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

Abstract

Synthetic DNA templates were transcribed by Escherichia coli RNA polymerase using nucleoside 5'-[gamma-S]triphosphates as one of the nucleotide substrates. Substitution of the thiol analogues for the normal nucleotides had no effect on the rate of RNA synthesis. RNA synthesized with either adenosine 5'-[gamma-S]triphosphate or guanosine 5'-[gamma-S]triphosphate was isolated with high efficiency on mercury-agarose columns prepared by activation with low concentrations of cyanogen bromide. Sulfur was shown to be incorporated at the 5' end of RNA by identification of the tetraphosphate HSpppA32p 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-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 analogues 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 throught the entire transcript can be isolated.