Identification of a region of Bacillus subtilis Ffh, a homologue of mammalian SRP54 protein, that is essential for binding to small cytoplasmic RNA.
Bacillus subtilis Ffh and scRNA are homologues of mammalian SRP54 and SRP RNA, respectively, which are components of the eukaryotic signal recognition particle (SRP). Ffh (446 amino acids) interacts with scRNA to form a stable complex in vivo. Here, we identified an RNA-binding domain of Ffh. The results obtained using a series of deletion mutants show that amino acid positions 364 to 432 in the C-terminal region of Ffh correlates with its ability to bind RNA. The amino acid sequence of this region is well conserved among members of the SRP54 protein family. This sequence contains two hydrophobic regions (h2, 364 to 391, and h3, 416 to 435), separated by the positively charged amino acid motif, 398RRKRIAKGSG407. Among the basic amino acid residues in this region, Arg-401 was essential for binding to scRNA, but Arg-399 and Lys-400 were not. The co-existence of Arg-398 and Lys-404 was necessary for the same affinity as wild type Ffh. The two glycine residues of the 405GSG407 were also essential. MH23 peptide (91 amino acids) encompassing from 356 to 446, consisting of h2-RRKRIAKGSG-h3, bound scRNA with the same affinity as wild type Ffh, whereas a 24-amino acid synthetic peptide 392DIINASRRKRIAKGSGTSVQEVNR415 did not. The region containing two hydrophobic segments separated by the positively charged motif is the minimal requirement of Ffh for RNA binding.
Kurita
K
K
Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan.
Honda
K
K
Suzuma
S
S
Takamatsu
H
H
Nakamura
K
K
Yamane
K
K
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D020733:RNA, Small Cytoplasmic; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D018271:Signal Recognition Particle / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D010455:Peptides; D020733:RNA, Small Cytoplasmic; D016601:RNA-Binding Proteins; D018271:Signal Recognition Particle; D012313:RNA