Specific polyadenylation and purification of total messenger RNA from Escherichia coli.
Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages. We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese. The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA. Withdrawal of Mg2+ from the polyadenylation reaction resulted in addition of poly(A) to 16S rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes. Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species. Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis. Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.
Amara
R R
RR
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Vijaya
S
S
D015152:Blotting, Northern; D015139:Blotting, Southern; D004926:Escherichia coli / Q000235:genetics*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D011061:Poly A / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D012329:RNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase
D012329:RNA, Bacterial; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D011061:Poly A; D008345:Manganese; D011062:Polynucleotide Adenylyltransferase; D012194:RNA-Directed DNA Polymerase; D008274:Magnesium