Expression and characterization of RNase III and Era proteins. Products of the rnc operon of Escherichia coli

Abstract

The synthesis rates of ribonuclease III (RNase III) and Era proteins are relatively low, and expression of the era gene is translationally coupled with expression of the rnc gene. Expression of both genes is negatively controlled by RNase III itself. We have constructed plasmids that overproduce RNase III and/or Era proteins under the control of the lambda PL promoter. A plasmid with the rnc gene under PL control expresses RNase III at levels greater than 40% of total cellular protein. Another plasmid with the era gene under PL control and a modified translation-initiation signal produces up to 80% of total cell protein as Era. Each protein has been purified using simple and rapid procedures. Purified RNase III protein specifically processes mRNA transcripts containing known RNase III sites. The purified Era protein binds GDP and GTP and has GTPase activity. Kinetic analysis shows that one molecule of GTP or GDP is bound/Era peptide with a Kd of 5.5 microM for GTP binding and 1.0 microM for GDP binding. The Km of the Era GTPase is 9.0 microM, and the maximum catalyzed rate of GTP hydrolyzed/min/mol of Era protein at 37 degrees C is 9.8 mmol.