News and Publications

mRNA-Protein Interaction in Light-Activated Translation

S.P. Mayfield, R. Bruick, A. Cohen, J. Kim, A. Lentz, E. Efuet, P. Choi

Translation of many plant mRNAs is regulated in response to light. Genetic analysis has revealed that RNA-binding proteins are required for this translational regulation and that these proteins interact with RNA elements contained within the 5´ untranslated region of specific mRNAs. We are examining how RNA-binding proteins recognize specific RNA elements and how this mRNA-protein interaction results in translational activation. These studies are being carried out in the green alga Chlamydomonas reinhardtii.

We have isolated a set of proteins that bind with high affinity and specificity to the 5´ untranslated region of the chloroplast psbA mRNA. Binding of these proteins to the mRNA is light regulated and is required for initiation of translation of this mRNA. Analysis of a cDNA that encodes one of these RNA-binding proteins indicated that the protein is a member of the poly(A)-binding proteins (PABPs). PABPs are RNA-binding proteins that facilitate the interaction of mRNAs and ribosomes.

Another of the psbA mRNA-binding proteins that we have detected is a protein disulfide isomerase, an enzyme involved in the oxidation and reduction of disulfide bonds associated with protein folding. The isomerase associated with psbA mRNA can alter the mRNA-binding activity of the PABP RNA-binding protein by changing sulfide bonds to sulfhydryl bonds in a redox-dependent manner. These data suggest that light-activated translation may involve activation of PABP binding to the psbA mRNA through changes in the light-generated redox potential of the cell.

Using in vitro selection, site-directed mutagenesis, and structural mapping of the 5´ untranslated region of the psbA mRNA, we determined that RNA elements located adjacent to a consensus ribosome-binding site are required for protein binding and light-activated translation. In addition, we analyzed nuclear mutants deficient in psbA mRNA translation. The results indicated that specific members of the psbA RNA-binding complex are required for psbA RNA binding, psbA mRNA-ribosome association, and psbA mRNA translation.

On the basis of these studies, a model for translational activation can be drawn in which redox potential, generated by the light reactions of photosynthesis, is used by chloroplast protein disulfide isomerase to activate the binding of a PABP to the 5´ untranslated region of the chloroplast psbA mRNA. Binding of these proteins to the RNA elements results in alterations of the RNA structure at the ribosome-binding site and allows increased ribosome association and initiation of translation. Structural analysis of the RNA-protein complex is being used to determine how RNA-protein interaction results in increased ribosome association and regulated translational activation.

PUBLICATIONS

Cohen, A., Yohn, C.B., Bruick, R., Mayfield, S.P. Translational regulation of chloroplast gene expression in Chlamydomonas reinhardtii. Methods Enzymol. 297:192, 1998.

Mayfield, S.P., Cohen A. Translational regulation in the chloroplast. Curr. Top. Plant Physiol. 19:174, 1998.

Yohn, C., Cohen, A., Danon, A., Mayfield, S.P. A poly(A) binding protein functions in the chloroplast as a message-specific translation factor. Proc. Natl. Acad. Sci. U.S.A. 95:2238, 1998.

Yohn, C., Cohen, A., Rosch, C., Kuchka, M., Mayfield, S.P. Translation of the chloroplast psbA mRNA requires the nuclear encoded poly(A) binding protein, RB47. J. Cell Biol. 142:435, 1998.