The 5 leader (⍀) of tobacco mosaic viral RNA functions as a translational enhancer. Sequence analysis of a 102-kD protein, identified previously as a specific ⍀ RNA-binding protein, revealed homology to the HSP101/HSP104/ClpB family of heat shock proteins and its expression in yeast complemented a thermotolerance defect caused by a deletion of the HSP104 gene. Up to a 50-fold increase in the translation of ⍀-luc, but not luc mRNA was observed in yeast expressing the tobacco HSP101 whereas ⍀ failed to enhance translation in the absence of HSP101. Therefore, HSP101 and ⍀ comprise a two-component translational regulatory mechanism that can be recapitulated in yeast. Analysis of HSP101 function in yeast translation mutants suggested that the initiation factor (eIF) 3 and specifically one (TIF4632) of the two eIF4G proteins were required for the HSP101-mediated enhancement. The RNA-binding and translational regulatory activities of HSP101 were inactive in respiring cells or in cells subject to nutrient limitation, but its thermotolerance function remained unaffected. This is the first identification of a protein required for specific translational enhancement of capped mRNAs, the first report of a translational regulatory function for any heat-shock protein, and the first functional distinction between the two eIF4G proteins present in eukaryotes. There is now substantial evidence showing the important regulatory role that an mRNA 5Ј leader can play during translation in eukaryotes. In addition to structural features, such as its length (Kozak 1988; Gallie and Walbot 1992) and higher order structure (Pelletier and Sonenberg 1985), the presence of cis-acting regulatory elements in a leader have been shown to influence protein synthesis including those present within ferritin mRNA (for review, see Rouault et al. 1996), GCN4 mRNA (for review, see Hinnebusch 1996) and picornaviruses (for review, see Ehrenfeld 1996). In each of these examples, the 5Ј leader serves as a regulator that enables translation to occur only under certain cellular conditions.Tobacco mosaic virus (TMV) is a single-stranded, positive-sense RNA virus for which the genomic RNA serves as an efficiently translated mRNA. The 68-nucleotide 5Ј leader (known as ⍀) is responsible, in part, for the efficient translation of TMV mRNA and is distinguished by the absence of guanosine residues and a central poly-(CAA) region required for the stimulation of translation (Gallie et al. 1987a,b;Gallie and Walbot 1992). A model frequently proposed to explain the translational enhancement afforded by ⍀ posits that the lack of secondary structure within the leader, in itself, facilitates 40S subunit scanning and thereby reduces the requirement for initiation factors (eIFs), such as eIF4E or eIF4A (Sleat et al. 1988;Altmann et al. 1990a Altmann et al. ,b, 1997Kozak 1991Kozak , 1992 Kozak , 1994. This model predicts that ⍀ functions passively by presenting no barrier to 40S ribosomal subunits as they scan the 5Ј leader in search of the initiation codon. In contrast, an ...

The internal light-regulatory element (iLRE) of ferredoxin (Fed-1) mRNA, comprising the 5' leader and at least the first 13 codons of the open reading frame, controls transcript abundance after illumination of the plant in a translation-dependent manner. We have characterized the RNA binding activities associated with the Fed-1 iLRE and have identified one activity as the heat shock protein HSP101, a protein shown to bind the 5' leader of tobacco mosaic virus. HSP101 was sufficient and necessary to mediate a high level of translational activity from a Fed-1 iLRE-containing mRNA in yeast. Moreover, the Fed-1 iLRE substantially enhanced translation of reporter mRNAs in plant protoplasts expressing HSP101. Expression of HSP101 was subject to developmental regulation in leaves in that expression was highest in young leaves. These data suggest that Fed-1 mRNA may use the HSP101 regulatory mechanism as a means of ensuring a high level of translation required for the light-mediated regulation of Fed-1 mRNA stability.

The internal light-regulatory element (iLRE) of ferredoxin ( Fed-1 ) mRNA, comprising the 5 Ј leader and at least the first 13 codons of the open reading frame, controls transcript abundance after illumination of the plant in a translationdependent manner. We have characterized the RNA binding activities associated with the Fed-1 iLRE and have identified one activity as the heat shock protein HSP101, a protein shown to bind the 5 Ј leader of tobacco mosaic virus. HSP101 was sufficient and necessary to mediate a high level of translational activity from a Fed-1 iLRE-containing mRNA in yeast. Moreover, the Fed-1 iLRE substantially enhanced translation of reporter mRNAs in plant protoplasts expressing HSP101. Expression of HSP101 was subject to developmental regulation in leaves in that expression was highest in young leaves. These data suggest that Fed-1 mRNA may use the HSP101 regulatory mechanism as a means of ensuring a high level of translation required for the light-mediated regulation of Fed-1 mRNA stability.