Proof that the bacterial primase hypothetical zinc finger sequence binds zinc

Mark A. Griep, Betsy J. Adkins, Daniel Hromas, Scott Johnson, Jennifer D. Miller

Research output: Contribution to journalArticle

Abstract

Primase is a special monomeric RNA polymerase that synthesizes RNA primers on the lagging strand DNA during replication. A 35-amino acid peptide corresponding to the putative 2zinc finger2 sequence of primase was prepared to study its zinc binding properties. When zinc was added to the peptide, it was found that the fluorescence quantum yield of the single tyrosine increased by 46% and the average lifetime by 34%. The binding stoichiometry was one zinc per peptide. The affinity at pH 7.5 was between 10 and 100 nM. The absorption spectrum of the cobalt(II)-peptide complex was consistent with tetrahedral metal coordination by three sulfur and one imidazole nitrogen ligands. The peptide affinity for cobalt was less than for zinc indicating metal specificity. Analysis of the fluorescence intensity pH profile, circular dichroism spectra, the effect of extrinsic quenchers indicated that at neutral pH: 1) the free peptide folded up into a structure to place the tyrosine in an environment protected from solvent, 2) the peptide bound zinc via its three cysteines and one of its histidines resulting in little change to the polypeptide secondary structure or to the tyrosine solvent accessibility, and 3) when the peptide bound zinc, it bound directly to or caused the immobilization of the groups that had been intramolecularly collisionally quenching the tyrosine which resulted in the observed increases in tyrosine quantum yield and lifetime. This work was supported by Council for Tobacco Research and the National Science Foundation.

Original languageEnglish (US)
Pages (from-to)A1365
JournalFASEB Journal
Volume11
Issue number9
Publication statusPublished - Dec 1 1997

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ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Griep, M. A., Adkins, B. J., Hromas, D., Johnson, S., & Miller, J. D. (1997). Proof that the bacterial primase hypothetical zinc finger sequence binds zinc. FASEB Journal, 11(9), A1365.