Expression of the tryptophan (trp) operon of Escherichia coli was shown to be regulated by attenuation in an in vitro DNA-dependent protein-synthesizing system. In extracts prepared from a temperature-sensitive tryptophanyl-tRNA synthetase mutant, plasmid-directed trpE enzyme synthesis was inhibited 2- to 3-fold by addition of purified wild type tryptophanyl-tRNA synthetase. When the extract used from a strain bearing a trpTts mutation that reduces charging of tRNATrp in vivo, a 2- to 3-fold increase in trpE enzyme synthesis was observed when an excess of uncharged wild type tRNATrp was added. Analysis of attenuation by measurement of trp mRNA synthesis was facilitated by constructing a plasmid (pAD1) containing the rpoC transcription terminator inserted early in trpE. Transcription proceeding past the trp attenuator of this plasmid terminates at this new terminator sequence and results in the production of a approximately 400-nucleotide long read-through transcript. Using this plasmid and extracts prepared from the tryptophanyl-tRNA synthetase mutant, a 4- to 8-fold decrease in relative read-through transcription was observed in response to exogenously added wild type tryptophanyl-tRNA synthase. Kinetic analyses of trp mRNA synthesis and studies using plasmid template DNAs bearing trp attenuator mutations indicate that translation of the leader peptide coding region of the transcript regulates transcription termination at the trp attenuator.