Related Experiment Video
Updated: Aug 12, 2026

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses
Published on: February 25, 2011
Conformational stability and activity of ribonuclease T1 and mutants. Gln25----Lys, Glu58----Ala, and the double
B A Shirley1, P Stanssens, J Steyaert
1Biochemistry Department, Texas A & M University, College Station 77843.
Abstract:
Ribonuclease T1 (RNase T1) and mutants Gln25----Lys, Glu58----Ala, and the double mutant were prepared from a chemically synthesized gene, cloned and expressed in Escherichia coli. The wild-type RNase T1 prepared from the cloned gene was identical in every functional and physical property examined to RNase T1 prepared from Aspergillus oryzae. Urea and thermal unfolding experiments show that Gln25----Lys is 0.9 kcal/mol more stable and Glu58----Ala is 0.8 kcal/mol less stable than wild-type RNase T1. In the double mutant, these contributions cancel and the stability does not differ significantly from that of wild-type RNase T1. For the double mutant, the dependence of delta G on urea concentration is significantly greater than for wild-type RNase T1 or the single mutants. This suggests that the double mutant unfolds more completely in urea than the other proteins. The activity of Gln25----Lys is identical with that of wild-type RNase T1. The activities of Glu58----Ala and the double mutant are 7% of wild-type when GpC hydrolysis is measured (due to a 35-fold decrease in kcat), and 37% of wild-type when RNA hydrolysis is measured. Thus, Glu58 is important, but not essential to the activity of RNase T1.
More Related Videos
Related Concept Videos
RNA Stability
Ligand Binding and Linkage
RNA Stability
tRNA Activation
Improving Translational Accuracy
Ribozymes
Ribozymes can be...

