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Báo cáo khoa học: RNA-binding properties of HCF152, an Arabidopsis PPR protein involved in the processing of chloroplast RNA

The nonphotosynthetic mutant ofArabidopsis hcf152is impaired in the processing of the chloroplast polycistronic transcript, psbB-psbT-psbH-petB-petD, resulting in non-production of the essential photosynthetic cytochromeb6 f complex. The nucleus-encodedHCF152gene was identified to encode a pentatricopeptide repeat (PPR) protein com-posed primarily of 12 PPR motifs, similar to other proteins of this family that were identified in mutants defected in chloroplast gene expression. | Eur. J. Biochem. 270 4070-4081 2003 FEBS 2003 doi 10.1046 j.1432-1033.2003.03796.x RNA-binding properties of HCF152 an Arabidopsis PPR protein involved in the processing of chloroplast RNA Takahiro Nakamura1 Karin Meierhoff2 Peter Westhoff2 and Gadi Schuster1 1 Department of Biology Technion - Israel Institute of Technology Haifa Israel 2 Heinrich-Heine-Universitat Institut fur Entwicklungs und Molekularbiologie der Pflanzen Universitatstrasse 1 Dusseldorf Germany The nonphotosynthetic mutant of Arabidopsis hcf152 is impaired in the processing of the chloroplast polycistronic transcript psbB-psbT-psbH-petB-petD resulting in nonproduction of the essential photosynthetic cytochrome b f complex. The nucleus-encoded HCF152 gene was identified to encode a pentatricopeptide repeat PPR protein composed primarily of 12 PPR motifs similar to other proteins of this family that were identified in mutants defected in chloroplast gene expression. To understand the molecular mechanism of how HCF152 modulates chloroplast gene expression the molecular and biochemical properties should be revealed. To this end HCF152 and several truncated versions were produced in bacteria and analyzed for RNA-binding and protein-protein interaction. It was found that two HCF152 polypeptides bind to form a homodimer and that this binding is impaired by a single amino acid substitute near the carboxyl terminus replacing leucine with proline. Recombinant HCF152 bound with higher affinity RNA molecules resembling the petB exon-intron junctions as well as several other molecules. The highest affinity was found to RNA composed of the poly A sequence. When truncated proteins composed of different numbers of PPR motifs were analyzed for RNA-binding it was found that two PPR motifs were required for RNA-binding but had very low affinity. The affinity to RNA increased significantly when proteins composed of more PPR motifs were analyzed displaying the highest affinity with the full-length protein composed .