FIG. 5.

Translation activity of sgRNA3a in vitro and in the presence of polysomes. (A) Translation in rabbit reticulocyte system. The RNAs were incubated in rabbit reticulocyte extract at two different concentrations of MgCl₂, as indicated, and the products were separated in 12% SDS-polyacrylamide gel. The template RNAs are BMV RNA, total encapsidated viral RNA extracted from purified BMV preparation; sgRNA3a, the fraction of sgRNA3a purified from the encapsidated BMV RNA by cutting a band off the agarose formaldehyde/formamide gel; trRNA3 and trRNA3a, the RNA3 and sgRNA3a preparations synthesized by transcription in vitro and purified from unincorporated NTPs (on RNeasy columns; QIAGEN) prior to the translation reaction. (B) Translation in the wheat germ system. The RNAs were incubated in the wheat germ system with [³⁵S]methionine, and the products were analyzed in polyacrylamide-SDS gel, as described for panel A. The synthetic RNA3 or sgRNA3a templates were either capped or uncapped (see Materials and Methods), as indicated (lanes 1 through 4), whereas standard BMV RNA (containing the same amount of the RNA3 component) was translated in lane 5. The migration of protein 3a is shown on the right. tr, transcribed. (C) Detection of BMV RNA3-related sequences in polysomes. The polysomal RNA was extracted from BMV-infected barley leaves as described in Materials and Methods and separated by ultracentrifugation in sucrose gradient. Individual fractions (numbered 1 to 8) were analyzed by Northern blotting in 1% denaturing agarose gel and with the RNA3-specific probe. The positions of RNA3 and sgRNA3a were confirmed by comigration with the corresponding size transcripts in the “Std.” lane on the right.