Synthesis of polyadenylate-containing RNA in vitro in permeable cells of Escherichia coli B.
As a starting point for the study of the biosynthesis of polyadenylated RNA in bacteria, the characteristics of RNA synthesis by cells of Escherichia coli B made permeable to small molecules by treatment with toluene were examined. Such cells mediated the incorporation of radiolabeled ribonucleoside triphosphates into RNA in a reaction that was sensitive to inhibitors of RNA polymerase and required the simultaneous presence of the four ribonucleoside triphosphates. Between 10 to 15% of the RNA synthesized under these conditions was polyadenylated as shown by affinity chromatography on oligo(dT)-cellulose. The presence of orthophosphate or dADP, inhibitors of polynucleotide phosphorylase, had no effect on the reaction and the rate of RNA synthesis was indistinguishable in the polynucleotide phosphorylase-deficient strain PR-7 and in its otherwise isogenic parent strain PR-100. The poly(A) tracts associated with the newly synthesized RNA could be isolated after exhaustive digestion with pancreatic and T1 ribonucleases and accounted for 14% of the poly(A)-RNA. At least 74% of the poly(A) sequences were located at the 3' ends of RNA molecules and their weight-average length was 48 nucleotide residues. The size distribution of total RNA and poly(A)-RNA synthesized in the toluenized cell system was similar to that of the corresponding pulse-labeled fractions derived from growing cultures. The sequence complexity of poly(A)-RNA and unadenylated RNA synthesized in toluenized cells with [alpha-32P]CTP as the labeled substrate was analyzed by hybridization to fragments of Escherichia coli B DNA generated by digestion with EcoRI restriction endonuclease and immobilized on nitrocellulose sheets. Both RNA fractions hybridized with many DNA fractions, the hybridization patterns being similar with poly(A)-RNA and unadenylated RNA. This indicated that many different types of RNA transcripts synthesized in toluenized cells were subject to polyadenylation, but that polyadenylation was incomplete so that each transcript was present in both an adenylated and an unadenylated state.
D001483:Base Sequence; D002463:Cell Membrane Permeability / Q000187:drug effects; D055598:Chemical Phenomena; D002621:Chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D011061:Poly A / Q000096:biosynthesis*; D012313:RNA / Q000096:biosynthesis*; D012329:RNA, Bacterial / Q000096:biosynthesis*; D012333:RNA, Messenger; D014050:Toluene / Q000494:pharmacology
D012329:RNA, Bacterial; D012333:RNA, Messenger; D011061:Poly A; D014050:Toluene; D012313:RNA