RNA-binding activities of barley stripe mosaic virus gamma b fusion proteins.
The barley stripe mosaic virus (BSMV) gamma-b gene encodes a 17 kDa cysteine-rich protein known to affect virulence and to have a role in regulating viral gene expression. We have constructed recombinant gamma-b-glutathione S-transferase fusion proteins in Escherichia coli and have determined the ability of the purified fusion proteins and various mutant derivatives to bind nucleic acids in vitro. Gel-shift analyses revealed that the wild-type gamma-b-fusion protein is able to bind RNA cooperatively. The binding affinity is highly selective for single-stranded RNA because double-stranded RNA, single-stranded and double-stranded DNA, and transfer RNA were unable to compete for binding with the labelled RNA probes. However, BSMV-specific sequence binding was not observed since a chloroplast RNA competed for binding with 32P-labelled transcripts derived from the BSMV genome. The first 44 amino acids of the 152 amino acid gamma-b fusion protein encompassing one of two cysteine-rich 'zinc finger-like' motifs, and a basic region separating the finger-like motifs are required for RNA binding. Site- specific amino acid substitutions within two groups of lysine and arginine residues located in the basic motif reduced the binding affinity of the fusion protein greatly, but cysteine and histidine substitutions designed to disrupt the finger-like motifs failed to have appreciable effects on binding. These findings indicate that the regulatory properties of gamma-b may be mediated in part by RNA binding activities.
Donald
R G
RG
Department of Plant Biology, University of California, Berkeley 94720, USA.
Jackson
A O
AO
D000595:Amino Acid Sequence; D001483:Base Sequence; D001467:Hordeum / Q000821:virology*; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D012313:RNA / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D012313:RNA