Studies on the bacteriophage MS2 6. The nucleoside 5'-triphosphate end groups of the replicative intermediate and the replicative form.

Abstract

Undegraded, uniformly 32P‐labelled replicative form and replicative intermediate were prepared and fractionated according to sedimentation rate. For each fraction the amount of 5′‐terminal residues, which after alkaline hydrolysis are released as 5′‐triphosphoryl, 3′(2′)‐phosphoryl nucleosides (nucleoside tetraphosphates), was quantitatively determined. The replicative form contained O, and the replicative intermediate 2.5 nascent chains, if the assumption is made that the latter are on the average half completed. The number of nascent chains apparently increases with increasing sedimentation constant of the replicative intermediate molecules. The nucleoside tetraphosphate, released from each fraction, was identified as exclusively pppGp. This is interpreted as evidence for a 5′ to 3′ polarity in the synthesis of viral RNA, i.e. a growing 3′OH end. The replicative form and replicative intermediate were also prepared from cells, in which the 32P‐label was chased for 5 min before harvest. The amount of nucleoside tetraphosphate released agreed with a single end group per complete chain. Presumably 50 to 100% of the residual labelled terminal was derived from the complementary strand, and again only pppGp was found. It follows that the 3′‐terminal adenosine residue of the viral RNA is presumably not part of the genetic information transferred during replication.