Reverse transcriptase with concomitant ribonuclease H activity in the cell-free synthesis of branched RNA-linked msDNA of Myxococcus xanthus

Abstract

msDNA is a peculiar molecule consisting of a branched RNA linked to single-stranded DNA via a 2',5' phosphodiester bond. A cell-free system, utilizing cells permeabilized with phenethyl alcohol, was established to study the synthesis of msDNA in M. xanthus. Permeablized cells labeled with [alpha-32P]dCTP in the presence of ddGTP, ddATP, or ddTTP produce a band that migrates at the same position as the full-sized msDNA in an polyacrylamide gel. However, when this band is treated with ribonuclease A prior to gel electrophoresis, it results in many different-sized bands. This indicates that during the labeling, intermediates are produced in which single-stranded DNAs of various lengths are associated with a compensatory length of RNA such that the total length for each intermediate is identical. These results provide evidence for the previously proposed model in which msDNA is synthesized by reverse transcriptase using a folded RNA precursor as a primer as well as a template. Furthermore, we found that there is a precise coupling mechanism of reverse transcriptase and ribonuclease H.