Binding of Ribosomes to Linear and Circular Forms of the 5′‐Terminal Leader Fragment of Tobacco‐Mosaic‐Virus RNA

Abstract

The sequence of the 5′‐terminal leader fragment preceding the AUG codon in the RNA of tobacco mosaic virus (TMV), tomato strain, SPS isolate, has been determined. This RNA, similarly to the RNAs of the U1 and Dahlemense strains of TMV [Kukla et al. (1979) Eur. J. Biochem. 98 , 61–66] has the 7‐methylguanosine(5′)triphospho(5′)guanosine cap separated from the initiation codon by a long stretch of nucleotides devoid of guanosine residues. The RNase‐T₁‐resistant 73‐nucleotide‐long leader fragment of TMV RNA from the SPS isolate was assayed for its ability to interact with eukaryotic and prokaryotic ribosomes. The linear fragment, labelled either at its 5′ or 3′ end, efficiently formed disome initiation complexes when incubated with wheat‐germ protein‐synthesis extract. In contrast to its linear counterpart, the circular covalently closed RNA leader fragment, obtained in a reaction catalysed by T4 RNA ligase, was unable to interact with wheat germ ribosomes. Both kinds of leader fragment bound equally well to Escherichia coli 70‐S ribosomes. The results offer further support to the notion that in eukaryotic initiation the free 5′ end (either capped or uncapped) is required for mRNA interaction with ribosomes. Furthermore, they suggest that both ribosomes found in disome initiation complexes with the TMV RNA leader fragment enter the mRNA sequentially via the free 5′ terminus.