Watts Joseph M, Gabruzsk J, Holmes Walter M
Department of Biochemistry, Medical College of Virginia campus of Virginia Commonwealth University, Institute for Structural Biology and Drug Discovery, 800 East Leigh Street, Suite 212, Richmond, Virginia 23219, USA.
Biochemistry. 2005 May 3;44(17):6629-39. doi: 10.1021/bi0481038.
Orthologs of TrmD, G37 tRNA methyltransferases, have been analyzed with regard to post-tRNA binding events required to move the residue G37 in proximity to bound AdoMet for catalysis. This was approached initially by probing tRNA with T2 nuclease or Pb acetate in the presence, then absence, of Escherichia coli TrmD protein. Cleavage patterns clearly show that portions of the anticodon loop phosphodiester backbone are protected from cleavage only in the presence of sinefungin, a potent AdoMet analogue. This demonstrates that there must be considerable movement of the loop region and/or protein as the AdoMet site is occupied. Florescence energy transfer experiments were employed to better assess the movement of the G37 and G36 base residues in response to occupancy of the AdoMet site. When the Streptococcus pneumoniae TrmD protein was bound to synthetic tRNA(1)(Leu) substituted with 2-aminopurine at positions 36 and 37, fluorescence energy transfer analysis showed that a decrease in 2-aminopurine fluorescence occurs only when AdoMet is present. Taken together, these results suggest that the base to be methylated by the TrmD protein is mobilized into the active center after tRNA binding only when the AdoMet site is occupied.
已对TrmD（G37 tRNA甲基转移酶）的直系同源物进行了分析，研究了在将G37残基移至与结合的腺苷甲硫氨酸（AdoMet）接近以进行催化所需的tRNA结合后事件。最初通过在存在和不存在大肠杆菌TrmD蛋白的情况下，用T2核酸酶或醋酸铅探测tRNA来进行研究。切割模式清楚地表明，只有在强效AdoMet类似物杀稻瘟菌素存在的情况下，反密码子环磷酸二酯主链的部分才受到保护不被切割。这表明当AdoMet位点被占据时，环区域和/或蛋白质必定有相当大的移动。采用荧光能量转移实验来更好地评估G37和G36碱基残基响应AdoMet位点占据的移动情况。当肺炎链球菌TrmD蛋白与在36和37位用2-氨基嘌呤取代的合成tRNA(1)(Leu)结合时，荧光能量转移分析表明，只有当AdoMet存在时，2-氨基嘌呤荧光才会降低。综上所述，这些结果表明，只有当AdoMet位点被占据时，TrmD蛋白要甲基化的碱基才会在tRNA结合后被转运到活性中心。
