The antisense RNA of the par locus of pAD1 regulates the expression of a 33-amino-acid toxic peptide by an unusual mechanism

TJ Greenfield; E Ehli; T Kirshenmann; T Franch; K Gerdes; KE Weaver

doi:10.1046/j.1365-2958.2000.02035.x

The antisense RNA of the par locus of pAD1 regulates the expression of a 33-amino-acid toxic peptide by an unusual mechanism

TJ Greenfield, E Ehli, T Kirshenmann, T Franch, K Gerdes, KE Weaver

58 Citations (Scopus)

Abstract

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA-regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA 1 encodes an open reading frame of 33 codons designated ist The results presented here demonstrate that the peptide encoded by fst is the par toxin, The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the ist-encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem-loop structure at the 5' end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300-fold increase in production of the fst-encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of ist in plasmid-free cells.

Original language	English
Journal	Molecular Microbiology
Volume	37
Issue number	3
Pages (from-to)	652-660
Number of pages	9
ISSN	0950-382X
DOIs	https://doi.org/10.1046/j.1365-2958.2000.02035.x
Publication status	Published - 2000
Externally published	Yes

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10.1046/j.1365-2958.2000.02035.x

Cite this

@article{770b65faefb54232afb4e270e8aeb2e2,

title = "The antisense RNA of the par locus of pAD1 regulates the expression of a 33-amino-acid toxic peptide by an unusual mechanism",

abstract = "The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA-regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA 1 encodes an open reading frame of 33 codons designated ist The results presented here demonstrate that the peptide encoded by fst is the par toxin, The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the ist-encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem-loop structure at the 5' end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300-fold increase in production of the fst-encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of ist in plasmid-free cells.",

author = "TJ Greenfield and E Ehli and T Kirshenmann and T Franch and K Gerdes and KE Weaver",

year = "2000",

doi = "10.1046/j.1365-2958.2000.02035.x",

language = "English",

volume = "37",

pages = "652--660",

journal = "Molecular Microbiology",

issn = "0950-382X",

publisher = "Wiley-Blackwell",

number = "3",

}

TY - JOUR

T1 - The antisense RNA of the par locus of pAD1 regulates the expression of a 33-amino-acid toxic peptide by an unusual mechanism

AU - Greenfield, TJ

AU - Ehli, E

AU - Kirshenmann, T

AU - Franch, T

AU - Gerdes, K

AU - Weaver, KE

PY - 2000

Y1 - 2000

N2 - The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA-regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA 1 encodes an open reading frame of 33 codons designated ist The results presented here demonstrate that the peptide encoded by fst is the par toxin, The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the ist-encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem-loop structure at the 5' end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300-fold increase in production of the fst-encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of ist in plasmid-free cells.

AB - The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA-regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA 1 encodes an open reading frame of 33 codons designated ist The results presented here demonstrate that the peptide encoded by fst is the par toxin, The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the ist-encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem-loop structure at the 5' end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300-fold increase in production of the fst-encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of ist in plasmid-free cells.

U2 - 10.1046/j.1365-2958.2000.02035.x

DO - 10.1046/j.1365-2958.2000.02035.x

M3 - Journal article

SN - 0950-382X

VL - 37

SP - 652

EP - 660

JO - Molecular Microbiology

JF - Molecular Microbiology

IS - 3

ER -