The 41 kDa protein component of the spinach chloroplast petD mRNA 3' stem-loop:protein complex is a nuclear encoded...

Spinach chloroplast petD gene encodes subunit IV of the cytochrome b6/f complex. Like many chloroplast mRNAs, the spinach petD mRNA contains a 3'UTR stem-loop structure that determines correct 3' processing of the pre-mRNA and stability of the mature mRNA. RNA-protein interactions with this structure may be involved in the regulation of petD mRNA processing and/or stability. In this report, a spinach chloroplast 41 kD protein has been identified as a component of the petD mRNA 3'stem-loop:protein complex. The 41 kD protein has been purified and four internal peptide sequences were obtained. Based on the peptide sequences, a 585 bp fragment was amplified from a spinach cDNA library by PCR. And, a 1404 bp cDNA clone was then isolated by using the initial PCR fragment as probe. Nucleic acid sequence analysis of the cDNA clone identified an ORF encoding a predicted 36 kD mature protein. An apparent N-terminal transit peptide in the coding region and a 3' poly(A) tail exist in the cDNA clone indicated that this chloroplast protein as nuclear encoded. The mature protein was expressed in E. coli, and the expressed product with a molecular mass of 41 kD was purified. A gel mobility shift assay demonstrated that the expressed 41 kD protein interacts with the petD mRNA 3' UTR to form an RNA-protein complex.