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|Title:||An in vitro transcription termination system to analyze spinach chloroplast promoters|
|Doctoral Committee Chair(s):||Orozco, Emil M.|
|Department / Program:||Agronomy|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||To determine whether chloroplast RNA polymerase would accurately terminate transcription in vitro, the 3$\sp\prime$ end of the rbcL gene as well as various factor-independent transcription terminators from E. coli were fused to the spinach chloroplast rbcL promoter. Transcription of the rbcL minigene did not result in production of the expected 265 nucleotide RNA. However, the spinach chloroplast RNA polymerase did terminate transcription with varying efficiency at the thra, rrnB, rrnC and gene 32 terminators. The most efficient transcription termination was observed for the thra terminator. For each of the prokaryotic terminators, the chloroplast enzyme ceased transcription at essentially the same position as the E. coli RNA polymerase.
To further characterize the soluble spinach chloroplast RNA polymerase, a second promoter from a spinach chloroplast gene, atpB, was cloned upstream from the thra terminator. The pTZ19-P scLSTa and pTZ19-P scBSTa "minigene" constructions were transcribed in vitro by a transcriptionally-active extract from spinach chloroplasts. The efficiency of transcription was up to 3.5 RNAs per template (pTZ19-P scLSTa). The temperature optimum $(30\sp\circ)$ and the Km for UTP (1.0 $\mu$M) for RNA synthesis in vitro (pTZ19-P scLSTa) were determined. Although the chloroplast RNA polymerase is sensitive to increasing ionic strength, for both pTZ19-P scLSTa and pTZ19-P scBSTa increasing concentrations of potassium chloride were much more inhibitory than corresponding increases in potassium glutamate. The relative transcriptional efficiencies of the rbcL and atpB promoters were compared under a variety of conditions. The ratio of rbcL:atpB transcripts varied from 2.0 to 9.9, depending on the length of time and the ionic environment of the reaction.
The efficiency of termination at the thra terminator by the chloroplast RNA polymerase was determined by a unique approach in which multiple copies of the thra terminator were cloned. The efficiency of termination at the thra terminator by the chloroplast RNA polymerase is 63 to 66%. The efficiency of termination at the thra terminator by the T7 RNA polymerase is 63% for either supercoiled or linearized DNA template. A much higher termination efficiency ($\sim$90%) was obtained for the T7 enzyme when only 10 $\mu$M UTP was used in the reaction.
One application of this in vitro transcription termination system will be the construction of a chloroplast promoter map. Twenty-eight promoter-containing recombinants were obtained by a shotgun cloning approach. Two of these clones contained the same 254 bp DNA fragment in opposite orientations, corresponding to the sequence $-4$ to $-257$ upstream from the protein coding region of the psbD gene. Two transcription initiation sites for the psbD gene were determined.
|Rights Information:||Copyright 1989 Chen, Liang-Jwu|
|Date Available in IDEALS:||2011-05-07|
|Identifier in Online Catalog:||AAI9010825|