Specific endonucleolytic cleavage of the mRNA for ribosomal protein S20 of Escherichia coli requires the product of the ams gene in vivo and in vitro

Abstract

Endonucleolytic cleavage is believed to initiate the degradation of most bacterial mRNAs, but with several exceptions, the enzymes responsible have yet to be identified. Crude (S-30) or partially fractionated extracts of Escherichia coli strains with reduced exonuclease activities catalyze the cleavage of a 372-residue RNA substrate containing the sequences coding for ribosomal protein S20 to yield a number of discrete products. The major product of 147 residues is obtained in 60 to 70% yield, is coterminal with the 3' end of the substrate, and is identical to an mRNA fragment previously characterized in vivo (G. A. Mackie, J. Bacteriol. 171:4112-4120, 1989). A number of other products of 150 to 340 residues are also formed, and the cleavage sites, typically N decreases AU sequences, have been identified in the S20 mRNA substrate by Northern (RNA) blotting and primer extension. All cleavages required a native rather than a denatured RNA substrate. The rate of cutting of the S20 mRNA substrate at the site yielding the prominent 147-residue product appears to be independent of cleavages at other sites. In addition, the activity of the putative endonuclease(s) depends strongly, both in vivo and in vitro, on the product of the ams gene, which is known to influence mRNA lifetimes in vivo. Taken together, the data show that the fractionated extract described here reproduces steps in the degradation of some mRNAs which occur in living cells.