Murakami, Shinya , Stern, David .
A protein complex stabilizes petD mRNA in Chloroplast.
Chloroplast gene expression is regulated at several levels, one of the most important being mRNA stability. To investigate this regulation, we have used the unicellular green alga Chlamydomonas reinhardtii, and focused on the chloroplast-encoded petD mRNA as a model. Chloroplast RNA stability is regulated primarily by nucleus-encoded polypeptides, and one such protein is MCD1. Strains lacking MCD1 are nonphotosynthetic because petD mRNA is rapidly degraded by a 5’ to 3’ exoribonuclease and its gene product, subunit IV of cytochrome b6/f complex, is therefore not synthesized. To understand the function of MCD1 in regulating petD stability, its cDNA sequence was determined. MCD1 encodes a 156.1 kDa protein, but contains no conserved motifs that would hint at its mechanism of action. We therefore constructed a variant of MCD1 encoding C-terminal double HA-, His-, and S-tags, and used this variant to complement an mcd1 mutant strain. The peptide tagged MCD1 restored normal accumulation of petD mRNA and rescued the non-photosynthetic phenotype of the mutant. We have used this strain for biochemical characterization and shown MCD1 to be a component of a protein complex by gel filtration. RNase treatment changed the elution pattern of this complex suggesting that the MCD1-containing complex interacts with RNA. We also have been able to purify this complex by Ni and S-protein affinity chromatography. The role of MCD1 complex in petD mRNA stability will be discussed.
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1 - Boyce Thompson Institute for Plant Research, Tower Rd., Ithaca, NY, 14853, USA
2 - Boyce Thompson Institute for Plant Research
Chloroplast gene expression
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM