Characterization of RNA-binding properties of three types of RNA-binding proteins in Anabaena sp. PPC 7120.
The Rbp proteins in cyanobacteria are RNA-binding proteins with a single RNA recognition motif or RRM. A comprehensive assembly of genomic data suggests that there are two major classes of Rbp proteins (classes I and II) that diverged before the diversification of cyanobacteria. Class I proteins are further classified into two types with or without a C-terminal glycine-rich domain. The results of selection from a random RNA pool suggest that RbpA1 (class I) has affinity to C-rich and G-rich sequences. In vitro RNA binding assay with homopolymers indicated that class II protein has low affinity to poly(G) in contrast with class I proteins. Site-specific mutagenesis analysis of the RRM in RbpA1 showed that the aromatic residues Tyr4 or Phe46 are important in RNA binding as well as maintenance of secondary structure. We also tested various truncated proteins lacking the C-terminal domain as well as point mutants. Most of these proteins exhibited decreased affinity to RNA. Circular dichroism analysis as well as chromatographic analysis showed that Tyr4 and Phe46 are also important in maintaining the structure of RbpA1 protein. The C-terminal glycine-rich domain itself does not contribute much to the RNA-binding, but Arg83 which is located close to the C-terminal end of RRM is important in the RNA-binding.
Hamano
T
T
Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-Ohkubo, Sakura-ku, Saitama, Saitama Prefecture, 338-8570, Japan.
Murakami
S
S
Takayama
K
K
Ehira
S
S
Maruyama
K
K
Kawakami
H
H
Morita
E H
EH
Hayashi
H
H
Sato
N
N
D000595:Amino Acid Sequence; D017033:Anabaena / Q000378:metabolism*; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
D016601:RNA-Binding Proteins; D010649:Phenylalanine; D012313:RNA