Abstract
The tobacco etch virus (TEV) 5'-leader promotes cap-independent translation in a 5'-proximal position and promotes internal initiation when present in the intercistronic region of a dicistronic mRNA, indicating that the leader contains an internal ribosome entry site. The TEV 143-nucleotide 5'-leader folds into a structure that contains two domains, each of which contains an RNA pseudoknot. Mutational analysis of the TEV 5'-leader identified pseudoknot (PK) 1 within the 5'-proximal domain and an upstream single-stranded region flanking PK1 as necessary to promote cap-independent translation. Mutations to either stem or to loops 2 or 3 of PK1 substantially disrupted cap-independent translation. The sequence of loop 3 in PK1 is complementary to a region in 18 S rRNA that is conserved throughout eukaryotes. Mutations within L3 that disrupted its potential base pairing with 18 S rRNA reduced cap-independent translation, whereas mutations that maintained the potential for base pairing with 18 S rRNA had little effect. These results indicated that the TEV 5'-leader functionally substitutes for a 5'-cap and promotes cap-independent translation through a 45-nucleotide pseudoknot-containing domain.
Highlights
Structure and Function of the tobacco etch virus (TEV) internal ribosome entry site (IRES)Ture of the TEV 5Ј-leader and its relationship with the function of the 5Ј-leader in promoting cap-independent translation is unknown
Because binding of eIF4E is critical for the subsequent binding of the 40 S subunit and assembly of a competent initiation complex, those mRNAs that naturally lack a 5Ј-cap must have evolved alternative mechanisms to promote translation
The tobacco etch virus (TEV) 5Ј-leader evolved to function in a 5Ј-proximal position in a monocistronic mRNA, it promoted translation of a second cistron in a dicistronic mRNA when the TEV leader sequence was introduced into the intercistronic region, indicating that the TEV 5Ј-leader contains an internal ribosome entry site (IRES) [22]
Summary
Ture of the TEV 5Ј-leader and its relationship with the function of the 5Ј-leader in promoting cap-independent translation is unknown. We show that a 5Ј-proximal, 45-nt RNA pseudoknot-containing domain within the TEV 5Ј-leader is required to promote cap-independent translation. Mutations that disrupted the pseudoknot reduced capindependent translation, including mutations to loop 3 that exhibits complementarity to a conserved region in eukaryotic 18 S rRNAs. Changes to the loop that maintained its potential for base pairing with 18 S rRNA had only a small effect, supporting the possibility that base pairing between the pseudoknot and the 18 S rRNA may be involved in promoting cap-independent translation
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