated pyrimidine nucleotides. The pattern of methylation of t R N A in P. aurelia is similar to that observed in other eukaryotes.

Introduction The methylation of bases in tRNA was first characterized in Escherichia coli [ 1 ]. Since then the examination of tRNAs from a large number of other biological systems has demonstrated the presence of a wide variety of modified bases including m e t h y l a t e d purines and pyrimidines [2,33]. The tRNAs from all species examined thus far have contained methylated bases. The identities and amounts of these modified nucleotides appear to be species specific. The various patterns of methylation of t R N A observed for different species probably reflect the amounts and specificities of the enzymes catalyzing the methylation reactions. In E. coli, for example, the average t R N A molecule contains about four methyl groups while in mammalian tRNAs there are 8--10 methyl groups

335 [3]. (E. coil) tRNA contains low levels of 1-methyladenine and few methylated guanines especially N 2 -methylguanine and N 2 ~ - d i m e t h y l g u a n i n e . In contrast, mammalian tRNAs contain substantial quantities of 1-methyladenine, N 2methylguanine and N 2~