Abstract
The antibiotic edenine induces binding of multiple 40 S ribosomes to reovirus messenger RNAs, producing complexes that sediment rapidly in glycerol gradients. Rapidly sedimenting complexes were also obtained with tobacco mosaic virus RNA and rabbit globin mRNA in the presence of edeine. Following ribonuclease digestion of the heavy complexes, nuclease-resistant 32P-labeled reovirus fragments protected by 40 S ribosomes in the presence of edeine were recovered and fingerprinted. The sequence complexity of the protected material supports the interpretation that 40 S subunits are distributed at many internal sites in each messenger RNA. Additional experiments indicate that binding of the multiple 40 S subunits occurs from a single "entry site" which involves the 5' terminus of the message. This, in turn, implies that in the presence of edeine 40 S ribosomes are able to move along the mRNA chain, attaching initially near the 5' end, then advancing to make room for the next subunit. We suggest that in the absence of antibiotics, also, a 40 S ribosome might bind near the 5' terminus and then advance, stopping where it encounters the first AUG triplet. The effect of edeine might be to interfere with the AUG recognition process, thus allowing the 40 S ribosome to continue unhalted along the message. The present experiments with edeine provide the first direct evidence that 40 S ribosomal subunits are capable of moving along the mRNA chain.
Highlights
Sedimenting complexes were obtained with tobacco mosaic virus RNA and rabbit globin mRNA in the presence of edeine
The above experiments reveal a number of unusual features about the abortive initiation complexes that accumulate in edeine-treated wheat germ extracts
This is indicated by the large size of the complexes induced by edeine, using either reovirus, globin, or tobacco mosaic virus mRNA, and by the sequence complexity of the protected mRNA fragments (Fig. 9)
Summary
It was suggested that the antibiotic affects a late step in initiation, allowing 40 S ribosomal subunits to bind to mRNA, but preventing attachment of the 60 S ribosomal subunit This effect has been observed in reticulocyte (3), wheat germ (4), and Artemia extracts (5). Based on the reports cited above, edeine seemed to be a promising candidate, but our experiments revealed that edeine induces the formation of highly abnormal initiation complexes in which multiple 40 S subunits bind to each messenger RNA molecule. The present studies with edeine indicate that each 40 S ribosome binds initially near the 5’ terminus of the message, and moves away from the entry site. In the absence of antibiotics, a 40 S ribosome may bind near the 5’ terminus and advance until it encounters an AUG initiator triplet. S complex, the complex of 40 S ribosomes bound to messenger RNA in the presence of edeine
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