Abstract
Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.
Highlights
C-nucleosides are a group of compounds that comprise the non-canonical nucleobases capable of Watson–Crick base pairing attached to the unaltered ribose moieties via a C– C bond
We characterized the inhibitory activity of the C-nucleoside antibiotic OZM on transcription by multisubunit RNA polymerases (RNAPs) from E. coli and S. cerevisiae and a single subunit RNAP from human mitochondria
We show that the OZM triphosphate is a good substrate for Escherichia coli (Eco) RNAP and is efficiently incorporated into RNA by Sce RNAPII and Hsa MT RNAP
Summary
C-nucleosides are a group of compounds that comprise the non-canonical nucleobases capable of Watson–Crick base pairing attached to the unaltered ribose moieties via a C– C bond (as opposed to an N–C bond in canonical nucleosides). Considering the known promiscuity of the nucleoside and nucleotide kinases [20], OZM may be converted into the OZM triphosphate upon entry into the target cell, compete with UTP as an RNAP substrate and inhibit transcription or post-transcriptional processes upon its incorporation into RNA. To test this hypothesis, we studied the effects of the OZM triphosphate on transcription by the classic representatives of DNA-dependent RNA polymerases: multisubunit RNAPs from Escherichia coli and Saccharomyces cerevisiae (RNA polymerase II) and a single subunit RNAP from human mitochondria. Our results suggest that OZM functions as a transcriptional inhibitor if present at similar or higher concentration than uridine in the cell, but do not rule out the possibility that OZM may interfere with post-transcriptional processes such as RNA folding and translation when the intracellular OZM/U is low
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