Ribosome-protected fragments from sindbis 42-S and 26-S RNAs

Sindbis virus 42-S and 26-S RNAs labeled with 32P were purified from infected chick embryo fibroblasts. The RNA's were incubated in the presence of a wheat germ cell-free translating system under conditions that yielded 40-S and 80-S initiation complexes. After digestion with RNase A, ribosome-protected fragments were isolated by polyacrylamide gel electrophoresis and compared with respect to number, size, cap content and oligonucleotide composition. The two RNA species yielded several fragments of chain length about 35--40 nucleotides from 80S complexes and up to 60--65 nucleotides from 40-S complexes. The 5'-terminal capped sequence, m7 GpppA-U-G that is present in both Sindbis virus RNA's, was not retained in any of the ribosome-protected fragments. Fingerprint analyses indicated that the fragments derived from 40S and 80-S initiation complexes of each species of RNA were overlapping, but the fragments from 42-S and 26-S RNAs were unrelated. The complexity of the fingerprints were consistent with protection of a single, different initiation site in each Sindbis virus RNA.