EFFECTS OF BICYCLOMYCIN ON RNA-BINDING AND ATP-BINDING ACTIVITIES OF TRANSCRIPTION TERMINATION FACTOR-RHO

Bicyclomgcin is a commercially important antibiotic that has been show n to be effective against many gramnegative bacteria. Genetic and bioc hemical evidence indicates that the antibiotic interferes with RNA met abolism in Escherichia coli by inhibiting the activity of transcriptio n termination factor Rho. However, the precise mechanism of inhibition is not completely known. In this study we have used in vitro transcri ption assays to analyze the effects of bicyclomycin on the termination step of transcription. The Rho-dependent transcription termination re gion located within the hisG cistron of Salmonella typhimurium has bee n used as an experimental system. The possible interference of the ant ibiotic with the various functions of factor Rho, such as RNA binding at the primary site, ATP binding, and hexamer formation, has been inve stigated by RNA gel mobility shift, photochemical cross-linking, and g el filtration experiments. The results of these studies demonstrate th at bicyclomycin does not interfere with the binding of Rho to the load ing site on nascent RNA. Binding of the factor to ATP is not impeded, on the contrary, the antibiotic appears to decrease the apparent equil ibrium dissociation constant for ATP in photochemical cross-linking ex periments. The available evidence suggests that this decrease might be due to an interference with the correct positioning of ATP within the nucleotide-binding pocket leading b an inherent block of ATP hydrolys is. Possibly, as a consequence of this interference, the antibiotic al so prevents ATP-dependent stabilization of Rho hexamers.