Studies on the altered rho factor in nitA mutants of Escherichia coli defective in transcription termination : II. Purification and molecular properties of the mutant rho

Abstract

The transcription-termination factor rho was purified from two termination-defective strains, nitA18 and nitA702 (temperature-sensitive), by the method of Roberts (1969) as modified due to the alteration of their molecular properties. The mutant rho factors are quite sensitive to temperature and proteolysis even after purification. The nitA702 rho shows a reduced affinity for phosphocellulose and a lower sedimentation rate (5.5 S) than does the wild-type (9.5 S). The transcription-termination activity is significantly decreased in both mutant rho factors; the maximum inhibition by the nitA18 and nitA702 rho being limited to 60% and 15%, respectively, whereas the wild-type inhibits 65% or more at 37 °C. The poly(C)-dependent ATPase activities (Richardson et al., 1975) of these mutant rho factors are apparently normal at 37 °C or below. However, they showed altered enzyme specificity with activator RNAs containing ribonucleosides other than cytidine. The ATPase activity of the nitA702 rho is much less than normal when activated with the nascent RNA produced by the transcription in vitro of various DNAs or with such synthetic ribohomopolymers as poly(A), poly(U) and poly(I). In contrast, the nitA18 rho exhibits significantly higher ATPase activity with all these RNA activators, in spite of its decreased activity in transcription termination. The altered functions of these mutant rho in transscription-termination regulation are discussed.