RHO-INDEPENDENT TERMINATORS WITHOUT 3' POLY-U TAILS FROM THE EARLY REGION OF ACTINOPHAGE-PHI-C31

C J INGHAM, Irene Hunter, Margaret Caroline MacHin Smith

Research output: Contribution to journalArticle

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Abstract

Previous work has identified three intergenic regions from the early region of actinophage phi C31 where transcription was either terminated or the mRNA was processed. Here we show using in vivo and in vitro approaches that these regions contain rho-independent terminators designated et(a), et(b) and et(c). Transcripts through et(a-c) would be expected to form stable RNA stem-loops but would lack poly-U tails. Et(a-c) contained part or all of the conserved sequences 5' AGCCCC and 5' GGGGCTT. A Streptomyces 'terminator probe' vector, pUGT1, was constructed and used to assay the efficiency of termination of transcription by et(a-c) from the thiostrepton-inducible tipA promoter by measuring the expression of a downstream reporter gene (aphll). In pUGT1 et(b) was at best a minor terminator in vivo whilst et(a) and et(c) exhibited strong termination activity. In vitro termination was assayed using templates containing a synthetic promoter recognised by E.coli RNA polymerase and fragments containing et(a-c) inserted downstream. All three terminators stimulated the formation of 3' ends in the promoter-distal arm of the inverted repeats with efficiencies et(a) > et(c) > et(b). As all three terminators either overlap with or lie close to sequences which interact with phage repressor proteins (conserved inverted repeats, CIRs) and these can potentially form stem-loop structures in RNA, the effect of CIRs on termination was also investigated. Termination at et(b) was unaffected by the presence or absence on the transcription template of CIR3. CIR4 forms the central 17 bp of et(c) and a 37 nt deletion which eliminated this stem-loop abolished termination in vivo and in vitro. Et(a) was investigated using an antisense oligonucleotide interference assay; an oligo designed to bind the 5' arm of et(a) inhibited termination whilst an oligo antisense to CIR5 was ineffective and an oligo targeted further upstream enhanced termination. Taken together these data show that et(a-c) are intrinsic, rho-independent terminators of varying efficiencies despite the absence of a poly-U tail.

Original languageEnglish
Pages (from-to)370-376
Number of pages7
JournalNucleic Acids Research
Volume23
Issue number3
Publication statusPublished - 11 Feb 1995

Keywords

  • ESCHERICHIA-COLI
  • TRANSCRIPTION TERMINATION
  • STREPTOMYCES-LIVIDANS
  • CLONING VECTORS
  • GENE-EXPRESSION
  • RNA-POLYMERASE
  • PHAGE PHI-C31
  • SEQUENCES
  • CONSTRUCTION
  • EFFICIENCY

Cite this

RHO-INDEPENDENT TERMINATORS WITHOUT 3' POLY-U TAILS FROM THE EARLY REGION OF ACTINOPHAGE-PHI-C31. / INGHAM, C J ; Hunter, Irene; Smith, Margaret Caroline MacHin.

In: Nucleic Acids Research, Vol. 23, No. 3, 11.02.1995, p. 370-376.

Research output: Contribution to journalArticle

INGHAM, C J ; Hunter, Irene ; Smith, Margaret Caroline MacHin. / RHO-INDEPENDENT TERMINATORS WITHOUT 3' POLY-U TAILS FROM THE EARLY REGION OF ACTINOPHAGE-PHI-C31. In: Nucleic Acids Research. 1995 ; Vol. 23, No. 3. pp. 370-376.
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abstract = "Previous work has identified three intergenic regions from the early region of actinophage phi C31 where transcription was either terminated or the mRNA was processed. Here we show using in vivo and in vitro approaches that these regions contain rho-independent terminators designated et(a), et(b) and et(c). Transcripts through et(a-c) would be expected to form stable RNA stem-loops but would lack poly-U tails. Et(a-c) contained part or all of the conserved sequences 5' AGCCCC and 5' GGGGCTT. A Streptomyces 'terminator probe' vector, pUGT1, was constructed and used to assay the efficiency of termination of transcription by et(a-c) from the thiostrepton-inducible tipA promoter by measuring the expression of a downstream reporter gene (aphll). In pUGT1 et(b) was at best a minor terminator in vivo whilst et(a) and et(c) exhibited strong termination activity. In vitro termination was assayed using templates containing a synthetic promoter recognised by E.coli RNA polymerase and fragments containing et(a-c) inserted downstream. All three terminators stimulated the formation of 3' ends in the promoter-distal arm of the inverted repeats with efficiencies et(a) > et(c) > et(b). As all three terminators either overlap with or lie close to sequences which interact with phage repressor proteins (conserved inverted repeats, CIRs) and these can potentially form stem-loop structures in RNA, the effect of CIRs on termination was also investigated. Termination at et(b) was unaffected by the presence or absence on the transcription template of CIR3. CIR4 forms the central 17 bp of et(c) and a 37 nt deletion which eliminated this stem-loop abolished termination in vivo and in vitro. Et(a) was investigated using an antisense oligonucleotide interference assay; an oligo designed to bind the 5' arm of et(a) inhibited termination whilst an oligo antisense to CIR5 was ineffective and an oligo targeted further upstream enhanced termination. Taken together these data show that et(a-c) are intrinsic, rho-independent terminators of varying efficiencies despite the absence of a poly-U tail.",
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AU - INGHAM, C J

AU - Hunter, Irene

AU - Smith, Margaret Caroline MacHin

PY - 1995/2/11

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N2 - Previous work has identified three intergenic regions from the early region of actinophage phi C31 where transcription was either terminated or the mRNA was processed. Here we show using in vivo and in vitro approaches that these regions contain rho-independent terminators designated et(a), et(b) and et(c). Transcripts through et(a-c) would be expected to form stable RNA stem-loops but would lack poly-U tails. Et(a-c) contained part or all of the conserved sequences 5' AGCCCC and 5' GGGGCTT. A Streptomyces 'terminator probe' vector, pUGT1, was constructed and used to assay the efficiency of termination of transcription by et(a-c) from the thiostrepton-inducible tipA promoter by measuring the expression of a downstream reporter gene (aphll). In pUGT1 et(b) was at best a minor terminator in vivo whilst et(a) and et(c) exhibited strong termination activity. In vitro termination was assayed using templates containing a synthetic promoter recognised by E.coli RNA polymerase and fragments containing et(a-c) inserted downstream. All three terminators stimulated the formation of 3' ends in the promoter-distal arm of the inverted repeats with efficiencies et(a) > et(c) > et(b). As all three terminators either overlap with or lie close to sequences which interact with phage repressor proteins (conserved inverted repeats, CIRs) and these can potentially form stem-loop structures in RNA, the effect of CIRs on termination was also investigated. Termination at et(b) was unaffected by the presence or absence on the transcription template of CIR3. CIR4 forms the central 17 bp of et(c) and a 37 nt deletion which eliminated this stem-loop abolished termination in vivo and in vitro. Et(a) was investigated using an antisense oligonucleotide interference assay; an oligo designed to bind the 5' arm of et(a) inhibited termination whilst an oligo antisense to CIR5 was ineffective and an oligo targeted further upstream enhanced termination. Taken together these data show that et(a-c) are intrinsic, rho-independent terminators of varying efficiencies despite the absence of a poly-U tail.

AB - Previous work has identified three intergenic regions from the early region of actinophage phi C31 where transcription was either terminated or the mRNA was processed. Here we show using in vivo and in vitro approaches that these regions contain rho-independent terminators designated et(a), et(b) and et(c). Transcripts through et(a-c) would be expected to form stable RNA stem-loops but would lack poly-U tails. Et(a-c) contained part or all of the conserved sequences 5' AGCCCC and 5' GGGGCTT. A Streptomyces 'terminator probe' vector, pUGT1, was constructed and used to assay the efficiency of termination of transcription by et(a-c) from the thiostrepton-inducible tipA promoter by measuring the expression of a downstream reporter gene (aphll). In pUGT1 et(b) was at best a minor terminator in vivo whilst et(a) and et(c) exhibited strong termination activity. In vitro termination was assayed using templates containing a synthetic promoter recognised by E.coli RNA polymerase and fragments containing et(a-c) inserted downstream. All three terminators stimulated the formation of 3' ends in the promoter-distal arm of the inverted repeats with efficiencies et(a) > et(c) > et(b). As all three terminators either overlap with or lie close to sequences which interact with phage repressor proteins (conserved inverted repeats, CIRs) and these can potentially form stem-loop structures in RNA, the effect of CIRs on termination was also investigated. Termination at et(b) was unaffected by the presence or absence on the transcription template of CIR3. CIR4 forms the central 17 bp of et(c) and a 37 nt deletion which eliminated this stem-loop abolished termination in vivo and in vitro. Et(a) was investigated using an antisense oligonucleotide interference assay; an oligo designed to bind the 5' arm of et(a) inhibited termination whilst an oligo antisense to CIR5 was ineffective and an oligo targeted further upstream enhanced termination. Taken together these data show that et(a-c) are intrinsic, rho-independent terminators of varying efficiencies despite the absence of a poly-U tail.

KW - ESCHERICHIA-COLI

KW - TRANSCRIPTION TERMINATION

KW - STREPTOMYCES-LIVIDANS

KW - CLONING VECTORS

KW - GENE-EXPRESSION

KW - RNA-POLYMERASE

KW - PHAGE PHI-C31

KW - SEQUENCES

KW - CONSTRUCTION

KW - EFFICIENCY

M3 - Article

VL - 23

SP - 370

EP - 376

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 3

ER -