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Synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase in anin vitro partially definedE. coli system

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Abstract

Thein vitro DNA- or RNA-directed synthesis of the large subunit (LS) of spinach chloroplast ribulose-1,5-biphosphate carboxylase (RuP2C) has been examined in a highly definedE. coli transcription-translation system. Spinach chloroplast DNA, RNA and recombinant plasmids containing the spinach chloroplast LS gene (rbcL) have been used as templates in thein vitro system and a quantitative assay has been developed to measure LS formation. Thein vitro formed product contains formylmethionine at the N-terminal position and sediments primarily as a monomer. There is no detectable enzymatic activity associated with thein vitro product. To determine where theE. coli RNA polymerase used in these systems initiates, we have examined the transcripts produced by this enzymein vitro. Measurements of run-off transcripts indicate thatE. coli RNA polymerase initiates at the same position on the gene as is seenin vivo. In addition, the complete nucleotide sequence of therbcL gene including previously unsequenced 3′ and 5′ flanking regions has been determined. The sequence agrees, except at two nucleotide positions, with previously published sequencing data for this gene (Zurawski, G, Perrot, B, Bottomley, W, Whitfeld, PR, 1981. Nucleic Acids Res. 9:3251–3270).

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Abbreviations

LS:

large subunit

RuP2C:

ribulose-1,5-bisphosphate carboxylase

rbcL:

gene for LS of RuP2C

EF:

elongation factor

IF:

initiation factor

cp:

chloroplast

NaDodSO4 :

sodium dodecyl sulfate

MDPF 1:

2-methoxy-2,3-diphenyl-3-(2H)-furanone

RuP2 :

ribulose-1,5-bisphosphate

PVP-40:

polyvinylpyrrolidone-40 000 MW

DNAse:

Deoxyribonuclease 1

EDTA:

ethylene-diaminetetraacetic acid

PMSF:

phenylmethylsulfonyl fluoride

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Author notes

  1. Joseph Tarnowski

    Present address: Hoffman-La Roche, Inc., 07110, Nutley, NJ, USA

Authors and Affiliations

  1. Roche Institute of Molecular Biology, Roche Research Center, 07110, Nutley, NJ, USA

    Joseph Tarnowski, Susan Peacock, Paul Caldwell, Betty Redfield, Nathan Brot & Herbert Weissbach

  2. ARCO Plant Cell Research Institute, 94566, Dublin, CA, USA

    Jack L. Erion

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  1. Jack L. Erion
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  2. Joseph Tarnowski
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  3. Susan Peacock
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  5. Betty Redfield
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  6. Nathan Brot
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  7. Herbert Weissbach
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Erion, J.L., Tarnowski, J., Peacock, S. et al. Synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase in anin vitro partially definedE. coli system. Plant Mol Biol 2, 279–290 (1983). https://doi.org/10.1007/BF01578646

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  • DOI: https://doi.org/10.1007/BF01578646

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