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Related Experiment Videos

Protection against 3'-to-5' RNA decay in Bacillus subtilis.

G A Farr1, I A Oussenko, D H Bechhofer

  • 1Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine of New York University, New York, New York 10029, USA.

Journal of Bacteriology
|November 26, 1999
PubMed
Summary

Polynucleotide phosphorylase (PNPase) activity can be stalled by specific RNA structures, revealing distinct intermediates in bacterial mRNA decay pathways. This study identifies RNA structural elements that impede PNPase and other exonucleases.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • RNA processing and degradation are crucial for gene regulation in bacteria.
  • Polynucleotide phosphorylase (PNPase) is a key enzyme in the 3'-to-5' exonucleolytic degradation of RNA.
  • Understanding RNA decay pathways involves identifying factors that regulate exonuclease activity.

Purpose of the Study:

  • To investigate the role of specific RNA structures in modulating bacterial RNA processing and degradation.
  • To determine how the absence of PNPase affects RNA processing and identify alternative degradation pathways.
  • To elucidate the mechanisms by which RNA structures can impede exonuclease activity.

Main Methods:

  • Expression of a synthetic 320-nucleotide RNA in Bacillus subtilis wild-type and pnpA deletion strains.

Related Experiment Videos

  • In vitro assays for phosphate-dependent degradation to assess PNPase activity.
  • Analysis of RNA processing intermediates and degradation products using molecular techniques.
  • Site-directed mutagenesis to map RNA structures affecting PNPase processivity.

    • Main Results:

    • PNPase activity was found to be stalled in vivo and in vitro by a specific stem-loop structure within the SP82 RNA sequence.
    • This stem-loop structure specifically blocked PNPase processivity, but not in a strain lacking PNPase.
    • An alternative 3'-to-5' exonuclease was implicated in RNA degradation in the pnpA deletion strain, leading to the accumulation of a distinct RNA intermediate.
    • These findings suggest the existence of RNA structural elements that confer specificity to different exonucleases.

    Conclusions:

    • Specific RNA secondary structures can act as specific impediments to bacterial exonucleases like PNPase.
    • The mRNA decay pathway involves discrete intermediates regulated by sequence- and structure-specific interactions with degrading enzymes.
    • Identifying these structural impediments provides insights into the regulation of RNA stability and turnover in bacteria.