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Journal of Bacteriology, November 2005, p. 7214-7221, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7214-7221.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Function of the Conserved S1 and KH Domains in Polynucleotide Phosphorylase

Leigh M. Stickney, Janet S. Hankins, Xin Miao, and George A. Mackie*

Department of Biochemistry & Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3

Received 7 June 2005/ Accepted 19 August 2005

We have examined the roles of the conserved S1 and KH RNA binding motifs in the widely dispersed prokaryotic exoribonuclease polynucleotide phosphorylase (PNPase). These domains can be released from the enzyme by mild proteolysis or by truncation of the gene. Using purified recombinant enzymes, we have assessed the effects of specific deletions on RNA binding, on activity against a synthetic substrate under multiple-turnover conditions, and on the ability of truncated forms of PNPase to form a minimal RNA degradosome with RNase E and RhlB. Deletion of the S1 domain reduces the apparent activity of the enzyme by almost 70-fold under low-ionic-strength conditions and limits the enzyme to digest a single substrate molecule. Activity and product release are substantially regained at higher ionic strengths. This deletion also reduces the affinity of the enzyme for RNA, without affecting the enzyme's ability to bind to RNase E. Deletion of the KH domain produces similar, but less severe, effects, while deletion of both the S1 and KH domains accentuates the loss of activity, product release, and RNA binding but has no effect on binding to RNase E. We propose that the S1 domain, possibly arrayed with the KH domain, forms an RNA binding surface that facilitates substrate recognition and thus indirectly potentiates product release. The present data as well as prior observations can be rationalized by a two-step model for substrate binding.

* Corresponding author. Mailing address: Department of Biochemistry & Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. Phone: (604) 822-5943. Fax: (604) 822-5227. E-mail: gamackie{at}interchange.ubc.ca.

Journal of Bacteriology, November 2005, p. 7214-7221, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7214-7221.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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