Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain

Takayuki Obita, Takafumi Iwura, Masayuki Su'etsugu, Yoichiro Yoshida, Yoshitsugu Tanaka, Tsutomu Katayama, Tadashi Ueda, Taiji Imoto

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

DnaA protein binds specifically to a group of binding sites collectively called as DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA. The DNA-binding domain of DnaA, domain IV, comprises the C-terminal 94 amino acid residues of the protein. We overproduced and purified a protein containing only this domain plus a methionine residue. This protein was stable as a monomer and maintained DnaA box-specific binding activity. We then analyzed its solution structure by CD spectrum and heteronuclear multi-dimensional NMR experiments. We established extensive assignments of the 1H, 13C, and 15N nuclei, and revealed by obtaining combined analyses of chemical shift index and NOE connectivities that DnaA domain IV contains six α-helices and no β-sheets, consistent with results of CD analysis. Mutations known to reduce DnaA box-binding activity were specifically located in or near two of the α-helices. These findings indicate that the DNA-binding fold of DnaA domain IV is unique among origin-binding proteins.

Original languageEnglish
Pages (from-to)42-48
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume299
Issue number1
DOIs
Publication statusPublished - Dec 2 2002

Fingerprint

Escherichia coli
DNA
Proteins
Replication Origin
Chemical shift
Methionine
Carrier Proteins
Monomers
Binding Sites
Nuclear magnetic resonance
Amino Acids
Mutation
Experiments

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain. / Obita, Takayuki; Iwura, Takafumi; Su'etsugu, Masayuki; Yoshida, Yoichiro; Tanaka, Yoshitsugu; Katayama, Tsutomu; Ueda, Tadashi; Imoto, Taiji.

In: Biochemical and Biophysical Research Communications, Vol. 299, No. 1, 02.12.2002, p. 42-48.

Research output: Contribution to journalArticle

Obita, Takayuki ; Iwura, Takafumi ; Su'etsugu, Masayuki ; Yoshida, Yoichiro ; Tanaka, Yoshitsugu ; Katayama, Tsutomu ; Ueda, Tadashi ; Imoto, Taiji. / Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain. In: Biochemical and Biophysical Research Communications. 2002 ; Vol. 299, No. 1. pp. 42-48.
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