POLYADENYLATED MESSENGER-RNA IN ESCHERICHIA-COLI - MODULATION OF POLY(A) RNA LEVELS BY POLYNUCLEOTIDE PHOSPHORYLASE AND RIBONUCLEASE-II

The effect of 3'-exoribonucleases on the polyadenylation of mRNA in Es cherichia coli was studied by comparing the synthesis and levels of po ly(A) RNA in wild-type E coli and mutant strains defective in the two major 3'-exoribonucleases: polynucleotide phosphorylase and ribonuclea se II. Mutations which substantially reduced the activity of these 3'- exonucleases caused a 10-fold increase in pulse-labeling of total poly (A) RNA in intact cells. When the net rate of RNA synthesis was measur ed in permeabilized cells, the mutant with defective 3'-exonucleases s howed 20- to 60-fold increased synthesis of total poly(A) RNA as well as of specific polyadenylated mRNAs, with less than two-fold changes i n non-poly(A) RNA. Measurement of mRNA polyadenylation in permeable ce lls under conditions when 3'-exoribonucleases were inactive showed a 6 -fold higher rate of poly(A) synthesis in the exonuclease-deficient mu tant strain, suggesting a higher concentration of mRNA 3'-ends amenabl e 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'-exonuclea ses were inactivated. Monitoring of the length of the poly(A) tracts b y denaturing polyacrylamide gel electrophoresis showed chain lengths o f up to 45 residues in the 3'-exonuclease-deficient mutant, whereas mo st of the poly(A) tracts in the parent strain were shorter than 12 res idues. These results show that 3'-exonucleases reduce the level of pol yadenylated mRNA in E coli not merely by causing its degradation but a lso by reducing its rate of synthesis, presumably by competing with po ly(A) polymerase for the 3'-ends of mRNA.