Sequence studies of satellite tobacco necrosis virus RNA: Isolation and characterization of a 5′-terminal trinucleotide

Abstract

Several approaches have been used to identify the 5′-terminal trinucleotide sequence of RNA from satellite tobacco necrosis virus. This RNA was shown to contain some degree of 5′-phosphorylation by its enhanced potential to be phosphorylated with polynucleotide kinase upon pretreatment with alkaline phosphatase. Alkaline digestion of STNV ‡ 5′-[ 32P]RNA prepared by such enzymic phosphorylation yielded stoichiometrically correct quantities of 5′- 32pAp, thus establishing the 5′-terminal nucleoside as adenosine. Extension of this approach through specific enzymic digestions of STNV 5′-[ 32P]RNA indicated a 5′-terminal sequence of ApGpPyp---. This sequence was confirmed by direct isolation of (ApGp)U from the 5′-end of STNV[ 14C]RNA. The isolation, on combination with the other experimental approaches, establishes the 5′-terminus of STNV RNA as ppApGpUp—--.