Highly Purified Colicin E3 Contains Immunity Protein

Jakes, Karen S.; Zinder, Norton D.

doi:10.1073/pnas.71.9.3380

Cited by 96 publications

(67 citation statements)

References 25 publications

(13 reference statements)

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“…described in Materials and Methods. Jakes and Zinder were unable to recover active E3* after 6 M guanidine.HCl treatment and hence used sodium dodecyl sulfate to obtain E3* (20). Using E3* prepared by our method (compare Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These authors demonstrated that the immunity protein could be separated from the E3 protein by preparative electrophoresis in sodium dodecyl sulfate/polyacrylamide gels and that E3 protein (called "ES*") free of immunity protein was much more active than the "complexed" ES preparation in ribosome inactivation in vitro (20). (21).…”

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confidence: 99%

“…Recently, Jakes and Zinder observed that the "purified" ES preparations obtained with the conventional purification methods (3) contained about one molar equivalent of the E3-immunity protein (20). These authors demonstrated that the immunity protein could be separated from the E3 protein by preparative electrophoresis in sodium dodecyl sulfate/polyacrylamide gels and that E3 protein (called "ES*") free of immunity protein was much more active than the "complexed" ES preparation in ribosome inactivation in vitro (20).…”

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confidence: 99%

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Colicin E2 is DNA endonuclease.

Schaller¹,

Nomura²

1976

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

133

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Colicin E2 purified by conventional methods contains a tightly bound low-molecular-weight protein, as has been found with purified colicin E3 [Jakes, N. & Zinder, N. D. (1974) Proc. Nat. Acad. Sci. USA 71, 3380-33841. Such E2 preparations do not cause DNA cleavage in vitro. After separation from the low-molecular-weight protein, colicin E2 retained the original in vivo killing activity, and in addition showed a high activity in vitro in cleaving various DNA molecules, such as a ColE1 hybrid plasmid and DNAs from Escherichia coli, X phage, 4X174 phage, and simian virus 40. The low-molecular-weight protein ("E2-immunity protein") specifically prevented this in vitro DNA cleavage reaction, i.e., had an "immunity function." The results demonstrate that colicin E2 itself is a DNA endonuclease and explain the in vivo effects caused by E2 in sensitive cells as well as the mechanism of immunity in E2-colicinogenic cells. Colicin E2 (E2) is a protein antibiotic synthesized by certain strains of coliform bacteria that carry the ColE2 plasmid, and is classified in the E group of colicins along with colicins El and E3 (1, 2). Although the physical properties of E2 and E3 are similar (3) and both colicins share the same receptor (1, 2), their apparent mode of action is different. It was first discovered that E2 causes degradation of DNA, while colicin E3 (E3) causes specific inhibition of protein synthesis in sensitive Escherichia coli cells treated with these colicins (4). Subsequent work on the mode of action of E3 has demonstrated that ES inactivates ribosomes in treated cells (5) by causing the cleavage of a fragment from the 3' end of the 16S RNA (6, 7). The same cleavage reaction has also been demonstrated in vitro using "purified" E3 protein (see below) and purified ribosomes (8,9). This in vitro demonstration of E3 action has strongly indicated that E3 acts directly on ribosomes rather than indirectly and that E3 itself is an RNase with a very stringent substrate specificity.In contrast to ES, the mechanism of action of E2 has not been clear. Since the initial discovery of DNA degradation in E2-treated sensitive cells (4), several authors have confirmed and extended the original observations on DNA cleavage reactions (10-13). However, others have suggested that the primary action of E2 is not on the cellular DNA, but on some other targets such as membranes (14) or tRNA (15).Since E2 has properties similar to E3 (3), and E3 causes ribosome inactivation in vitro, one might expect some kind of effects of E2 on cellular DNA in vitro, if DNA is in fact the primary target of E2. Several attempts have been made to demonstrate such effects in vitro using purified E2 (e.g., refs. 16-19). However, published results were either negative or too uncertain to allow a definitive conclusion regarding such direct effects.Recently, Jakes and Zinder observed that the "purified" ES Colicin E2* was separated from the immunity protein by dissociation of the complexed E2 with guanidine-HCl at about 4°. Lyophilized E2 was di...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Colicin E2 is DNA endonuclease.

Schaller¹,

Nomura²

1976

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

133

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“…The assays (in triplicate) were done as described in Fig. 1 Role of BtuB and OmpF in the uptake of colicins E2 and E3 Colicins E2 and E3 are very tightly associated with their respective immunity proteins (Jakes & Zinder, 1974). These two colicins are highly homologous except for their catalytic domains (Ohno-Iwashita & Imahori, 1980).…”

Section: Role Of Btub In Colicin El Uptakementioning

confidence: 99%

Comparison of the Uptake Systems for the Entry of Various BtuB Group Colicins into Escherichia coli

Bénédetti¹,

Frenette²,

Baty³

et al. 1989

Microbiology

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Colicins A, El, E2 and E3 belong to the BtuB group of colicins. The NH,-terminal region of colicin A is required for translocation, and defects in this region cannot be overcome by osmotic shock of sensitive cells. In addition to BtuB, colicin A requires OmpF for efficient uptake by sensitive cells. The roles of BtuB and OmpF in translocation and binding to the receptor of the colicins A, El, E2 and E3 were compared. The results suggest that for colicin A OmpF is used both as a receptor and for translocation across the outer membrane. In contrast, for colicin El, OmpF is used neither as a receptor nor for translocation. For colicins E2 and E3, the situation is intermediate: only BtuB is used as a receptor but both BtuB and OmpF are involved in the translocation step.

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“…Recent studies have shown that colicins E2 and E3 have some similar molecular features: each consists of two components, A and B [2,7,8] where A is the active component and B is the so-called immunity protein [7], which neutralizes the in vitro activity of protein A of homologous but not of heterologous colicin. Digestion of protein A with trypsin leads in each case to two fragments, E2-Tl, E2-T2A and E3-Tl, E3-T2A, respectively [9,10].…”

Section: Introductionmentioning

confidence: 99%