Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein.
The RNA-binding properties of the cell-to-cell movement protein (MP) of Apple chlorotic leaf spot virus were analysed. MP was expressed in Escherichia coli and was used in UV-crosslinking analysis, using a digoxigenin-UTP-labelled RNA probe and gel-retardation analysis. The analyses demonstrated that MP bound cooperatively to single-stranded RNA (ssRNA). When analysed for NaCl dependence of the RNA-binding activity, the majority of the MP could bind ssRNA even in binding buffer with 1 M NaCl. Furthermore, competition binding experiments showed that the MP bound preferentially to ssRNA and single-stranded DNA without sequence specificity. MP deletion mutants were used to identify the RNA-binding domain by UV-crosslinking analysis. Amino acid residues 82-126 and 127-287 potentially contain two independently active, single-stranded nucleic acid-binding domains.
Isogai
Masamichi
M
Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda 3-chome 18-8, Morioka 020-8550, Japan.
Yoshikawa
Nobuyuki
N
Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda 3-chome 18-8, Morioka 020-8550, Japan.
D000595:Amino Acid Sequence; D004076:Digoxigenin; D015180:Electrophoresis, Gel, Two-Dimensional; D008969:Molecular Sequence Data; D053830:Plant Viral Movement Proteins; D010942:Plant Viruses / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D053830:Plant Viral Movement Proteins; D012367:RNA, Viral; D014764:Viral Proteins; D004076:Digoxigenin