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Journal of Bacteriology, May 2006, p. 3614-3621, Vol. 188, No. 10
0021-9193/06/$08.00+0     doi:10.1128/JB.188.10.3614-3621.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Role of Conserved Amino Acids in the Catalytic Activity of Escherichia coli Primase

Anna Rodina{dagger} and G. Nigel Godson*

Biochemistry Department, New York University Medical Center, New York, New York 10016

Received 2 December 2005/ Accepted 1 March 2006

The role of conserved amino acid residues in the polymerase domain of Escherichia coli primase has been studied by mutagenesis. We demonstrate that each of the conserved amino acids Arg146, Arg221, Tyr230, Gly266, and Asp311 is involved in the process of catalysis. Residues Glu265 and Asp309 are also critical because a substitution of each amino acid irreversibly destroys the catalytic activity. Two K229A and M268A mutant primase proteins synthesize only 2-nucleotide products in de novo synthesis reactions under standard conditions. Y267A mutant primase protein synthesizes both full-size and 2-nucleotide RNA, but with no intermediate-size products. From these data we discuss the significant step of the 2-nucleotide primer RNA synthesis by E. coli primase and the role of amino acids Lys229, Tyr267, and Met268 in primase complex stability.

* Corresponding author. Mailing address: Biochemistry Department, New York University Medical School, 550 First Avenue, New York, NY 10016. Phone: (212) 263-5622. Fax: (212) 263-8166. E-mail: gn.godson{at}med.nyu.edu.

{dagger} Present address: Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

Journal of Bacteriology, May 2006, p. 3614-3621, Vol. 188, No. 10
0021-9193/06/$08.00+0     doi:10.1128/JB.188.10.3614-3621.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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