Allosteric stimulatory effect of S-adenosylmethionine on the RNA polymerase in cytoplasmic polyhedrosis virus. A model for the positive control of eukaryotic transcription.

Abstract

The stimulatory effect of S-adenosylmethionine (AdoMet) on cytoplasmic polyhedrosis virus transcription in vitro has been studied in detail. AdoMet affects the initiation of transcription but not chain elongation. It is required for each reinitiation step during multiple transcription cycles. Under limited conditions of synthesis in reaction mixtures depleted of UTP and CTP, a greater amount of initiator oligonucleotides was formed in the presence of AdoMet than in its absence. These small oligonucleotides were also formed along with the completed mRNA under conditions of mRNA formation. Although these capped (or uncapped) oligonucleotides corresponded to the 5'-terminal sequence of cytoplasmic polyhedrosis virus mRNA, they failed to substitute for AdoMet in promoting mRNA synthesis. Efficient synthesis of mRNA containing 5'-m7GpppAmpG was dependent on the concentrations of AdoMet and the initiating nucleotide, ATP. There was also an inverse relationship between AdoMet and ATP concentrations such that the apparent Km for ATP was decreased strikingly from 10 to 0.25 mM as the AdoMet concentration was increased from 5 to 500 microM. This effect was observed only for ATP. Double reciprocal plots for estimating the Km of ATP formed concave upward curves but became linear upon addition of a higher concentration of AdoMet, indicating a positive cooperativity of ATP and allosteric effect of AdoMet in the initiation process. Stimulation of transcription by lowering the Km for the initiating nucleotide at the promoter site is proposed as a model for positive regulation of eukaryotic gene expression.