Identification of features in 5' terminal fragments from reovirus mRNA which are important for ribosome binding

Four types of experiments were carried out with reovirus messenger RNAs or with 5′ terminal fragments of known sequence to identify features in mRNA which appear to be important for formation of initiation complexes with ribosomes. With a number of reovirus mRNAs, 40S initiation complexes had been previously shown to protect a significantly larger segment of the RNA (including the 5′ terminal m7G) than that protected by 80S initiation complexes. Each 80S-protected sequence had an AUG codon and was a subset of the 40S-protected sequence from the same message. When 40S- and 80S-protected fragments were tested for ability to rebind to ribosomes, the 80S-protected fragments showed considerably lower binding ability, implying that the “extra” sequences protected by 40S initiation complexes contribute to ribosome attachment. Nevertheless, wheat germ ribosomes select the same 5′ terminal initiation site in each reovirus mRNA, irrespective of the presence or absence of m7G on the message. This was demonstrated by comparing fingerprints of the ribosome-protected regions obtained with methylated versus unmethylated RNA. The contribution of m7G to formation of initiation complexes is therefore quantitative rather than qualitative. Limited T1 RNAase digestion of isolated 5′ terminal fragments from several reovirus messages generated a series of smaller fragments which were analyzed for ability to rebind to ribosomes. Partial digestion products up to 30 nucleotides in length which retained the 5′ cap but not the AUG codon were unable to associate stably with ribosomes, whereas every AUG-containing fragment that was analyzed was able to form initiation complexes. The efficiency of binding of certain AUG-containing fragments, however, was reduced by removal of either the 5′ terminal region, including the cap, or of sequences comprising the beginning of the coding region, on the 3′ side of the AUG. Complex formation between messenger RNA and ribosomes was inhibited by the trinucleotide AUG, but not by various other oligonucleotides. Although the inhibition was specific, a vast excess of trinucleotide was required for moderate inhibition of 80S complex formation, and the same concentration of AUG failed to inhibit formation of 40S initiation complexes.