Characterization of ribosome-protected fragments from reovirus messenger RNA

The 5'-terminal methylated cap (m7G(5')ppp(5')Gm) in reovirus messenger RNA comprises part of the ribosomes binding site, since attachment of 40 S wheat germ ribosomal subunits to reovirus small (s), medium (m), and large (l) RNA classes conferred almost complete protection of the cap against RNase digestion. After joining of the 60 S ribosomal subunits, however, the cap continued to be protected against T1 RNase within the 80 S initiation complexes formed with only some messenger species; namely the three l-messages, one of the m-messages, and one or two of the s-messages. When protected fragments were recovered from 40 S and 80 S complexes and tested for ability to rebind to ribosomes those fragments which retained the cap were able to rebind most efficiently. The protected fragments recovered from 40 S initiation complexes with several of the s- and m-RNA species were larger than the messenger fragments recovered from 80 S complexes. The medium size class of reovirus RNA, which consists of three messenger species, gave rise to three discrete 5'-terminal fragments after digestion of 40 S complexes with T1 RNase, and to three somewhat smaller fragments after T1 RNase digestion of 80 S complexes. Fingerprints of the T1 oligonucleotides derived from these fragments are consistent with the interpretation that each messenger species within the m-RNA class gives rise to a protected fragment of a unique size and that, with each message, there is extensive overlap between the regions of the message protected by 40 S and 80 S ribosomes. The ratio of the three protected fragments recovered from 40 S complexes with m-RNA was highly reproducible under a given set of binding conditions, but could be shifted by varying the messenger/ribosome ratio in the binding reaction. Thus, one of the fragments, which was preferentially recovered when the ribosome concentration was limiting, could be tentatively identified as the binding site of the most efficiently translated message within the m-RNA class.