Preparation of Escherichia coli Rne Protein and Reconstitution of RNA Degradosome

Mackie, George A.; Coburn, Glen A.; Miao, Xin; Briant, Douglas J.; Prud'homme-Genereux, Annie

doi:10.1016/s0076-6879(01)42557-2

Cited by 13 publications

(14 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Assay of minimal degradosomes reconstituted with truncated PNPase derivatives. Rne⌬408, RhlB, and derivatives of PNPase were purified, reconstituted, and assayed on the 375-nt malEF RNA substrate as described previously (6,15). Portions of the incubation mixture were removed at the time indicated above each lane, denatured with 3 volumes of 90% formamide, separated electrophoretically on a 6% polyacrylamide gel containing 8 M urea, and quantified using a PhosphorImager.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Function of the Conserved S1 and KH Domains in Polynucleotide Phosphorylase

et al. 2005

Self Cite

View full text Add to dashboard Cite

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 multipleturnover 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.The processing and/or degradation of RNAs, including rRNA, tRNA and mRNA, is a critical posttranscriptional regulatory step. In Escherichia coli, several enzymes which participate in RNA processing and degradation are organized into a macromolecular complex, the RNA degradosome (3,18,22). Major components of the degradosome include RNase E, an 5Ј-end-dependent endonuclease, polynucleotide phosphorylase (PNPase), a phosphate-dependent 3Ј exonuclease, RhlB, a DEAD box RNA helicase, and enolase, an abundant glycolytic enzyme (3,5,27). Other proteins associate with the degradosome in apparently substoichiometric quantities, but only RNase E, PNPase, and RhlB are required to reconstitute the activity of the RNA degradosome in vitro (6).Although their activities are quite different, both RNase E and PNPase share a common structural motif, an S1 (or oligonucleotide/oligosaccharide binding fold) domain, as do RNase G and RNase II (2). The relative locations of the S1 domain in these RNases are shown in Fig. 1a. Its location provides no clue to its function(s) in any of these enzymes. S1 domains are also found in a number of unrelated proteins whose principal common feature is interaction with singlestranded nucleic acids (1,20,30). The solution structure of the S1 domain in PNPase has been determined and consists of five antiparallel ␤-strands with surface-exposed hydrophobic and basic residues (2). The structure of the S1 domain of RNase E has also been determined recently and displays a similar overall fold (8,25). Both of the best-characterized mutations in RNase E, rne-1 (G66S) and rne-3071 (...

show abstract

Section: Discussionmentioning

confidence: 99%

“…In all cases, products were quantified using a PhosphorImager (Molecular Dynamics). Minimal degradosomes were reconstituted and assayed on the 375-nucleotide (nt) malEF RNA substrate as described previously (6,15).…”

Section: Methodsmentioning

confidence: 99%

Function of the Conserved S1 and KH Domains in Polynucleotide Phosphorylase

et al. 2005

Self Cite

View full text Add to dashboard Cite

show abstract

“…"Standard" degradosomes were purified from strain CF881 grown in LB medium with vigorous aeration at 37°C as described previously (26). "Cold shock" degradosomes were purified from the same strain grown at 30°C in LB medium to an A 600 of 0.4 and then mixed with an equal volume of ice-cold LB and shifted to a water bath at 15°C.…”

Section: Strainsmentioning

confidence: 99%

Role of RNA Structure and Susceptibility to RNase E in Regulation of a Cold Shock mRNA, cspA mRNA

et al. 2007

Self Cite

View full text Add to dashboard Cite

Degradation of the cspA mRNA in vivo is very rapid at temperatures greater than 30°C and is moderately dependent on RNase E. Investigations in vitro show that degradosomes prepared from normal or cold-shocked cultures cleave the cspA mRNA preferentially at a single site in vitro between two stem-loops ϳ24 residues 3 to the termination codon and ϳ31 residues from the 3 end. The site of cleavage is independent of the temperature and largely independent of the phosphorylation status of the 5 end of cspA mRNA. A 5 stem-loop, potential occlusion of the initiation and termination codons, temperature-dependent translational efficiency, and the position of the RNase E cleavage site can explain the differential stability of the cspA mRNA.

show abstract

“…In addition, PNPase may exist as a single homotrimeric enzyme, associated with the RNA helicase RhlB (32), and in a multiprotein machine, the RNA degradosome, together with the endoribonuclease RNase E, which also serves as a scaffold for the assembly of the complex, RhlB, and enolase. In such heteromultimeric associations, PNPase can degrade otherwise refractory double-stranded RNA regions in an ATP-dependent manner (32)(33)(34)(35)(36), a property that has been used as a degradosome functional assay (37).…”

mentioning

confidence: 99%

Regulation of Escherichia coli Polynucleotide Phosphorylase by ATP

Favero

Mazzantini

Briani

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

Polynucleotide phosphorylase (PNPase), an enzyme conserved in bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA, releasing nucleotide diphosphates, and the reverse polymerization reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either poly(A) or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Here, we report that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPasedependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase.Polynucleotide phosphorylase (PNPase), 3 a polyribonucleotide nucleotidyltransferase (EC 2.7.7.8), is a homotrimeric enzyme involved in RNA turnover in bacteria and eukaryotic organelles (1). PNPase processively catalyzes the phosphorolysis of RNA in the 3Ј-to 5Ј-direction, thus releasing nucleoside diphosphates, and the reverse 5Ј-to 3Ј-template-independent polymerization of nucleoside diphosphates (2-7). PNPase also binds RNA via its KH and S1 RNA-binding domains located at the C terminus of the polypeptide (6, 8 -11).The monomeric subunit exhibits a five-domain structure that is widely conserved from bacteria to plants and mammals (12, 13). The structural core of the subunit appears to be a duplication of an RNase PH domain, with the two RNase PHlike domains connected by a poorly conserved linker domain. In the doughnut-shaped homotrimeric protein, the subunits form a central channel where catalysis is thought to occur; the KH and S1 RNA-binding domains are located on top of the enzyme, with the former immediately above the central channel and the latter facing outward away from the channel (14).PNPase was originally implicated in the synthesis of cellular RNA before the template-dependent RNA polymerase was discovered (2, 15, 16); later on, because of its phosphorolytic activity, it was implicated in RNA degradation (6). It has long been assumed that, because of the high P i intracellular concentration, PNPase would act in vivo mainly phosphorolytically (17). More recently, however, it was shown that PNPase can add heteropolymeric tails to RNA 3Ј-ends (18,19). Because in Escherichia coli PNPase-dependent heteropolymeric failing, like polyadenylation by polyadenyl polymerase (PAP), targets bacterial RNAs to degradation (20), both PNPase phosphorolytic and polymerization activities participate in PNPase-dependent RNA decay. Finally, it was suggested that PNPase plays a central role in the biosynthetic pathway of dCTP by providing the CDP precursor (21), thus linking RNA turnover to DNA replication.Although widely conserved in Bacteria and Eukarya, the pnp gene does not seem to be essential for s...

show abstract

Preparation of Escherichia coli Rne Protein and Reconstitution of RNA Degradosome

Cited by 13 publications

References 17 publications

Function of the Conserved S1 and KH Domains in Polynucleotide Phosphorylase

Function of the Conserved S1 and KH Domains in Polynucleotide Phosphorylase

Role of RNA Structure and Susceptibility to RNase E in Regulation of a Cold Shock mRNA, cspA mRNA

Regulation of Escherichia coli Polynucleotide Phosphorylase by ATP

Contact Info

Product

Resources

About