Mutational Analysis of Splicing Activities of Ribonucleotide Reductase alpha Subunit Protein from Lytic Bacteriophage P1201

Abstract

A CP1201 RIR1 intein is found in the ribonucleotide reductase alpha subunit (RNR alpha subunit) protein of lytic bacteriophage P1201 from Corynebacterium glutamicum NCHU 87078. This intein can be over-expressed and spliced in Escherichia coli NovaBlue cells. Mutations of C539, the N-terminal residue of the C-extein in the CP1201 RIR1 protein, led to the changes of pattern and level of protein-splicing activities. A G392S variant was found to be a temperature-sensitive protein with complete splicing activity at 17 and 28 degrees C but not at 37 degrees C or higher. We also found that the cleavage at the CP1201 RIR1 intein C-terminus of the double mutant G392S/C539G was blocked, but other cleavage activities could be efficiently performed at 17 degrees C. G392S/C539G variant possessed the properties of low-temperature-induced cleavage at the intein N-terminus.