Abstract
Isolated mature L-A viral particles from yeast have a transcriptase activity that uses endogenous L-A double-stranded RNA (dsRNA) as template. We have previously demonstrated that empty particles derived from mature L-A viral particles have replicase activity capable of synthesizing minus strand single-stranded RNA (ssRNA) on an added plus strand ssRNA template to form dsRNA. We report here that empty particles also have transcriptase activity that uses added viral dsRNA as template. The newly synthesized ssRNA was the plus strand, and some of these transcripts were converted to the dsRNA form by the replicase activity associated with the empty particles. This transcriptase activity, however, required a much higher concentration of polyethylene glycol than that used previously for the replicase activity. The mode of transcription was conservative. The enzyme transcribed ssRNA from L-A, M1, or X (a deletion mutant of L-A) dsRNAs but not from other yeast dsRNAs (L-BC, T, or W), bacteriophage Phi6 dsRNAs, or animal rotavirus dsRNAs, indicating the same template specificity as that expected for the in vivo reaction. This assay system, and the replicase assay system, will allow us to study in vitro all the enzymatic reactions essential for the viral replication cycle.
Highlights
Mature L-A viral particles contain one L-A dsRNA molecule/particle (Esteban and Wickner, 1986) and have a transcriptase activity whose product is L-A (+) singlestranded RNA (ssRNA) (Herring and Bevan, 1977; Bruenn et al, 1980)
When mature L-A viral particles are exposed to low ionic strength conditions, these particles release L-A dsRNA and Because host cells have no activity that synthesizes singlestranded RNAf’romdouble-stranded
We show that these empty particles, in addition totheir replicase activity, have atranscriptase activity capable of using externally added dsRNA as template
Summary
Vol 264, No 18, Issue of June 25, pp. 101897829-10877, Printed in U.S.A. Reconstitution of Template-dependent in Vitro Transcriptase Activity of a Yeast Double-strandedRNA Virus*. Isolated mature L-A viral particles from yeast have 1988b; Icho and Wickner, 1989) of the 39-nm viral particles a transcriptase activity that uses endogenousL-A double-stranded RNA (dsRNA) as template. The newly synthesized ssRNA was the plus strand, and some of these transcripts were converted to the dsRNA form by the replicase activity associated with the empty particles. This transcriptase activity, ,required a much higher concentration of polyethylene glycol than that used previously for the replicase activity. The (+) strand transcripts are packaged to form that expected for the in vivoreaction Analysis of template specificity by altering the dsRNA substrate and study of the enzymes involved in the transcriptase reaction requires a completely in vitro system in which enzymes and template areadded separately
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