On the interaction of colicin E3 with the ribosome

BtuB is a TonB-dependent outer-membrane transporter for vitamin B 12 (or cyanocobalamin, CN-Cbl) in Escherichia coli. The binding of CN-Cbl is believed to promote an unfolding or undocking of the Ton box, the conserved N-terminal energy coupling motif at the periplasmic surface of the transporter. This structural change may facilitate the interaction of BtuB with the inner membrane protein TonB. In this work, the effect of the receptor binding fragment of colicin E3 (E3R) on the conformation of the Ton box was examined with site-directed spin labeling. Addition of E3R reverses the undocking of the Ton box that is promoted by CN-Cbl, consistent with a competitive binding between the substrate and the colicin fragment. EPR spectroscopy indicates that the Ton box is in a two-state equilibrium between docked and undocked conformations. In the absence of substrate, the docked conformation is the predominant state; however, the equilibrium can be shifted to the undocked state by the addition of detergents or site-specific proline substitutions. Even when the undocking is induced by detergents or by certain proline mutations, E3R binding shifts the equilibrium to the docked conformation. Thus, two competitive extracellular ligands, CN-Cbl and ER3, transduce opposite conformations of the N-terminal Ton box. Substrate binding stabilizes an undocked conformation, whereas E3R binding stabilizes a docked conformation of the Ton box.T he outer membrane of Gram-negative bacteria such as Escherichia coli contains a family of membrane proteins that actively transport large and scarce nutrients, such as iron siderophores or vitamin B 12 (cyanocobalamin, CN-Cbl) into the periplasmic space. These proteins derive energy for transport from the proton chemical potential across the inner membrane by coupling to the transperiplasmic protein TonB and are therefore termed TonB-dependent. High-resolution structures for the vitamin B 12 transporter, BtuB, have recently been obtained (1). These structures are similar to other TonBdependent transporters, such as FepA, FhuA, and FecA (2-5), and consist of a ␤-barrel formed from 22 antiparallel strands with an N-terminal (core) region that is folded into the lumen of the barrel. However, BtuB, which is the smaller than FepA, FhuA, or FecA, has shorter extracellular loops.The molecular mechanism of TonB-dependent transport is presently not known, but it likely involves a major structural rearrangement or unfolding of the N-terminal core region of the transporter during the transport cycle (6). Site-directed spin labeling (SDSL) is a technique that is particularly sensitive to the changes in tertiary contact made by a spin-labeled side chain (7), and this method indicates that there are significant conformational changes in the Ton box on CN-Cbl binding. The Ton box is a highly conserved region near the N terminus of the transporter that is believed to participate in interactions between TonB and the TonB-dependent transporter. SDSL indicates that the Ton box segment of BtuB (residues 6-12; see ...

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“…target cells by different activities. E9 kills its targets by DNA degradation (17), whereas E3 acts as a highly specific RNase (21,22).…”

mentioning

confidence: 99%

Competing ligands stabilize alternate conformations of the energy coupling motif of a TonB-dependent outer membrane transporter

Fanucci

Cadieux²,

Kadner³

et al. 2003

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

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“…Colicin E3 cleaves with high specificity the phosphodiester bond located between A1493 and G1494 of the 16S rRNA (E. coli numbering) [24][25]. -sarcin cleaves a GAGA tetra-loop (after the second G) [26][27].…”

Section: Sequence-specificitymentioning

confidence: 99%

RNA Recognition and Cleavage by Sequence-Specific Endoribonucleases

Saïda¹,

Odaert²

2007

PPL

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Inactivation of RNA molecules by sequence-specific endoribonucleolytic cleavage is a subtle mechanism by which cells regulate gene expression. Sequence-specific endoribonucleases can recognize and cleave particular phosphodiester bonds confined within hundreds/thousands of chemically similar bonds. Here, we present a comparative analysis of the mechanisms used by endoribonucleases to select and cleave their target RNA molecules. This analysis is based on the very recent molecular details obtained from the structural and/or biochemical studies of nine sequence-specific ribonucleases that target messenger, ribosomal, and transfer RNA molecules. This analysis shows that despite the absence of sequence homologies and the wide diversity of biological sources (prokaryotes, archaea and eukaryotes), the sequence-specific ribonucleases studied here adopt limited structural folds, catalyze their cleavage reactions using a common chemistry and involve a very limited set of amino acids for both RNA binding and processing.

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“…For example, colicin E3 is a well-known ribonuclease that specifically cleaves 16S rRNA at the 3'-end of the coding sequence both in vivo and in vitro , which leads to the abolishment of protein synthesis resulting in death of the susceptible cell [9-12]. Previous reports indicate that colicin E3 consists of a killer protein with three domains (i.e., a translocation domain [T domain], receptor binding domain [R domain], and nuclease domain) and an immunity protein that retards antibiotic activity [13,14].…”

Section: Introductionmentioning

confidence: 99%

Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum

Chan

et al. 2011

BMC Microbiol

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BackgroundMost isolates of Pectobacterium carotovorum subsp. carotovorum (Pcc) produce bacteriocins. In this study, we have determined that Pcc strain F-rif-18 has a chromosomal gene encoding the low-molecular-weight bacteriocin, Carocin S2, and that this bacteriocin inhibits the growth of a closely related strain. Carocin S2 is inducible by ultraviolet radiation but not by mutagenic agents such as mitomycin C.ResultsA carocin S2-defective mutant, TF1-2, was obtained by Tn5 insertional mutagenesis using F-rif-18. A 5706-bp DNA fragment was detected by Southern blotting, selected from a genomic DNA library, and cloned to the vector, pMS2KI. Two adjacent complete open reading frames within pMS2KI were sequenced, characterized, and identified as caroS2K and caroS2I, which respectively encode the killing protein and immunity protein. Notably, carocin S2 could be expressed not only in the mutant TF1-2 but also in Escherichia coli DH5α after entry of the plasmid pMS2KI. Furthermore, the C-terminal domain of CaroS2K was homologous to the nuclease domains of colicin D and klebicin D. Moreover, SDS-PAGE analysis showed that the relative mass of CaroS2K was 85 kDa and that of CaroS2I was 10 kDa.ConclusionThis study shown that another nuclease type of bacteriocin was found in Pectobacterium carotovorum. This new type of bacteriocin, Carocin S2, has the ribonuclease activity of CaroS2K and the immunity protein activity of CaroS2I.

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On the interaction of colicin E3 with the ribosome

Cited by 17 publications

References 32 publications

Competing ligands stabilize alternate conformations of the energy coupling motif of a TonB-dependent outer membrane transporter

Competing ligands stabilize alternate conformations of the energy coupling motif of a TonB-dependent outer membrane transporter

RNA Recognition and Cleavage by Sequence-Specific Endoribonucleases

Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum

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