RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

Template-independent labeling of RNAs by Pol II. (A) Pol II was incubated for 15 min at 30°C with 1 μg each of pre-tRNA^Tyr, half tRNA^Tyr, 5S rRNA, and the 250-nt ORF RNA. α-³²P-UTP was added in to Pol II-RNA complexes, with no DNA present in the reactions, and reactions proceeded for 30 min at 30°C and were run out on a 10% denaturing gel. All RNAs tested got radiolabel added in the absence of template DNA. (B) All four α-³²P-labeled NTPs add label onto the RNAs (labeled 5S rRNA shown here). (C) Treatment with the Pol II-specific inhibitor, α-amanitin, prior to addition of RNA abolishes this labeling activity (5S rRNA shown here).