The tobacco etch virus (TEV) 5-leader promotes capindependent 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.Virtually all eukaryotic cellular mRNAs contain a 5Ј-cap structure (m 7 G(5Ј)ppp(5Ј)N) that, during translation, serves as the binding site for the eukaryotic initiation factor (eIF) 1 4E, a subunit of eIF4F that also contains eIF4G and eIF4A (1, 2). eIF4F promotes binding of 40 S ribosomal subunits through the interaction of eIF4G and eIF3, the latter of which is associated with the 40 S ribosomal subunit. The interaction between eIF4G and eIF3 directs 40 S subunit binding at or close to the 5Ј terminus. Following binding, the 40 S subunit scans down the RNA in search of the first AUG codon in a good context at which translation will initiate. The rate of 40 S subunit binding and initiation can be slowed or prevented altogether by the presence of a highly stable secondary structure (3, 4).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 only known mRNAs that naturally lack a cap are viral in origin. Poliovirus, encephalomyocarditis virus (EMCV), and foot-and-mouth disease virus are examples of animal picornaviruses whose polyadenylated genomic mRNA lacks a 5Ј-cap structure and instead contains a VPg, i.e. a viral protein genome linked to the 5Ј terminus that is removed prior to RNA recruitment into polysomes (5-9). The 5Ј-leader of picornaviruses is long (610 to Ͼ1200 nucleotides), highly structured, and contains multiple AUGs upstream of the initiation codon of the polyprotein coding region (10), features that should inhibit normal 40 S ribosomal scanning. Despite these apparent barriers to translation, picornaviral mRNAs are efficiently translated as a consequence of its 5Ј-leader, which contains an internal ribosome entry site (IRES) to which...

The response to amino acid starvation involves the global decrease of protein synthesis and an increase in the translation of some mRNAs that contain an internal ribosome entry site (IRES). It was previously shown that translation of the mRNA for the arginine/lysine amino acid transporter Cat-1 increases during amino acid starvation via a mechanism that utilizes an IRES in the 5 untranslated region of the Cat-1 mRNA. It is shown here that polypyrimidine tract binding protein (PTB) and an hnRNA binding protein, heterogeneous nuclear ribonucleoprotein L (hnRNP L), promote the efficient translation of Cat-1 mRNA during amino acid starvation. Association of both proteins with Cat-1 mRNA increased during starvation with kinetics that paralleled that of IRES activation, although the levels and subcellular distribution of the proteins were unchanged. The sequence CUUUCU within the Cat Cationic amino acid transporter 1 (Cat-1) is a high-affinity Na ϩ -independent transporter of L-arginine and L-lysine belonging to system yϩ (12, 62). Growth factors, hormones, and nutrients can modulate its expression level (20,53). Expression of the Cat-1 gene increases during stress in a manner that involves phosphorylation of translation initiation factor 2␣ (eIF2␣) (24). During such conditions, expression of the Cat-1 gene is regulated at the level of (i) mRNA synthesis via the transcription factor ATF4, which binds an amino acid response element in the first exon of the gene (54); (ii) mRNA stability via the binding of the nucleocytoplasmic protein HuR to an AU-rich element present within its 3Ј untranslated region (UTR) (94); and (iii) translation via a cap-independent mechanism of initiation via an internal ribosome entry site (IRES) (23).IRES-dependent translation involves the recruitment of ribosomes independently of the m 7 G cap at the 5Ј end of the mRNA followed by initiation downstream of the ribosome binding site (46). We showed that increased translation of the Cat-1 mRNA during amino acid starvation requires the translation of a 48-amino-acid open reading frame (ORF) in the 5Ј UTR of the mRNA, introducing the concept of a "dynamic IRES" (23,93). In the absence of upstream ORF (uORF) translation, the Cat-1 IRES remains in an inactive conformation. During starvation, translation of the uORF unwinds this secondary structure, leading to formation of a conformation that has IRES activity. Previous studies have also proposed that the active conformation is stabilized by proteins (IRES transactivating factors [ITAFs]) that are either synthesized or modified by processes that require eIF2␣ phosphorylation. We also showed that decreased translation elongation rates of the uORF in fed cells result in a prolonged half-life of this active remodeled structure, mimicking the role of ITAFs in amino acid-starved cells (21). Ribosome stalling within the uORF can occur physiologically during stresses that cause increased phosphorylation of elongation factor 2 and therefore decreased translation elongation rates (63). These two mechanisms o...

The genomic RNA of tobacco mosaic virus (TMV), like that of other positive-strand RNA viruses, acts as a template for both translation and replication. The highly structured 3 untranslated region (UTR) of TMV RNAs plays an important role in both processes; it is not polyadenylated but ends with a tRNA-like structure (TLS) preceded by a conserved upstream pseudoknot domain (UPD). The TLS of tobamoviral RNAs can be specifically aminoacylated and, in this state, can interact with eukaryotic elongation factor 1A (eEF1A)/GTP with high affinity. Using a UV cross-linking assay, we detected another specific binding site for eEF1A/GTP, within the UPDs of TMV and crucifer-infecting tobamovirus (crTMV), that does not require aminoacylation. A mutational analysis revealed that UPD pseudoknot conformation and some conserved primary sequence elements are required for this interaction. Its possible role in the regulation of tobamovirus gene expression and replication is discussed.Tobacco mosaic virus (TMV), a positive-strand plant RNA virus, is the type member of the tobamovirus family in the alphavirus-like superfamily. The genomic RNA of TMV strain vulgare (U1) is 6,395 nucleotides (nt) long and encodes at least four proteins. The full-length RNA is used to produce 126-and 183-kDa RNA-dependent replicase proteins, while the 30-kDa movement protein (MP) and the 17.5-kDa coat protein are translated from 3Ј-coterminal subgenomic mRNAs. The coding region is flanked by the 5Ј untranslated region (5Ј-UTR, or ⍀) and the 3Ј-UTR, both of which are required for viral replication. TMV RNA is capped, but it lacks a 3Ј poly(A) tail. Instead, the 3Ј-UTR contains a highly structured and conserved sequence composed of several pseudoknots (PKs) of the hairpin loop type (Fig. 1) (50,54,61). The TMV U1 3Ј-UTR is comprised of two structural domains: a 3Ј-terminal domain containing two PKs important for formation of a tRNA-like structure (TLS) (21, 53), linked to an upstream PK domain (UPD)-a quasicontinuous double-helical stalk comprising three consecutive PKs (61). A similar tandem arrangement of structural units (UPD-TLS) is found in all tobamoviruses and satellites of TMV, although the sizes and numbers of PKs of the UPD are variable (26). The TLS of TMV can be aminoacylated and binds to several tRNA-specific enzymes (reviewed in reference 42). Interestingly, two of the PKs in the U1 UPD are phylogenetically conserved among all tobamoviruses, TMV satellites, and hordeiviruses in location, in structure, and even in several positions of primary sequence, strongly suggesting their functional importance (38, 61).Whether ending with a poly(A) tail or a TLS, the 3Ј terminus of the genomic RNA of positive-strand RNA viruses is generally thought to contain elements of the promoter for initiation of minus-strand viral RNA synthesis. The minimal 3Ј cis-acting element required for the initiation of negative-strand RNA synthesis on TMV genomic RNA includes the 3Ј-terminal TLS and the 3Ј-most-proximal PK structure (PK3) of the UPD (12,49,59). Destabiliza...

In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2a on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to-Ala mutation in eIF2a (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 mg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 59-end cap (m 7 GpppN) and a 39-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from b-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.