Regulated in Vitro Synthesis of Escherichia coli Tryptophan Operon Messenger Ribonucleic Acid and Enzymes

Abstract

DNA-dependent synthesis of tryptophan (trp) operon mRNA and enzymes was studied in vitro. With φ80 trpEDCBA template DNA trp operon transcription was initiated primarily at a phage promoter estimated to be approximately 7400 to 8000 nucleotides from trpE. Sequential transcription of trp mRNA in the correct direction proceeded at a rate of about 19 nucleotides per s. Trp mRNA resulting from read-through transcription from the phage promoter was larger than 23 S. Coupled sequential synthesis of trp operon enzymes occurred at a rate of approximately 4 amino acids per s. Experiments were conducted to determine if ribosomes discharge from polycistronic trp mRNA following translation termination at natural chain termination codons. Kasugamycin was used to inhibit reinitiation of translation. Kasugamycin shut down of steady state translation required 6.5 min for trpE enzyme, 7 min for trpD enzyme, and 3 min for trpA enzyme. These results indicate that ribosomes which translate trpE mRNA must discharge and reattach in order to translate trpD mRNA, and that ribosomes that translate trpD mRNA must discharge and reattach in order to translate trpA mRNA. Kasugamycin inhibition of trpD enzyme and trpA enzyme synthesis under appropriate conditions of sequential transcription and coupled translation provided further evidence for obligatory discharge and reattachment of ribosomes. Enzyme synthesis coupled to trp operon-promoted transcription from λh80 trp template DNA was repressible by tryptophan in reaction mixtures containing S-30 extract from trpR+ cells. In contrast, enzyme synthesis coupled to read-through transcription from φ80 trp template DNA was essentially irrepressible. It is concluded that read-through transcription is not repressible in vitro. Trp operon mRNA transcribed from a λh80 trpEDCBA DNA template was smaller than 23 S. The polar mutation trpC6 was expressed in vivo in phage λh80 trpEDCBA 190-9 but not in phage φ80 trpEDCBA 190. Read-through transcription of the trp operon occurs in the latter phage but not in the former. Polarity was not expressed in vitro using template DNA from either phage. Trp operon mRNA was extremely stable in vitro; the half-life varied between 30 min and more than 2 hours.