Polyadenylated mRNA 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 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'-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 measured in permeabilized cells, the mutant with defective 3'-exonucleases showed 20- to 60-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 rate of 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.
Cao
G J
GJ
Boston Biomedical Research Institute, MA 02114, USA.
Kalapos
M P
MP
Sarkar
N
N
D002463:Cell Membrane Permeability; D018076:DNA, Complementary / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005095:Exoribonucleases / Q000378:metabolism*; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011117:Polyribonucleotide Nucleotidyltransferase; D005095:Exoribonucleases; C022535:exoribonuclease II