Mediated Sulfur-containing Peptide Antibiotic Resistance 23s Rrna Methyltransferase Biochemical Function,

The main object of this project is to study the biochemical properties of 23S rRNA methyltransferase, and discuss the mechanism of thiopeptin antibiotics resistance.Here we used following methods: a. Constructed the clones, expressed and purified TSR and NHR protein in vitrob. Transcripted RNA substrate in vitro via T7 polymerase system, include two kinds of RNA substrates:58 bp and 29 bpc. Set up in vitro detect system of RNA methyltransferase activity, measured the methylation extent of RNA substrate via liquid scintillation counter with existence of 14C-SAMd. Observed the binding between RNA substrate and TSR or NHR protein via Gel shifte. Measured the Kd value of bind between TSR or NHR protein and RNA substrates via AnisotropyWe obtained following results:a. Obtained TSR and NHR protein, RNA substrate expressed and purified in vitrob. both TSR and NHR can methylate RNA substrate, and NHR protein showed better activity; 58 bp RNA is better than 29 bp RNA as substratec. both TSR and NHR can bind with RNA substrate, and the binding intensity enhanced with the protein concentration.d. The Kd values of bind between TSR or NHR protein and RNA substrates are similar...