Conformational studies of reovirus single-stranded RNAs synthesized in vitro

Abstract

The reovirion contains ten distinct double-helical RNA molecules packaged together with an RNA polymerase activity capable of synthesizing milligram amounts of 10 single-stranded RNA transcripts of the genome by an asymmetric, conservative mechanism. These in vitro transcription products can be resolved into 14-S, 19-S and 25-S size classes containing four, three and three distinct molecular species, respectively. Thermal-denaturation studies and ethidium bromide binding assays were employed to investigate the conformational state of reovirus single-stranded RNA. Molecules resolved into size classes and unresolved mixtures possessed similar T m values and percent hyperchromicities as well as characteristic hysteresis during single and repeated thermal denaturation-renaturation cycles. Interaction of ethidium bromide with reovirus single-stranded RNA altered the visible spectrum of the dye with pronounced hyperchromicity at 480 nm. Estimates of the relative binding capacities derived from this hyperchromic shift indicated that the RNA molecules within the 14-S and 19-S size classes possess structural content proportional to their lengths whereas those within the 25-S size class do not. The thermal-denaturation studies and ethidium bromide binding assays provide evidence for extensive secondary structure in the single-stranded RNA molecules synthesized in vitro by subviral cores of reovirus.