RNA-binding activities of the different domains of a spinach chloroplast ribonucleoprotein.
An RNA-binding protein of 28 kD (28RNP) has been previously isolated from spinach chloroplasts and was found to be required for 3' end processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic and glycine-rich amino terminal domain. Each domain by itself, as well as in combination with other domains, was expressed in bacterial cells and the polypeptides were purified to homogeneity. We have investigated the RNA-binding properties of the different structural domains using UV-crosslinking, saturation binding and competition between the different domains on RNA-binding. It was found that the acidic domain does not bind RNA, but that each of the RNA-binding domains, expressed either individually or together, do bind RNA, although with differing affinities. When either the first or second RNA-binding domain was coupled to the acidic domain, the affinity for RNA was greatly reduced. However, the acidic domain has a positive effect on the binding of the full-length protein to RNA, because the mature protein binds RNA with a better affinity than the truncated protein which lacks the acidic domain. In addition, it was found that a stretch of two or three G residues is enough to mediate binding of the 28RNP, whereas four U residues were insufficient. The implications of the RNA-binding properties of 28RNP to its possible function in the processing of chloroplast RNA is discussed.
Lisitsky
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I
Department of Biology, Technion-Israel Institute of Technology, Haifa.
Liveanu
V
V
Schuster
G
G
D001483:Base Sequence; D001667:Binding, Competitive; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000378:metabolism*; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D011068:Poly G / Q000378:metabolism; D018747:RNA, Chloroplast / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion / Q000502:physiology; D018724:Spinacia oleracea
D060365:Chloroplast Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D018747:RNA, Chloroplast; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D011068:Poly G