Participation of 5'-terminal leader sequences in in vitro translation of Rous sarcoma virus RNA.

The cell-free translation of genome RNA from Rous sarcoma virus was examined following hybridization to selected fragments of viral DNA. Single-stranded fragments, generated by t-RNATrp primed transcription of 70 S RNA from the Schmidt-Ruppin D strain, were isolated and purified by electrophoresis. These included DNA complementary to the 5'-terminal 101 nucleotides (DNA100) of virion 38 S RNA and a collection of prematurely terminated transcripts which lack the complement to the extreme 5'-terminal 7-20 nucleotides (DNA less than 100). In addition, DNA encompassing the viral leader sequences was purified from a cloned copy of proviral DNA. Under hybrid-arrested translation conditions, the leader DNA as well as DNA100 inhibited translation of all protein products generated from the 70 S RNA, while hybridization to the shorter transcripts (DNA less than 100) did not affect in vitro protein synthesis. All single-stranded DNAs were shown to hybridize with equal efficiency to viral RNA under hybrid-arrested translation conditions and inhibition of protein synthesis by DNA100 was concentration-dependent. These results document the participation of noncoding leader RNA in Rous sarcoma virus protein synthesis and demonstrate that a free, single-stranded 5' terminus is necessary for cell free translation of 70 S RNA.