Nucleic Acids Research, 1995, Vol. 23, No. 22 4690-4697
© 1995
Articles |
Transcription termination at the Escherichia coli thra terminator by spinach chloroplast RNA polymerase in vitro is influenced by downstream DNA sequences
Institute of Molecular Biology, National Chung Hsing University Taichung 40227, Taiwan, Republic of China 1Department of Botany, National Taiwan University Taipei, Taiwan, Republic of China 2DEKALB Plant Genetics 62 Maritime Drive, Mystic, CT 06355, USA 3Department of Biochemistry, College of Medicine and School of Chemical Sciences, University of Illinois Urbana, IL 61801, USA
*To whom correspondence should be addressed
Received July 24, 1995. Accepted October 20, 1995.
We have investigated the mechanism of transcription termination in vitro by spinach chloroplast RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. Fourteen 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 stem-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 role in termination efficiency, but base pairing between the A and U tract of the transcript does not. From the studies using deletion variants and a series of mutants that alter the sequences immediately downstream from the transcription termination site, we found that termination of transcription by spinach chloroplast RNA polymerase was also modulated by downstream DNA sequences in a sequence-specific manner. The second base immediately following the poly(T) tract is crucial for determining the termination efficiency by chloroplast RNA polymerase, but not of the T7 or E.coli enzymes.