Identity elements for N2-dimethylation of guanosine-26 in yeast tRNAs

N2,N2-dimethylguanosine (m2(2)G) is a characteristic nucleoside that is found in the bend between the dihydro-uridine (D) stem and the anticodon (AC) stem in over 80% of the eukaryotic tRNA species having guanosine at position 26 (G26). However, since a few eukaryotic tRNAs have an unmodified G in that position, G26 is a necessary but not a sufficient condition for dimethylation. In yeast tRNA(Asp) G26 is unmodified. We have successively changed the near surroundings of G26 in this tRNA until G26 became modified to m2(2)G by a tRNA(m2(2)G26)methyltransferase in Xenopus laevis oocytes. In this way we have identified the two D-stem basepairs C11-G24, G10-C25 immediately preceding G26 as major identity elements for the dimethylating enzyme modifying G26. Furthermore, increasing the extra loop in tRNA(Asp) from four to the more usual five bases influenced the global structure of the tRNA such that the m2(2)G26 formation was drastically decreased even if the near region of G26 had the two consensus basepairs. We conclude that not only are the two consensus base pairs in the D-stem a prerequisite for G26 modification, but also is any part of the tRNA molecule that influence the 3D-structure important for the recognition between nuclear coded tRNAs and the tRNA(m2(2)G26)methyltransferase.