TRANSCRIPTION TERMINATION AT THE ESCHERICHIA-COLI THRA TERMINATOR BY SPINACH CHLOROPLAST RNA-POLYMERASE IN-VITRO IS INFLUENCED BY DOWNSTREAM DNA-SEQUENCES

We have investigated the mechanism of transcription termination in vit ro by spinach chloroplast RNA polymerase using templates encoding vari ants of the transcription-termination structure (attenuator) of the re gulatory region of the threonine (thr) operon of Escherichia coil. Fou rteen sequence variants located within its d(G+C) stem-loop and d(A+T) -rich regions were studied. We found that the helix integrity in the s tem-loop structure is necessary for termination but that its stability is not directly correlated with termination efficiency. The sequence of the G+C stem-loop itself also influences termination. Moreover, the dA template stretch at the 3' end of the terminator plays a major rol e in termination efficiency, but base pairing between the A and U trac t of the transcript does not. From the studies using deletion variants and a series of mutants that after the sequences immediately downstre am from the transcription termination site, we found that termination of transcription by spinach chloroplast RNA polymerase was also modula ted by downstream DNA sequences in a sequence-specific manner. The sec ond base immediately following the poly(T) tract is crucial for determ ining the termination efficiency by chloroplast RNA polymerase, but no t of the T7 or E. coli enzymes.