ESCHERICHIA-COLI RNASE-T FUNCTIONS IN-VIVO AS A DIMER DEPENDENT ON CYSTEINE-168

It was shown that Cys-168 is required for RNase T function and thermos tability and that its hydrophobic properties are important for this ro le (Li, Z., Zhan, L., and Deutscher, M. P. (1996) J. Biol Chem. 271, 1 127-1132). To understand the molecular basis for these findings, furth er studies of Cys-168 and RNase T structure were carried out. Treatmen t of RNase T with the sulfhydryl-modifying agent 5,5'-dithiobis-(2-nit robenzoic acid) leads not only to inactivation, but also to monomeriza tion of the protein. Similarly, specifically converting Cys-168 to eit her serine or asparagine leads to loss of activity and to monomer form ation at 37 degrees C. However, at 10 degrees C the serine mutant rema ins as a dimer and retains full RNase T activity, whereas the asparagi ne derivative shows only a low level of activity and of dimer formatio n. These data show a strong correlation between activity and the dimer form of RNase T. The importance of dimer formation was also shown in vivo using genetic studies. An inactive mutant of RNase T, termed HA2, which exists as a dimer at 37 degrees C in vitro, completely suppress es endogenous RNase T activity in vivo and in vitro when introduced in to a RNase T+ cell on a multicopy phagemid, most likely as a consequen ce of inactive heterodimer formation. Introduction of the HA2 gene on a single copy plasmid, as expected, leads to a propertionally smaller effect on endogenous activity. The dominant negative effect displayed by the HA2 protein can be relieved by an additional mutation in HA2 RN ase T that abolishes its ability to dimerize. An inactive mutant aspar agine derivative of Cys-168, which also does not dimerize, also shows little of the dominant negative phenotype. Thus, these data demonstrat e that RNase T dimerizes in vine, that the dimer form is required for RNase T activity, and that Cys-168 is needed for dimerization of the e nzyme.