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 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.
Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan, Republic of China.
L J
LJ
Chen
Y J
YJ
Liang
S T
ST
Jeng
E M
EM
Orozco
R I
RI
Gumport
C H
CH
Lin
M T
MT
Yang
D001482:Base Composition; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D014644:Genetic Variation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009876:Operon; D016133:Polymerase Chain Reaction; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D018724:Spinacia oleracea / Q000201:enzymology; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D013912:Threonine / Q000096:biosynthesis; D014158:Transcription, Genetic
D013912:Threonine; D012321:DNA-Directed RNA Polymerases