Structural requirements for tRNA methylation. Action of Escherichia coli tRNA(guanosine-1)methyltransferase on tRNA(1Leu) structural variants.
The Escherichia coli enzyme tRNA(m1G)methyltransferase, one of a group of post-transcription tRNA-modifying enzymes, shows remarkable specificity in selecting the tRNA species and the specific guanosine base to be methylated. To examine the structural basis of this specificity, we synthesized a total of 15 modifications of tRNA(1Leu) and measured their methylation reaction kinetics in vitro. Elimination of any one of the three tRNA side loops, the V loop, the T loop, or the D loop, reduced the Vmax for methylation by about 1 order of magnitude. Elimination of all three side loops reduced Vmax by about 2 orders of magnitude. Clearly, gross tRNA structure is important for full enzyme activity. At the bottom of the stem proximal to the anticodon loop, in the pair at positions 31-39, substitution of a G-C for a C-G, a change that should not weaken the helical structure, had little effect on Vmax or Km. However, substitution of a G for a C increased Vmax and Km, whereas substitution of a C for G sharply reduced Vmax and, to a lesser extent, Km. These results appear to be a consequence of the principle that purines are better than pyrimidines in the stacking of adjacent bases for stability. Stacking in the stem structure appears to be important for methylation enzyme activity. In the anticodon loop itself, changing a U to a C had little effect, but changing the G of the anticodon to a C reduced Vmax over 20-fold, demonstrating the importance of the presence of the anticodon G adjacent to the G being methylated for enzyme recognition.
Holmes
W M
WM
Department of Microbiology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0678.
Andraos-Selim
C
C
Roberts
I
I
Wahab
S Z
SZ
D000926:Anticodon; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D012356:RNA, Transfer, Leu / Q000737:chemistry / Q000378:metabolism*; D012359:tRNA Methyltransferases / Q000378:metabolism*
D000926:Anticodon; D012356:RNA, Transfer, Leu; D012343:RNA, Transfer; D012359:tRNA Methyltransferases