Presence of polyriboadenylate sequences in pulse-labeled RNA of Escherichia coli.

Abstract

Pulse-labeled RNA isolated from E. coli cells grown on limiting phosphate medium and phosphate-containing medium was analyzed by oligo(dT-cellulose chromatography and by Millipore binding assay for polyriboadenylate-containing RNA. Whereas poly(A)containing RNA amounted to as much as 15% of the total pulse-labeled RNA from cells grown on limiting phosphate medium, pulse-labeled RNA from cells grown on phosphate medium Extraction of RNA. The labeled cells were mixed with about 0.5 g of unlabeled cells grown under the same conditions, ground with 1 g of alumina, suspended in 2-3 ml of 0.05 M sodium acetate buffer containing 0.5 M NaCl, and clarified by centrifugation.The supernatant solution was mixed with 10% sodium dodecylsulfate to give a final concentration of 0.5%, treated with an equal volume of freshly distilled phenol, agitated vigorously for 30 sec, allowed to stand for 10 min, and agitated for 30 sec. The suspension was treated with 1 volume of CHC13, mixed vigorously for 30 sec, and allowed to stand for 5 min, and mixed for 30 sec. The aqueous layer obtained after centrifugation at 12,000 X g for 5 min was again deproteinized with CHC13, mixed with 2 volumes of 95% C2H5OH, and allowed to stand overnight at -20°. Precipitated RNA was collected by centrifugation, dissolved in 1 ml of 0.01 M Tris-Cl buffer (pH 7.5) containing 2 mM MgCl2, treated with electrophoretically pure DNase (10,g), incubated for 30 min at 40, and the solution was subjected twice to deproteinization with CHC13. RNA in the aqueous layer was precipitated with C2H5OH, collected by centrifugation at 12,000 X g, dissolved in 1 ml of Tris-Cl buffer (pH 7.5) containing 0.5 M KCl, and dialyzed twice against 100 volumes of the same buffer for a period of 1 hr. The yield of RNA was 30-40% based on radioactivity, and had a A260/280 ratio over 2.0.Oligo(dT)Cellulose Column Chromatography. The procedure described by Aviv and Leder (23)

Detection of high levels of polyadenylate-containing RNA in bacteria by the use of a single-step RNA isolation procedure

“…The high level of poly(A)-RNA found in bacterial pulse-labeled RNA contrasts with the much lower and variable levels seen by other workers. These have ranged from less than 1% of pulse-labeled RNA in E. coZi (5) to 15% when the same organism was grown with limiting phosphate (6). In B. subtiZis, one group of workers (8) reported a poly(A)-RNA content of 5-11% during growth and 3-6% during sporulation, while others (9) were unable to detect poly(A)-RNA in growing B. subtiZis but found up to 20% during sporulation.…”

All things must pass: contrasts and commonalities in eukaryotic and bacterial mRNA decay

“…Despite the widespread belief that 3’ polyadenylation was a characteristic unique to eukaryotic messages, E. coli had long been known to contain a poly(A) polymerase 37, 38 and polyadenylated RNA 39, 40 . However, it was not until many years later that the important role of polyadenylation in bacterial RNA decay was recognized 41, 42 .…”

RNA Processing and Degradation inBacillus subtilis

“…RNAs from both E. coli and B. subtilis are polyadenylated at their 3Ј end (74,75,164,216). Although the lengths of the tails are similar (10 to 20 A residues), B. subtilis contains intrinsically higher levels of poly(A) RNA than E. coli does (reviewed in reference 206).…”