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

Mol Microbiol. 2001 Jun;40(5):1155-64. doi: 10.1046/j.1365-2958.2001.02457.x.

Abstract

The Streptomyces coelicolor genome sequence was searched for open reading frames (ORFs) similar to Escherichia coli poly(A) polymerase I, revealing an ORF with 36% amino acid sequence identity to that protein. Mycelial extracts prepared from S. coelicolor cultures incorporated radioactive ATP into an acid-insoluble form, and some of the products of this incorporation had the properties expected of poly(A). [3H]-uridine and [3H]-adenosine were used to label the RNA in S. coelicolor cultures of different ages, and total RNA was fractionated by oligo dT cellulose chromatography. Approximately 3% of the total uridine-labelled RNA and 11% of the adenosine-labelled RNA were retained by the oligo dT cellulose columns. Enzymatic digestion of the retained RNA supported the conclusion that a significant fraction of the adenosine label was present in 3'-poly(A) chains. Measurement of poly(A) tail lengths by end labelling of total RNA and RNase digestion revealed a maximum length of approximately 18 residues. Radioactive cDNA prepared from the RNA fraction retained by oligo dT cellulose hybridized to the 16S and 23S genes from a streptomycete ribosomal RNA operon but not to the 5S gene. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the presence of mRNAs in the RNA fraction retained by oligo dT cellulose.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Blotting, Southern
  • Escherichia coli Proteins*
  • Molecular Sequence Data
  • Operon
  • Poly A / chemistry
  • Poly A / metabolism
  • Polynucleotide Adenylyltransferase / genetics*
  • Polynucleotide Adenylyltransferase / metabolism*
  • RNA, Bacterial / metabolism*
  • RNA, Messenger / isolation & purification
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Streptomyces / genetics
  • Streptomyces / metabolism*

Substances

  • Escherichia coli Proteins
  • RNA, Bacterial
  • RNA, Messenger
  • Poly A
  • Polynucleotide Adenylyltransferase
  • pcnB protein, E coli