Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus

Akio Kanai, Hanako Oida, Nana Matsuura, Hirofumi Doi

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

We systematically screened a genomic DNA library to identify proteins of the hyperthermophilic archaeon Pyrococcus furiosus using an expression cloning method. One gene product, which we named FAU-1 (P. furiosus AU-binding), demonstrated the strongest binding activity of all the genomic library-derived proteins tested against an AU-rich RNA sequence. The protein was purified to near homogeneity as a 54 kDa single polypeptide, and the gene locus corresponding to this FAU-1 activity was also sequenced. The FAU-1 gene encoded a 472-amino-acid protein that was characterized by highly charged domains consisting of both acidic and basic amino acids. The N-terminal half of the gene had a degree of similarity (25%) with RNase E from Escherichia coli. Five rounds of RNA-binding-site selection and footprinting analysis showed that the FAU-1 protein binds specifically to the AU-rich sequence in a loop region of a possible RNA ligand. Moreover, we demonstrated that the FAU-1 protein acts as an oligomer, and mainly as a trimer. These results showed that the FAU-1 protein is a novel heat-stable protein with an RNA loop-binding characteristic.

Original languageEnglish
Pages (from-to)253-261
Number of pages9
JournalBiochemical Journal
Volume372
Issue number1
DOIs
Publication statusPublished - 2003 May 15

Fingerprint

Pyrococcus furiosus
RNA-Binding Proteins
Cloning
Organism Cloning
Genes
Proteins
RNA
Genomic Library
Escherichia coli
Acidic Amino Acids
Basic Amino Acids
Site selection
Archaea
Gene Library
Oligomers
Hot Temperature
Binding Sites
Ligands
Amino Acids
Peptides

Keywords

  • Archaea
  • RNA-binding protein
  • Stem-loop structure

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression cloning and characterization of a novel gene that encodes the RNA-binding protein FAU-1 from Pyrococcus furiosus. / Kanai, Akio; Oida, Hanako; Matsuura, Nana; Doi, Hirofumi.

In: Biochemical Journal, Vol. 372, No. 1, 15.05.2003, p. 253-261.

Research output: Contribution to journalArticle

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