The role of 5-methylcytidine in the anticodon arm of yeast tRNA(Phe) site-specific Mg2+ binding and coupled conformational transition in DNA analogs

The tDNA(PheAC), d(CCAGACTGAAGAU13m(5)C14U15GG), with a DNA sequence similar to that of the anticodon stem and loop of yeast tRNA(Phe), forms a stem and loop structure and has an Mg(2+) induced structural transition that was not exhibited by an unmodified tDNA(PheAC)d(T13C14T15) [Guenther, R. H., Hardin, C. C., Sierzputowska-Gracz, H., Dao, V., and Agris, P. F. (1992) Biochemistry (preceding paper in this issue)]. Three tDNA(PheAC) molecules having m(5)C14,tDNA(PheAC)d(Ul3m(5)Cl4U15),d(Ul3m(5)Cl4T15), and D(T13m(5)C14UI5), also exhibited Mg(2+)-induced structural transitions and biphasic thermal transitions (Tm approximately equal to 23.5 and 52 degrees C), as monitored by CD and UV spectroscopy. Three other tDNA(PheAC),d(T13C14T15), d(U13C14U15), and d(A7;U13m(5)C14U15) in which T7 was replaced with an A, thereby negating the T7.A10 base pair across the anticodon loop, had no Mg(2+) -induced structural transitions and only monophasic thermal transitions (Tm of approximately equal to -52 degrees C). The tDNA(PheAC), d(U13m(5)C14U15) had a single, strong Mg(2+) binding site with a Kd of 1.09 x 10-6 M and a AG of -7.75 kcal/mol associated with the Mg(2+) -induced structural transition. In thermal denaturation of tDNA(PheAC), d(U13m(5)C14U15), the 1H NMR signal assigned to the imino proton of the A5.dU13 base pair at the bottom of the anticodon stem could no longer be detected at a temperature corresponding to that of the loss of the Mg(2+)-induced conformation from the CD spectrum. Therefore, we place the magnesium in the upper part of the tDNA hairpin loop near the A5.dU13 base pair, a location similar to that in the X-ray crystal structure of native, yeast tRNA(Phe). We conclude that m(5)C in the anticodon stems of the tDNA analog, and of tRNA, facilitates a site-specific Mg(2+) binding without 2'-OHs and a conformational change in the anticodon loop, which for tRNA has important functional implications