In vitro transcription of the supB-E tRNA operon of Escherichia coli. Characterization of transcription products.

The seven tRNA genes clustered in the supB-E region of the Escherichia coli chromosome were transcribed in vitro with purified RNA polymerase, using a restriction fragment from lambda psu degrees 2, a transducing phage carrying the chromosome region, as template. A single major transcript was synthesized, which was about 770 nucleotides long and contained all seven tRNA sequences. The terminal sequences of the transcript were determined and mapped on the DNA sequence of the supB-E region previously determined. The transcription start site is seven base pairs downstream from the Pribnow box sequence, as expected from the DNA sequence analysis and consistent with the findings on the trimeric tRNA precursor (pppG--tRNAMETM-tRNALeu-tRNAGln1) which was detected in an RNase P mutant and shown to be coded for by the supB-E region. Cleavage of the restriction fragment at the -35 region with another restriction endonuclease abolished the template activity of the fragment. Transcription of the supB-E tRNA operon was relatively unaffected by the presence of rho factor. Transcription termination occurs within a region of three bases between positions 770 and 772 from the transcription start site. Immediately upstream from the termination sites, there is a region of 26 nucleotides that could form a stem structure, thereby consistent with the general feature of rho-independent termination sites.