Abstract
A DNA-directed coupled transcription-translation system has been developed in cell-free extracts from the facultative phototroph, Rhodopseudomonas sphaeroides. The in vitro protein-synthesizing system was active when prepared from either chemoheterotrophically or photoheterotrophically grown cells. Optimal activity was dependent upon: use of extracts prepared freshly from early exponential phase cells, the method of cell breakage, and the length of preincubation of the extract (S-30), as well as the concentrations of S-30, template, and cations. The R. sphaeroides cell-free system was compared to one prepared from Escherichia coli. DNA templates tested included R. sphaeroides phage RS1 DNA and E. coli phages T4 and T7 DNA, as well as plasmids RSF1010, pBR322, pSL25 (a pBR322 derivative), and a chimeric plasmid of pSL25 and RSF1010. One RNA template, phage R17, was also employed to test translational fidelity. Transcriptional-translational specificity was observed between R. sphaeroides and E. coli and these observations are discussed in terms of differential gene expression among phylogenetically distinct groups of bacteria.
Highlights
The control of ICM biosynthesis in vivo has been the derivative), and achimericplasmidof pSL25and subject of numerous studies and considerable information has RSF1010
When culture conditions are altered from high to low oxygen tensions, the bacterium develops an extensive intracellular membrane system called the intracytoplasmic membrane; the ICM’ is the photosynthetic organelle.Further, therelative composition of the various protein constituents of the ICM, as well as the amount of cellular ICM, is governed by the available light intensity [4,5]. precise data arelacking, there areat least 70-100 genes involved in photosynthesis and photosynthetic membrane formation and control
The broad host range plasmid,RSF1010, that is known to replicate in Pseudomonas spp. [43],E . coli [44], and R. sphaeroides was used to optimize conditions for preparation of active S-30 extracts
Summary
~-[’~C]Leucin(e343mCi/mmol) Dependence ofprotein synthesis on thepreincubation time of the S-. 30 extract dinetriphosphate (50 Ci/mmol) werefrom New England Nuclear. Materials for cell-free protein synthesis were purchased from Calbiochem. AII other chemicals were of reagent grade. Reduction of endogenous proteinsynthesis activity with the length of the preincubation time. The conditions for assay were those described under “ExperimentalProcedures.”
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