Related Experiment Videos
Poly(A) polymerase activity and RNA polyadenylation in Streptomyces coelicolor A3(2)
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.
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.