Incorporation of purine nucleoside 5'-[γ-S]triphosphates as affinity probes for initiation of RNA synthesis in vitro

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Abstract
Synthetic DNA templates were transcribed by Escherichia coli RNA polymerase using nucleoside 5''-[.gamma.-S]triphosphates as 1 of the nucleotide substrates. Substitution of the thiol analogs for the normal nucleotides had no effect on the rate of RNA synthesis. RNA synthesized with adenosine 5''[.gamma.-S]triphosphate or guanosine 5''-[.gamma.-S]triphosphate was isolated with high efficiency on Hg-agarose columns prepared by activation with low concentrations of CNBr. S was shown to be incorporated at the 5'' end of RNA chains by identification of the tetraphosphate HSpppA 32p liberated after alkaline hydrolysis of HS(A-32pU)n (alternating copolymer synthesized by the action of E. coli RNA polymerase on d(A-T)n.cntdot.d(A-T)n with adenosine 5''[.gamma.-S]triphosphate and uridine 5''-[.alpha.-32P] triphosphate as substrates). Transcripts elongated but not initiated with these thiol analogs did not bind to the affinity column. This technique provides an extremely sensitive assay for RNA synthesis initiation in vitro, since initiated transcripts containing radiolabel through the entire transcripts can be isolated.