Mutant rho factors with increased transcription termination activities. I: Functional correlations of the primary and secondary polynucleotide binding sites with the efficiency and site-selectivity of rho-dependent termination

Abstract

We have characterized rho proteins from mutants of Escherichia coli, rho s-81 and rho s -82, which are hyperactive in termination. The two mutant rho proteins are differentially altered both in termination activities and in RNA interactions. rho s-81 generally elicits enhanced termination on various templates such as phage T7 DNA and a DNA restriction fragment containing the trpE intracistronic rho-dependent terminators, either measured as a whole or examined for individual sites. On the other hand, rho s-82 has strikingly different preferences toward individual termination sites, exhibiting overall termination activities higher or lower than normal, depending on templates. From measurements of the rho ATPase activity with T7 RNA and various homoribopolymers as cofactors, both mutant rho proteins are shown to have broadened RNA base specificities in contrast to the stringent requirement for cytosine observed with the wild-type rho. Functional tests on the two kinds of polynucleotide binding sites known for rho have indicated that rho s-81 is mainly altered in the primary site, whereas rho s-82 is simultaneously affected in the secondary binding site as well as the primary site. Thus, we conclude that the primary and secondary sites contribute distinctly in determining the overall efficiency and site-specificity of termination, respectively. Further analysis of detailed termination points at the trpE and λt R1 terminators has revealed that major RNA transcripts generated by the wild-type rho and rho s-81 are notably rich in adenine and poor in cytosine for the 3′-terminal five to ten nucleotides, whereas those preferentially terminated by rho s-82 are conversely richer in cytosine than adenine. This finding suggests that rho may recognize the RNA-DNA hybrid region at the 3′ end of a nascent transcript in its secondary binding reaction.