Polyadenylated mRNA in Escherichia coli: Modulation of poly(A) RNA levels by polynucleotide phosphorylase and ribonuclease II

Add time:08/22/2019    Source:sciencedirect.com

The effect of 3′-exoribonucleases on the polyadenylation of mRNA in Escherichia coli was studied by comparing the synthesis and levels of poly(A) RNA in wild-type E coli and mutant strains defective in the two major 3′-exoribonucleases: polynucleotide phosphorylase and ribonuclease II. Mutations which substantially reduced the activity of these 3′-exonuclease caused a 10-fold increase in pulse-labeling of total poly(A) RNA in intact cells. When the net rate of RNA synthesis was measured in permeabilized cells, the mutant with defective 3′-exonucleases showed 20- to 6-fold increased synthesis of total poly(A) RNA as well as of specific polyadenylated mRNAs, with less than two-fold changes in non-poly(A) RNA. Measurement of mRNA polyadenylation in permeable cells under conditions when 3′-exoribonucleases were inactive showed a 6-fold higher rateof poly(A) synthesis in the exonuclease-deficient mutant strain, suggesting a higher concentration of mRNA 3′-ends amenable to polyadenylation. Steady-state levels of poly(A) RNA, measured by the ability to serve as template for oligo(dT)-dependent complementary DNA synthesis, also increased more than 40-fold when the 3′-exonucleases were inactivated. Monitoring of the length of the poly(A) tracts by denaturing polyacrylamide gel electrophoresis showed chain lengths of up to 45 residues in the 3′-exonuclease-deficient mutant, whereas most of the poly(A) tracts in the parent strain were shorter than 12 residues. These results show that 3′-exonucleases reduce the level of polyadenylated mRNA in E coli not merely by causing its degradation but also by reducing its rate of synthesis, presumably by competing with poly(A) polymerase for the 3′-ends of mRNA.

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