According to the information on the hammerhead structure of ribozyme known so far,the cleavage occurs at the 3′-side of the specific position.The 3′-half molecule of yeast alanyl tRNA with GTΨ sequence is chosen as...According to the information on the hammerhead structure of ribozyme known so far,the cleavage occurs at the 3′-side of the specific position.The 3′-half molecule of yeast alanyl tRNA with GTΨ sequence is chosen as substrate.Consequently,a 38-mer ribozyme is designed by Haseloff and Gerlach mod- el.In the presence of Mg^(2+),the expected cleavage really occurs at the 3′-side of GTΨ in vitro.The Michaelis-Menten kinetic parameters for the reaction are:K_m,6-7 μmol/L,k_(cat),3.4×10^(-3)/min.At the same time,a 17-mer substrate analogue has been synthesized,with a GUU-sequence instead of GTΨ men- tioned above.The steady-state kinetic analyses reveal that K_m values are nearly the same for both sub- strates.But k_(cat) values decrease by 294 fold.These observations demonstrate that the modified nucleotide Ψ can be the cleavage site of hammerhead structure of ribozyme with a much lower k_(cat).展开更多
基金the National Natural Science Foundation of China National High Technology Program
文摘According to the information on the hammerhead structure of ribozyme known so far,the cleavage occurs at the 3′-side of the specific position.The 3′-half molecule of yeast alanyl tRNA with GTΨ sequence is chosen as substrate.Consequently,a 38-mer ribozyme is designed by Haseloff and Gerlach mod- el.In the presence of Mg^(2+),the expected cleavage really occurs at the 3′-side of GTΨ in vitro.The Michaelis-Menten kinetic parameters for the reaction are:K_m,6-7 μmol/L,k_(cat),3.4×10^(-3)/min.At the same time,a 17-mer substrate analogue has been synthesized,with a GUU-sequence instead of GTΨ men- tioned above.The steady-state kinetic analyses reveal that K_m values are nearly the same for both sub- strates.But k_(cat) values decrease by 294 fold.These observations demonstrate that the modified nucleotide Ψ can be the cleavage site of hammerhead structure of ribozyme with a much lower k_(cat).
