tRNA recognition for modification: solution probing of tRNA complexed with Escherichia coli tRNA (guanosine-1) methyltransferase.
The interaction of Escherichia coli tRNA (guanosine-1) methyltransferase and tRNA(1Leu) transcripts has been probed using cleavage with iodine of phosphorothioate-substituted transcripts, lead acetate, and enzymes specific for single- and double-stranded RNA. All lytic agents protect the anticodon stem-loop and variable loop regions against cleavage, and some protection is also seen in core structures of the tRNA. Residues from both strands of the anticodon stem are protected against cleavage with iodine and lead by enzyme, yet positions G37 and G36, which are crucial for catalysis and binding, are not. This suggests that these residues may undergo structural perturbation in the presence of S-adenosyl methionine. Occupancy of the AdoMet site by the product S-adenosyl-homocysteine, a potent inhibitor of the enzyme, has little or no effect on tRNA binding or protection. Enhanced reactivity with lead is seen at residues located in the anticodon stem-loop, extra-loop, and core (C34, U47c, and G49), which suggests some perturbations in RNA structure might accompany binding.
Gabryszuk
J
J
Department of Microbiology/Immunology, and The Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond 23298, USA.
Holmes
W M
WM
D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007202:Indicators and Reagents; D007455:Iodine / Q000737:chemistry; D007854:Lead / Q000737:chemistry; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012356:RNA, Transfer, Leu / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012996:Solutions; D012359:tRNA Methyltransferases / Q000378:metabolism*
D007202:Indicators and Reagents; D015335:Molecular Probes; D012356:RNA, Transfer, Leu; D016601:RNA-Binding Proteins; D012996:Solutions; D007854:Lead; D007455:Iodine; D012359:tRNA Methyltransferases; C036950:tRNA (guanine-N1-)-methyltransferase