L. Carrano et al., EFFECTS OF BICYCLOMYCIN ON RNA-BINDING AND ATP-BINDING ACTIVITIES OF TRANSCRIPTION TERMINATION FACTOR-RHO, Antimicrobial agents and chemotherapy, 42(3), 1998, pp. 571-578
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.