Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria

Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria

Divisions of ^*Biological Science and ^†Material Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan

Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved November 10, 1999 (received for review August 3, 1999)

Abstract

A negative feedback control of kaiC expression by KaiC protein has been proposed to generate a basic oscillation of the circadian clock in the cyanobacterium Synechococcus sp. PCC 7942. KaiC has two P loops or Walker's motif As, that are potential ATP-/GTP-binding motifs and DXXG motifs conserved in various GTP-binding proteins. Herein, we demonstrate that in vitro KaiC binds ATP and, with lower affinity, GTP. Point mutation by site-directed mutagenesis of P loop 1 completely nullified the circadian rhythm of kaiBC expression and markedly reduced ATP-binding activity. Moreover, KaiC can be autophosphorylated in vitro. These results suggest that the nucleotide-binding activity of KaiC plays important roles in the generation of circadian oscillation in cyanobacteria.

Footnotes

↵ ‡ Present address: Center for Gene Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
↵ § To whom reprint requests should be addressed. E-mail: kondo{at}bio.nagoya-u.ac.jp or ishiura{at}bio.nagoya-u.ac.jp.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

CI,

tandem duplicated domain of KaiC (N-terminal half);

CII,

tandem duplicated domain of KaiC (C-terminal half);

LL,

continuous light;

GST,

glutathione S-transferase