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

Abstract

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.

5 S rRNA and tRNA Import into Human Mitochondria

“…Synthetic RNAs were excised from a 40-cm-long 12% denaturing gel permitting single base resolution, and were refolded by one cycle of heat denaturing-refolding in the presence of 0.5 mM Mg 2ϩ . We identified 5Ј-terminal nucleotides of the tranzymes r2 and r8 by P1 nuclease hydrolysis (39) with subsequent thin layer chromatography (40). To verify 3Ј-termini of synthetic tRNAs, we used aminoacylation tests.…”

The open reading frame TTC1157 of Thermus thermophilus HB27 encodes the methyltransferase forming N2-methylguanosine at position 6 in tRNA

“…The gene encoding this tRNA (m 2 2 G26) MTase was identified in lower as well as in higher Eukaryota (Ellis et al 1986;Reinhart et al 1986;Edqvist et al 1992Edqvist et al , 1994Liu et al 1999;Niederberger et al 1999;Liu and Straby 2000). In yeast, the TRM1 gene is responsible for m 2 2 G formation in both the cytoplasmic and mitochondrial tRNAs (Hopper et al 1982;Ellis et al 1986).…”

Effects of N2,N2-dimethylguanosine on RNA structure and stability: Crystal structure of an RNA duplex with tandem m22G:A pairs

“…It is hypothesized that methylation either serves the formation of a hydrophobic contact with protein or RNA or a structural purpose such as the prevention of base triples involving the minor groove (Lesnyak et al 2007;Sergiev et al 2007). The m 2 2 G nucleotide is found in the bend between the dihydro-uridine stem and the anticodon stem in the vast majority of eukaryotic tRNAs (Edqvist et al 1992;Grosjean and Benne 1998). At that location, m 2 2 G26 is paired with A44 and flanked by C27:G43 on one side and the m 2 G10-C25-G45 triple on the other.…”