Oxazinomycin arrests RNA polymerase at the polythymidine sequences

Area of Science:

• Molecular Biology
• Microbiology
• Biochemistry

Background:

• Oxazinomycin is a C-nucleoside antibiotic produced by Streptomyces hygroscopicus.
• It structurally resembles the nucleoside uridine.

Purpose of the Study:

• To investigate the interaction of oxazinomycin triphosphate with bacterial and eukaryotic RNA polymerases (RNAPs).
• To elucidate the mechanism of transcriptional arrest induced by oxazinomycin incorporation.
• To compare the effects of oxazinomycin with the C-nucleoside pseudouridine on transcription.

Main Methods:

• Assessing oxazinomycin triphosphate as a substrate for RNAPs.
• Analyzing transcriptional elongation and arrest upon oxazinomycin incorporation.
• Investigating the role of Gre RNA cleavage factors in mitigating transcriptional arrest.
• Comparing oxazinomycin with pseudouridine in transcription assays.

Main Results:

• Oxazinomycin triphosphate is a suitable substrate for both bacterial and eukaryotic RNAPs.
• While single incorporations are extended, successive oxazinomycins or specific sequence contexts cause RNAP arrest.
• Gre factors resolve arrest at single oxazinomycin sites and shorten arrested RNAs, indicating RNAP backtracking.
• Pseudouridine is also a substrate but does not inhibit transcription.

Conclusions:

• Oxazinomycin acts as a Trojan horse substrate, inhibiting transcription through a mechanism involving RNAP backtracking.
• The oxygen atom at the C5 position of the uracil ring in oxazinomycin is crucial for its inhibitory effect.
• Gre factors can overcome oxazinomycin-induced transcriptional arrest.