Functional inactivation of lac alpha-peptide mRNA by a factor that purifies that Escherichia coli RNase III.
Using RNA-directed synthesis of the alpha-peptide of beta-galactosidase as an assay, a factor was purified that inactivated further function of the mRNA. In the presence of Ca2+ ions to inhibit most nuclease activity, inactivation of mRNA occurred during incubation with ribosomes or with a 1 M KCl wash of ribosomes. The inactivation activity required Mg2+ ions, and purified as a single factor which did not bind to DEAE-cellulose, but bound reversibly to phosphocellulose. The factor eluted from Sephadex G-150 with an apparent molecular weight of about 43,000. Purified 700-fold, it showed no detectable exonuclease activity, and little or no cleavage of a variety of single-stranded substrates, including full length lac operon mRNA; but repurified inactivated mRNA was still inactive for protein synthesis. The factor did not inhibit poly(U)-directed polyphenylalanine synthesis. When proteins isolated from the ribosomal wash were individually tested, highly purified RNase III, which purifies in the same way and has the same size, also inactivated lac mRNA. The ribosomal wash from an RNase III- strain showed little if any activity compared to that from an isogenic RNase III+ strain. The possibility of a site-specific inactivating cleavage of mRNA by RNase III at or near the 5' end is considered.
D002118:Calcium / Q000494:pharmacology; D004720:Endonucleases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D043244:Ribonuclease III; D012260:Ribonucleases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification; D001616:beta-Galactosidase / Q000096:biosynthesis
D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D004720:Endonucleases; D012260:Ribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli; D001616:beta-Galactosidase; D008274:Magnesium; D002118:Calcium