Detalle de: Identification of temperature-sensitive mutations and characterization of thermolabile RNase II variants

The RN ase II family of ribonucleases is ubiquitous and critical for RNA metabolism. The rnb500 allele has been widely used for over 30 years; however, the underlying genetic changes which result in RN ase II thermolabile activity remain unknown. Here, we combine molecular and biophysical studies to carry out an in vivo and in vitro investigation of RN ase II mutation(s) that confer the rnb500 phenotype. Our findings indicate that RN ase II thermolability is due to the Cys284Tyr mutation within the RNB domain, which abolishes activity by increasing protein kinetic instability at the nonpermissive temperature. These findings have important implications for the design of temperature‐sensitive variants of other RN ase II enzymes, namely those with yet unknown functions.