Function of the conserved S1 and KH domains in polynucleotide phosphorylase.
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.
Stickney
Leigh M
LM
Department of Biochemistry & Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
Hankins
Janet S
JS
Miao
Xin
X
Mackie
George A
GA
D001665:Binding Sites / Q000235:genetics; D053487:DEAD-box RNA Helicases; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D009097:Multienzyme Complexes / Q000378:metabolism; D011117:Polyribonucleotide Nucleotidyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology*; D020365:RNA Helicases / Q000378:metabolism; D012329:RNA, Bacterial / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D012329:RNA, Bacterial; C111244:degradosome; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; C022537:ribonuclease E; C502431:RhlB protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases