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|Title:||Analysis of tandemly and divergently oriented spinach chloroplast promoters|
|Author(s):||Rogers-Werneke, Sharon Anne|
|Doctoral Committee Chair(s):||Orozco, Emil M.|
|Department / Program:||Plant Biology|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
Biology, Plant Physiology
|Abstract:||Chloroplast genes in higher plants frequently synthesize multiple RNA transcripts. Part of this heterogeneity in RNA is due to posttranscriptional RNA processing events; however, some chloroplast genes such as the $\beta$ subunit of the ATP synthase (atpB) may contain multiple promoters. Ba131 deletion analyses have determined that 3 out of 4 of the previously observed spinach atpB $5\sp\prime$ ends represent transcription initiation sites and not processing sites. A previously undetected transcript situated one codon 3$\sp\prime$ to the translation initiation site was also identified.
A direct assay for chloroplast in vitro transcription was developed using the strong factor-independent transcription terminator from the threonine attenuator (thra) of the threonine biosynthetic operon in Escherichia coli. Factor-independent transcription terminators in prokaryotes contain GC-rich regions of dyad symmetry followed by consecutive thymidines. A second prokaryotic transcription terminator that is efficiently recognated by the chloroplast RNA polymerase was identified, the terminator from the histidine attenuator (hisa) from Salmonella typhimurium. Both thra and hisa will stop transcription when present in either orientation 3$\sp\prime$ to the ribolose-1,5-bisphosphate carboxylase (rbcL) promoter.
Genes in the chloroplast are closely spaced in either tandem, converging or diverging orientation. In order to analyze the closely spaced divergently oriented spinach rbcL and the atpB -455 promoters (this atpB promoter is situated nearest the rbcL promoter), 3 bidirectional transcription plasmids were constructed. These vectors each contained a multiple cloning region flanked by either 2 tandem copies of the thra terminators or by divergently oriented hisa and thra terminators. The divergently oriented rbcL and atpB promoters were inserted into the multiple cloning region of each of these vectors. Simultaneous transcription from both promoters was observed.
In order to measure interaction between the rbcL and atpB promoters, deletion mutants were constructed that increased or decreased the intergenic region between the promoters. Fewer RNA transcripts were produced from the mutants that altered the spacing, compared to the wild-type constructions. Therefore, the optimal spacing between the two promoters is the 152 bp region present in the wild-type DNA. Additional mutants were constructed which either removed the rbcL promoter or removed regions of the rbcL gene at the 3$\sp\prime$ end of the promoter. Transcription from the atpB promoter increased substantially when the rbcL promoter was removed. Deletions located 11 bp 5$\sp\prime$ to the -35 region of the atpB promoter did not transcribe this promoter with fidelity; instead, multiple transcripts were produced. These data indicated that the spinach atpB promoter and rbcL promoters interact transcriptionally and that sequences with the intergenic region are necessary for accurate transcription initiation.
|Rights Information:||Copyright 1990 Rogers-Werneke, Sharon Anne|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9026306|
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