S.D. Zorov - Ph.D. (Biol.), Senior Lecturer, Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia. E-mail: zorov@inbox.ru

The effect of antibiotic streptolydigin (stl) [1] оn RNA polymerase (RNAP) from Thermus aquaticus was under study. Here, we show that RNAP bound to stl remains active, but the rates of RNAP-mediated catalysis are significantly retarded. This is the first mentioning for RNAP inhibitors proceeding by such unusual mechanism instead of conventional totally blocking RNAP activity after their binding. In the presence of stl elongation complexes were divided into two fractions: slow fraction representing complexes bound to inhibitor, and fast one with free complexes. The elongation rate of the slow fraction was 11,000 lower than that for the fast fraction. We also found that Mn2+ activate only RNAP bound to stl, while not having any effect on the inhibitor binding. Half-inhibition concentration for the intrinsic cleavage was 5 fold lower than that for elongation. Obviously, this difference was due to the facts that rate of elongation is significantly higher than that for intrinsic RNA cleavage; and that binding of stl to RNAP requires an external Mg2+ ion [2], which was added at the time of start of the reaction. The incorporation of modified NTP (thioCTP) into RNA was 1250 fold slower than with a normal NTP (CTP), while with stl the reaction slowed down 125 fold only. These data point to the difference in properties of slow and fast fraction, supporting a hypothesis that RNAP bound to stl is active. Stl binding does not change Кm for incoming NTP, i.e. the inhibition by stl is not due to the change of RNAP affinity to the substrate. Given data is fully consistent with previously published structure of RNAP complex with bound stl and related mechanism of RNAP active site functioning [3].

 

1. Siddhikol C., Erbstoeszer J.W., Weisblum B. Mode of action of streptolydigin // J. Bacteriol. 1969. V. 99. P. 151-155.
2. Zorov S., Yuzenkova Y., Nikiforov V., Severionov K., Zenkin N. Antibiotic streptolydigin requires noncatalytic Mg2+ for binding to RNA polymerase // Antimicrob Agents Chemother. 2014. V. 58. № 3. P. 1420-1424.
3. Temiakov D., Zenkin N., Vassylyeva M.N., Perederina A., Tahirov T.H., Kashkina E., Savkina M., Zorov S., Nikiforov V., Igarashi N., Matsugaki N., Wakatsuki S., Severinov K., Vassylyev D.G. Structural basis of transcription inhibition by antibiotic streptolydigin // Mol. Cell. 2005. Sep. 2. V. 19. № 5. P. 655-666.