Poly(A) polymerase activity and RNA polyadenylation in Streptomyces coelicolor A3(2)

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P Bralley1, G H Jones

  • 1Department of Biology, Emory University, Atlanta, GA 30322, USA.

Molecular Microbiology
|June 13, 2001
PubMed

Insights

Streptomyces coelicolor possesses polyadenylation activity, indicated by the presence of poly(A) tails on RNA molecules. This finding suggests a role for polyadenylation in gene regulation within this bacterium.

Area of Science:

  • Molecular biology
  • Genomics
  • Microbiology

Background:

  • The Streptomyces coelicolor genome was analyzed for similarities to Escherichia coli poly(A) polymerase I.
  • An open reading frame (ORF) with 36% amino acid identity was identified.

Purpose of the Study:

  • To investigate the presence and characteristics of polyadenylation in Streptomyces coelicolor.
  • To determine if S. coelicolor possesses poly(A) polymerase activity and to characterize the poly(A) tails.

Main Methods:

  • Bioinformatic search for homologous ORFs.
  • Enzymatic assays using radioactive ATP to detect poly(A) synthesis.
  • RNA labeling with [3H]-uridine and [3H]-adenosine.
  • Oligo (dT) cellulose chromatography for RNA fractionation.
  • RNase digestion and end-labeling to measure poly(A) tail length.
  • Hybridization studies with ribosomal RNA genes.
  • Reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA.

Main Results:

  • Mycelial extracts demonstrated poly(A) polymerase activity, incorporating ATP into acid-insoluble products.
  • Oligo (dT) cellulose chromatography retained significant portions of adenosine-labeled RNA (11%), indicating polyadenylated sequences.
  • Enzymatic digestion confirmed the presence of 3'-poly(A) chains.
  • Poly(A) tail lengths were measured to be approximately 18 residues.
  • Hybridization studies showed poly(A) containing RNA associated with 16S and 23S rRNA genes, but not 5S rRNA.
  • RT-PCR confirmed the presence of mRNA in the polyadenylated RNA fraction.

Conclusions:

  • Streptomyces coelicolor exhibits polyadenylation activity, with identified poly(A) polymerase activity and poly(A) tails on RNA.
  • The presence of poly(A) tails on mRNA and association with rRNA genes suggest a role in gene regulation or RNA processing in S. coelicolor.

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