Ribosome‐Protected Fragments from Sindbis 42‐S and 26‐S RNAs

Abstract

Sindbis virus 42-S and 26-S RNA labeled with 32P were purified from infected chick embryo fibroblasts. The RNA 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 2 RNA species yielded several fragments of chain length about 35-40 nucleotides from 80-S complexes and up to 60-65 nucleotides from 40-S complexes. The 5''-terminal capped sequence, m7GpppA-U-G that is present in both Sindbis virus RNA, was not retained in any of the ribosome-protected fragments. Fingerprint analyses indicated that the fragments derived from 40-S and 80-S initiation complexes of each species of RNA were overlapping, but the fragments from 42-S and 26-S RNA were unrelated. The complexity of the fingerprints were consistent with protection of a single, different initiation site in each Sinbis virus RNA.