Specific ribonuclease activities in spinach chloroplasts promote mRNA maturation and degradation.
We have used an in vitro system to characterize ribonuclease activities present in spinach chloroplasts. We show that 3' end maturation of petD mRNA, which encodes subunit IV of the cytochrome b6/f complex, is affected by a 33-kDa protein that binds to a hairpin structure at the 3' end of the mature mRNA. Binding of the 33-kDa protein to the petD hairpin structure decreases the efficiency of 3' end maturation, probably by impeding the progress of the processive 3'-5' exonuclease activity involved in chloroplast mRNA processing. A two-base mutation in the stem of the petD hairpin structure creates a novel recognition site for a ribonuclease which competes with the normal processing exonuclease activity. This mutation results in a very low 3' end processing efficiency for mutant petD transcripts, and instead generates a second processing product that lacks a complete hairpin structure. An endonuclease activity which is biochemically distinct from the previously characterized exonuclease activities has also been identified. This endonuclease activity is EDTA-insensitive, and cleaves petD RNA both at the termination codon and at the mature RNA 3' end. Cleavage of petD mRNA at the termination codon leads to rapid degradation of upstream RNA. The possible roles of these ribonuclease activities in chloroplast mRNA decay in vivo are discussed.
Chen
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Boyce Thompson Institute for Plant Research, Ithaca, New York 14853-1801.
Stern
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DB
D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D003573:Cytochrome b Group / Q000235:genetics*; D045346:Cytochrome b6f Complex; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D012313:RNA / Q000138:chemical synthesis / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*
D003573:Cytochrome b Group; D012333:RNA, Messenger; D012313:RNA; D045346:Cytochrome b6f Complex; D012260:Ribonucleases