Phylogenetic conservation of RNA secondary and tertiary structure in the trpEDCFBA operon leader transcript in Bacillus

Research output: Contribution to journalArticle

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Abstract

Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. We recently determined that the B. subtilis trp leader readthrough transcript can adopt a Mg2+-dependent tertiary structure that appears to interfere with TRAP-mediated translation control of trpE. In the present study, sequence comparisons to trp leaders from three other Bacillus sp. were made, suggesting that RNA secondary and tertiary structures are phylogenetically conserved. To test this hypothesis, experiments were carried out with the trp leader transcript from Bacillus stearothermophilus. Structure mapping experiments confirmed the predicted secondary structure. Native gel experiments identified a faster mobility species in the presence of Mg2+, suggesting that a Mg2+-dependent tertiary structure forms. Mg2+-dependent protection of residues within the first five triplet repeats of the TRAP binding target and a pyrimidine-rich internal loop were observed, consistent with tertiary structure formation between these regions. Structure mapping in the presence of a competitor DNA oligonucleotide allowed the interacting partners to be identified as a single-stranded portion of the purine-rich TRAP binding target and the large downstream pyrimidine-rich internal loop. Thermal denaturation experiments revealed a Mg2+- and pH-dependent unfolding transition that was absent for a transcript missing the first five triplet repeats. The stability of several mutant transcripts allowed a large portion of the base-pairing register for the tertiary interaction to be determined. These data indicate that RNA secondary and tertiary structures involved in TRAP-mediated translation control are conserved in at least four Bacillus species.

Original languageEnglish (US)
Pages (from-to)1502-1515
Number of pages14
JournalRNA
Volume9
Issue number12
DOIs
StatePublished - Dec 1 2003

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Trinucleotide Repeats
Operon
Bacillus subtilis
Bacillus
RNA
Geobacillus stearothermophilus
Oligonucleotides
Base Pairing
Hot Temperature
Gels
DNA
pyrimidine
purine

All Science Journal Classification (ASJC) codes

  • Molecular Biology

Cite this

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title = "Phylogenetic conservation of RNA secondary and tertiary structure in the trpEDCFBA operon leader transcript in Bacillus",
abstract = "Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. We recently determined that the B. subtilis trp leader readthrough transcript can adopt a Mg2+-dependent tertiary structure that appears to interfere with TRAP-mediated translation control of trpE. In the present study, sequence comparisons to trp leaders from three other Bacillus sp. were made, suggesting that RNA secondary and tertiary structures are phylogenetically conserved. To test this hypothesis, experiments were carried out with the trp leader transcript from Bacillus stearothermophilus. Structure mapping experiments confirmed the predicted secondary structure. Native gel experiments identified a faster mobility species in the presence of Mg2+, suggesting that a Mg2+-dependent tertiary structure forms. Mg2+-dependent protection of residues within the first five triplet repeats of the TRAP binding target and a pyrimidine-rich internal loop were observed, consistent with tertiary structure formation between these regions. Structure mapping in the presence of a competitor DNA oligonucleotide allowed the interacting partners to be identified as a single-stranded portion of the purine-rich TRAP binding target and the large downstream pyrimidine-rich internal loop. Thermal denaturation experiments revealed a Mg2+- and pH-dependent unfolding transition that was absent for a transcript missing the first five triplet repeats. The stability of several mutant transcripts allowed a large portion of the base-pairing register for the tertiary interaction to be determined. These data indicate that RNA secondary and tertiary structures involved in TRAP-mediated translation control are conserved in at least four Bacillus species.",
author = "Schaak, {Janell E.} and Babitzke, {Paul Lee} and Bevilacqua, {Philip C.}",
year = "2003",
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Phylogenetic conservation of RNA secondary and tertiary structure in the trpEDCFBA operon leader transcript in Bacillus. / Schaak, Janell E.; Babitzke, Paul Lee; Bevilacqua, Philip C.

In: RNA, Vol. 9, No. 12, 01.12.2003, p. 1502-1515.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Phylogenetic conservation of RNA secondary and tertiary structure in the trpEDCFBA operon leader transcript in Bacillus

AU - Schaak, Janell E.

AU - Babitzke, Paul Lee

AU - Bevilacqua, Philip C.

PY - 2003/12/1

Y1 - 2003/12/1

N2 - Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. We recently determined that the B. subtilis trp leader readthrough transcript can adopt a Mg2+-dependent tertiary structure that appears to interfere with TRAP-mediated translation control of trpE. In the present study, sequence comparisons to trp leaders from three other Bacillus sp. were made, suggesting that RNA secondary and tertiary structures are phylogenetically conserved. To test this hypothesis, experiments were carried out with the trp leader transcript from Bacillus stearothermophilus. Structure mapping experiments confirmed the predicted secondary structure. Native gel experiments identified a faster mobility species in the presence of Mg2+, suggesting that a Mg2+-dependent tertiary structure forms. Mg2+-dependent protection of residues within the first five triplet repeats of the TRAP binding target and a pyrimidine-rich internal loop were observed, consistent with tertiary structure formation between these regions. Structure mapping in the presence of a competitor DNA oligonucleotide allowed the interacting partners to be identified as a single-stranded portion of the purine-rich TRAP binding target and the large downstream pyrimidine-rich internal loop. Thermal denaturation experiments revealed a Mg2+- and pH-dependent unfolding transition that was absent for a transcript missing the first five triplet repeats. The stability of several mutant transcripts allowed a large portion of the base-pairing register for the tertiary interaction to be determined. These data indicate that RNA secondary and tertiary structures involved in TRAP-mediated translation control are conserved in at least four Bacillus species.

AB - Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. We recently determined that the B. subtilis trp leader readthrough transcript can adopt a Mg2+-dependent tertiary structure that appears to interfere with TRAP-mediated translation control of trpE. In the present study, sequence comparisons to trp leaders from three other Bacillus sp. were made, suggesting that RNA secondary and tertiary structures are phylogenetically conserved. To test this hypothesis, experiments were carried out with the trp leader transcript from Bacillus stearothermophilus. Structure mapping experiments confirmed the predicted secondary structure. Native gel experiments identified a faster mobility species in the presence of Mg2+, suggesting that a Mg2+-dependent tertiary structure forms. Mg2+-dependent protection of residues within the first five triplet repeats of the TRAP binding target and a pyrimidine-rich internal loop were observed, consistent with tertiary structure formation between these regions. Structure mapping in the presence of a competitor DNA oligonucleotide allowed the interacting partners to be identified as a single-stranded portion of the purine-rich TRAP binding target and the large downstream pyrimidine-rich internal loop. Thermal denaturation experiments revealed a Mg2+- and pH-dependent unfolding transition that was absent for a transcript missing the first five triplet repeats. The stability of several mutant transcripts allowed a large portion of the base-pairing register for the tertiary interaction to be determined. These data indicate that RNA secondary and tertiary structures involved in TRAP-mediated translation control are conserved in at least four Bacillus species.

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