Crystal Structure of Escherichia coli Polynucleotide Phosphorylase Core Bound to RNase E, RNA and Manganese: Implications for Catalytic Mechanism and RNA Degradosome Assembly

Nurmohamed, Salima; Vaidialingam, Bhamini; Callaghan, Anastasia J.; Luisi, Ben F.

doi:10.1016/j.jmb.2009.03.051

Cited by 99 publications

(175 citation statements)

References 60 publications

(88 reference statements)

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“…In E. coli, RNase E interacts with PNPase via a fragment of 23 residues (GAAGGHTAT HHASAAPARPQPVE, residues 1039-1061) (Callaghan et al 2004;Nurmohamed et al 2009), while in C. crescentus, the site is demonstrated to be the GWW motif (EKPRRGWW RR, residues 889-898) (Hardwick et al 2011(Hardwick et al , 2012. We showed that a highly conserved motif of cyanobacterial RNase E within the noncatalytic domain of AnaRne could bind to AnaPnp.…”

Section: Discussionmentioning

confidence: 88%

“…In terms of residue composition, these PNPase recognition sites display great variation. Accordingly, the regions on E. coli and C. crescentus PNPases that determine RNase E recognition are also distinct (Nurmohamed et al 2009;Hardwick et al 2012). These differences probably reflect the long coevolution history of PNPase-RNase E interactions in different species.…”

Section: Discussionmentioning

confidence: 99%

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RNase E forms a complex with polynucleotide phosphorylase in cyanobacteria via a cyanobacterial-specific nonapeptide in the noncatalytic region

Zhang

Deng

et al. 2014

RNA

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RNase E, a central component involved in bacterial RNA metabolism, usually has a highly conserved N-terminal catalytic domain but an extremely divergent C-terminal domain. While the C-terminal domain of RNase E in Escherichia coli recruits other components to form an RNA degradation complex, it is unknown if a similar function can be found for RNase E in other organisms due to the divergent feature of this domain. Here, we provide evidence showing that RNase E forms a complex with another essential ribonuclease-the polynucleotide phosphorylase (PNPase)-in cyanobacteria, a group of ecologically important and phylogenetically ancient organisms. Sequence alignment for all cyanobacterial RNase E proteins revealed several conserved and variable subregions in their noncatalytic domains. One such subregion, an extremely conserved nonapeptide (RRRRRRSSA) located near the very end of RNase E, serves as the PNPase recognition site in both the filamentous cyanobacterium Anabaena PCC7120 and the unicellular cyanobacterium Synechocystis PCC6803. These results indicate that RNase E and PNPase form a ribonuclease complex via a common mechanism in cyanobacteria. The PNPase-recognition motif in cyanobacterial RNase E is distinct from those previously identified in Proteobacteria, implying a mechanism of coevolution for PNPase and RNase E in different organisms.

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Section: Discussionmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

RNase E forms a complex with polynucleotide phosphorylase in cyanobacteria via a cyanobacterial-specific nonapeptide in the noncatalytic region

Zhang

Deng

et al. 2014

RNA

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“…87,116 PNPase has been shown to be associated with endonuclease RNase E and other proteins in the RNA degradosome. [117][118][119] In vitro, PNPase catalyzes phosphorolytic degradation of RNA, releasing nucleoside ©2 0 1 1 L a n d e s B i o s c i e n c e .…”

Section: Discussionmentioning

confidence: 99%

RNA remodeling and gene regulation by cold shock proteins

2010

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“…PNPase and the truncated core of PNPase lacking the S1 and KH domains were purified as described (42). Ribonuclease R (RNR07250; Epicentre) was stored at −20°C.…”

Section: Methodsmentioning

confidence: 99%

Direct observation of processive exoribonuclease motion using optical tweezers

Fazal

Koslover

Luisi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial RNases catalyze the turnover of RNA and are essential for gene expression and quality surveillance of transcripts. In Escherichia coli, the exoribonucleases RNase R and polynucleotide phosphorylase (PNPase) play critical roles in degrading RNA. Here, we developed an optical-trapping assay to monitor the translocation of individual enzymes along RNA-based substrates. Single-molecule records of motion reveal RNase R to be highly processive: one molecule can unwind over 500 bp of a structured substrate. However, enzyme progress is interrupted by pausing and stalling events that can slow degradation in a sequence-dependent fashion. We found that the distance traveled by PNPase through structured RNA is dependent on the A+U content of the substrate and that removal of its KH and S1 RNA-binding domains can reduce enzyme processivity without affecting the velocity. By a periodogram analysis of single-molecule records, we establish that PNPase takes discrete steps of six or seven nucleotides. These findings, in combination with previous structural and biochemical data, support an asymmetric inchworm mechanism for PNPase motion. The assay developed here for RNase R and PNPase is well suited to studies of other exonucleases and helicases.single-molecule biophysics | optical trap | exoribonuclease | processivity | step size B oth polynucleotide phosphorylase (PNPase) and RNase R are responsible for degrading a variety of transcripts in bacteria, such as mRNAs, defective rRNAs, and antisense regulatory RNAs (1-4). Although strains of Escherichia coli remain viable with a loss of either enzyme, the elimination of both results in an accumulation of rRNA fragments and cell death (3). To degrade RNA, both ribonucleases require an unstructured binding site of at least 7-10 nt at the 3′ end of the substrate (5-7), a sequence that often is supplied by posttranscriptional polyadenylation (8-10). After binding RNA, PNPase and RNase R processively digest the substrate in the 3′-to-5′ direction (11-13).PNPase consists of a trimer that degrades RNA at catalytic sites within a central channel (9,14). The enzyme is homologous to eukaryotic and archaeal exosomes and, like these hexameric proteins, possesses a core of six RNase PH domains arranged in a ringlike configuration (two per protomer) (6, 15). Each protomer also carries a KH and an S1 RNA-binding domain at its C terminus, both of which are connected to the body of the enzyme by flexible linkers (6, 16). These domains are thought to bind RNA upstream of the catalytic sites, and recent structural evidence suggests that they may guide the substrate into the central channel through a "hands gripping a rope" mechanism (16,17). By itself, PNPase may stall on structured RNA, particularly on G:C hairpins comprising more than six base pairs (18). PNPase often associates with the DEAD-box helicase RhlB to move through structured regions and generally does so in the context of the multienzyme degradosome (2, 19). However, there is some evidence that PNPase can digest structured tra...

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Crystal Structure of Escherichia coli Polynucleotide Phosphorylase Core Bound to RNase E, RNA and Manganese: Implications for Catalytic Mechanism and RNA Degradosome Assembly

Cited by 99 publications

References 60 publications

RNase E forms a complex with polynucleotide phosphorylase in cyanobacteria via a cyanobacterial-specific nonapeptide in the noncatalytic region

RNase E forms a complex with polynucleotide phosphorylase in cyanobacteria via a cyanobacterial-specific nonapeptide in the noncatalytic region

RNA remodeling and gene regulation by cold shock proteins

Direct observation of processive exoribonuclease motion using optical tweezers

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