News and Publications

mRNA-Protein Interaction in Light-Activated Translation

S.P. Mayfield, D. Barnes, K. Bieza, E. Brown, A. Caragliotti, E. Efuet, K. Espina, S. Franklin, D. Hambly, B. Ngo, J. Schultz, A. Somanchi, K. Yamaguchi

Gene expression in most organisms is keyed to environmental signals. In plants and algae, expression of key genes is regulated in response to exposure to light. Biochemical and genetic analyses revealed that light activates translation of mRNAs encoding proteins of the photosynthetic apparatus and that both RNA elements and corresponding RNA-binding proteins are required for this translational regulation.

A number of proteins that bind with high affinity and specificity to RNA elements contained within the untranslated regions of chloroplast mRNAs have been isolated. We identified 4 major, and several minor, proteins that bind to the 5´ untranslated region of the chloroplast psbA mRNA in the green alga Chlamydomonas reinhardtii. One of these proteins is homologous to 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 has homology to protein disulfide isomerases, enzymes involved in the oxidation and reduction of disulfide bonds within proteins. We showed that the chloroplast protein disulfide isomerase can modulate the mRNA-binding activity of chloroplast PABP by changing the sulfide bonds of cysteine residues within the RNA-binding domains in a redox-dependent manner. The 2 other major psbA-associated proteins are novel RNA- binding proteins.

Using in vitro selection, site-directed mutagenesis, and reporter gene constructs, we identified several RNA elements required for protein binding and light-activated translation. We found that a ribosome-binding sequence similar to sequences found in bacteria is essential for ribosome association of chloroplast mRNAs. The ribosome-binding sequence of chloroplast mRNAs is positioned farther upstream than is the ribosome-binding sequence of bacterial mRNAs, suggesting a fundamental difference between chloroplasts and bacteria in the mechanism by which a ribosome-binding sequence facilitates initiation of translation.

We also analyzed a number of nuclear mutants deficient in psbA mRNA translation. In these mutants, specific members of the RNA-binding complex, such as the PABP, are required for initiation of translation of psbA mRNA. This genetic analysis has also defined a unique set of proteins, not previously identified in other species, as required for 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 the chloroplast PABP, and other RNA-binding proteins, to the 5´ untranslated region of the chloroplast psbA mRNA. Binding of these proteins to RNA elements allows increased ribosome association and initiation of translation. Binding of the chloroplast PABP to these mRNAs also allows association of the message with photosynthetic membranes, possibly targeting the mRNA to the correct site of protein insertion. We are using proteomic and genetic analysis to identify the complete set of proteins and RNA elements required for chloroplast translation. Structural analysis of the identified RNA-protein complex is being used to determine how RNA-protein interactions enhance ribosome binding and membrane association and regulate translational activation.

PUBLICATIONS

Brown, E.C., Somanchi, A., Mayfield, S.P. Interorganellar crosstalk: New perspectives on signaling from the chloroplast to the nucleus. Genome Biol. 2:1021, 2001.

Cohen, A., Yohn, C., Mayfield, S.P. Translation of the chloroplast-encoded psbD mRNA is arrested in a nuclear mutant of Chlamydomonas reinhardtii. J. Plant Physiol. 158:1069, 2001.

Somanchi, A., Mayfield, S.P. Regulation of chloroplast translation. In: Regulation of Photosynthesis. Aro, E.-M., Anderson, R. (Eds.). Kluwer, Boston, 2001, p. 121. Vol. 11 in Advances in Photosynthesis and Respiration.

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