Poly(A) polymerase activity and RNA polyadenylation in Streptomyces coelicolor A3(2).
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.
Bralley
P
P
Department of Biology, Emory University, Atlanta, GA 30322, USA.
Jones
G H
GH
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009876:Operon; D011061:Poly A / Q000737:chemistry / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics* / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011061:Poly A; D011062:Polynucleotide Adenylyltransferase; C063643:pcnB protein, E coli