Protection of <i>E. coli</i> ribosomes against colicin E3‐induced inactivation by bound aminoacyl‐tRNA

Kaufmann, Yael; Zamir, Ada

doi:10.1016/0014-5793(73)80390-4

Cited by 11 publications

(7 citation statements)

References 8 publications

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“…Similarly, tRNA and colE3 binding are mutually exclusive. These predictions are in agreement with experimental results that mRNA as well tRNA confers protection of ribosomes from colE3 attack [33,34]. In contrast, the P-site is free to bind tRNA in the presence of colE3.…”

Section: Protection From Cole3 Cleavage By Mrna or Trnasupporting

confidence: 91%

On the interaction of colicin E3 with the ribosome

Zarivach

Ben-Zeev

et al. 2002

Biochimie

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Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone

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Section: Protection From Cole3 Cleavage By Mrna or Trnasupporting

confidence: 91%

On the interaction of colicin E3 with the ribosome

Zarivach

Ben-Zeev

et al. 2002

Biochimie

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“…When colicin E3 was not removed from the ribosomes by immunity protein Im3, not only the rate of A-site binding was strongly affected, but the binding was also much less efficient, reaching a plateau at about 40%. This effect is in agreement with the notion that colicin E3 and [ 14 C]Phe-tRNA Phe compete for the A site ( Kaufmann and Zamir, 1973 ). For further experiments, only ribosomes treated with colicin E3 followed by removal of E3 with immunity protein Im3 were used.…”

Section: Resultssupporting

confidence: 91%

“…Previous studies of the mechanism of colicin E3 action on the ribosome focused on the structure of ribosomes, experimental conditions required for cleavage to occur and the specific cleavage site ( Boon, 1972 ; Bowman, 1972 ; Dahlberg et al ., 1973 ; Kaufmann and Zamir, 1973 ; 1975 ; Ohno-Iwashita and Imahori, 1977 ; Jakes and Zinder, 1974b ). Very few investigations into the stage of translational arrest have been performed.…”

Section: Introductionmentioning

confidence: 99%

Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes

Lancaster¹,

Savelsbergh²,

Kleanthous³

et al. 2008

Molecular Microbiology

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SummaryThe cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon-anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon-recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA-mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin.

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“…The results may reflect a general feature distinguishing between synthetic and natural mRNAs, a specific difference between the particular polynucleotides em-ployed in the study, or may be unique to Phe-tRNA alone or in combination with poly(U). It should be noted that previous experiments have shown that poly(U) in itself exerted no protection against E3 action [8]. Binding complexes formed with natural mRNAs may be generally vulnerable to E3, as polysomes isolated from bacterial cell lysates were inactivated by E3 [5].…”

Section: Discussionmentioning

confidence: 99%

The Effect of tRNA Derivatives Bound with Natural or Synthetic mRNA on the Interaction of Escherichia coli Ribosomes with Colicin E3

Kaufmann

Zamir

1975

Eur J Biochem

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Ribosomal binding complexes directed by poly(U) or T4 mRNA were formed with aminoacyl-tRNA or its derivatives bound to predominantly the P or A binding site. The defined binding complexes were reacted with colicin E3 and the reaction was assessed by the ability of the complexes to proceed with polypeptide synthesis. The results indicated that only one of the four complexes tested was completely resistant to colicin E3-induced inactivation: that of Phe-tRNA bound in the presence of poly(U) to the A-site. The poly(U) directed complex of AcPhe-tRNA and the T4-mRNA-directed complex at the A-site appeared slightly resistant, while the T4 mRNA initiation complex was inactivated by colicin E3 in a manner similar to non-complexed ribosomes. Colicin E3 added to ribosomes after protein synthesis had been initiated affected the subsequent polymerization in a manner corresponding to the response of the binding complexes. Thus, poly(U)-translating ribosomes were less affected than ribosomes translating the viral mRNA. The vulnerability of natural-mRNA-directed binding complexes to inactivation by colicin E3 is in accord with the mode of inactivation by the colicin in vivo.

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Protection of E. coli ribosomes against colicin E3‐induced inactivation by bound aminoacyl‐tRNA

Cited by 11 publications

References 8 publications

On the interaction of colicin E3 with the ribosome

On the interaction of colicin E3 with the ribosome

Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes

The Effect of tRNA Derivatives Bound with Natural or Synthetic mRNA on the Interaction of Escherichia coli Ribosomes with Colicin E3

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