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

Erion, Jack L.; Tarnowski, Joseph; Peacock, Susan; Caldwell, Paul E.; Redfield, Betty; Brot, Nathan; Weissbach, Herbert

doi:10.1007/bf01578646

Cited by 24 publications

(16 citation statements)

References 42 publications

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“…Transcription in procaryotes has been used as a model to describe chloroplast RNA synthesis because of similarities between plastid and bacterial genomes. However, evidence supporting this hypothesis is indirect, based on DNA sequence homology (60) and expression of chloroplast genes in bacterial systems (20,22,24,52). Only a few studies have examined directly the synthesis of chloroplast RNAs at the molecular level by using plastid enzymes in vitro (16,27,28,34,45).…”

Section: Discussionmentioning

confidence: 99%

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

Hanley‐Bowdoin¹,

Orozco²,

Chua³

1985

Mol. Cell. Biol.

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The large subunit gene (rbcL) of ribulose 1,5-bisphosphate carboxylase was transcribed in vitro by using maize and pea chloroplast extracts and a cloned plastid DNA template containing 172 base pairs (bp) of the maize rbcL protein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maize rbcL RNAs with 5' termini positioned 300, 100 to 105, and 63 nucleotides upstream of the protein-coding region. A deletion of 109 bp, including the "-300" 5' end (the 5' end at position -300), depressed all rbcL transcription in vitro. A plasmid DNA containing this 109-bp fragment was sufficient to direct correct transcription initiation in vitro. A cloned template, containing 191 bp of plastid DNA which includes the -105 and -63 rbcL termini, did not support transcription in vitro. Exogenously added -300 RNA could be converted to the -63 transcript by maize chloroplast extract. These results established that the -300 RNA is the primary maize rbcL transcript, the -63 RNA is a processed form of the -300 transcript, and synthesis of the -105 RNA is dependent on the -300 region. The promoter for the maize rbcL gene is located within the 109 bp flanking the -300 site. Mutagenesis of the 109-bp chloroplast sequence 11 bp upstream of the -300 transcription initiation site reduced rbcL promoter activity in vitro.Ribulose 1,5-bisphosphate carboxylase (RUBISCO) catalyzes CO2 fixation, the first step of the Calvin cycle (40). In higher plants, the chloroplast enzyme is composed of eight identical, catalytic polypeptides of 50,000 to 60,000 kilodaltons and eight smaller polypeptides of 12,000 to 20,000 kilodaltons (2). The small subunit, whose function is unknown, is encoded in the nucleus by a small multigene family (7,14,19). The large subunit gene (rbcL) is present as a single copy on the multicopy chloroplast genome (5). Under most conditions, the synthesis of the two RUBISCO subunits is coordinated (55) and regulated by light (15, 50) and cytokinins (23). In maize and other C4 plants, RUBISCO expression is leaf cell-type specific (30). The maize mRNAs encoding the two subunits are present in bundle sheath cells but not in mesophyll cells, which assimilate CO2 via ribulose 1,5-bisphosphate and C4 dicarboxylic acids, respectively (11,35).In chloroplast genomes of higher plants, the genes for the large subunit of RUBISCO and the beta subunit of ATP synthetase (atpB) are adjacent and transcribed divergently (31, 60). The 5' ends of the two genes are separated by approximately 150 base pairs (bp) (52). Northern hybridization analyses of transcripts from a broad spectrum of angiosperms indicate that the rbcL gene is generally transcribed as a 1.6-kilobase mRNA, although larger transcripts are also observed (46). Si nuclease protection experiments have revealed the presence of two 5' termini for the rbcL RNAs of maize, barley, spinach, and peas (16,21,41,49,62.) Tobacco has a single rbcL RNA with its 5' end at position -180 relative to the coding region (53). In maize ...

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Section: Discussionmentioning

confidence: 99%

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

Hanley‐Bowdoin¹,

Orozco²,

Chua³

1985

Mol. Cell. Biol.

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“…1, peak 1). The lower line is the amino acid sequence derived from the DNA sequence for the LS of RbuP2 carboxylase from tobacco (14), spinach (15), and wheat (8 (6), which identified this peptide as the penultimate N-terminal tryptic peptide of the LS of RbuP2 carboxylase. Position 10 in the LS of RbuP2 carboxylase from spinach, tobacco, and muskmelon leaves was a serine residue, but this was substituted by glycine in the enzyme from wheat.…”

Section: Fig 2 Amino Acid Composition Of Rbup2 Carboxylase Lsmentioning

confidence: 99%

Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

Houtz

Stults

Mulligan

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of NE-trimethylation.Ribulose bisphosphate carboxylase/oxygenase [RbuP2 carboxylase, 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] is a hexadecameric plant protein with eight large subunits (LSs) and eight small subunits (SSs) (for a recent review, see ref.

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“…This in vivo start site has been found to be preceded by a region which is similar to the consensus sequence determined for promoter sites recognized by E. coli RNA polymerase (26). This similarity has been further substantiated by showing that E. coli RNA polymerase initiates RNA transcripts from this position in vitro using the rbcL gene from either spinach (8) or tobacco (23). In maize, however, there is considerable divergence in the nucleotides upstream from the coding region of the gene compared to the same region in spinach and tobacco.…”

Section: Introductionmentioning

confidence: 83%

“…For higher plants, the most detailed information, including the nucleotide sequence, is available for the rbcL gene from tobacco (23,24), maize (5,10,11,17) and spinach (7,8,30,31). The results of these studies indicate that the gene is transcribed as a single 1.6 kilobase (Kb) mRNA species, colinear with the DNA sequence in all three plants (17,23,31).…”

Section: Introductionmentioning

confidence: 93%

Characterization of the mRNA transcripts of the maize, ribulose-1,5-bisphosphate carboxylase, large subunit gene

Erion¹

1985

Plant Mol Biol

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The analysis of RNA isolated from maize leaves indicates that there are two mRNA transcripts which are homologous to the chloroplast encoded gene for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL). The 5' end of the smaller transcript, 1.62 Kb in length, begins at a position which is 60 nucleotides upstream from the coding sequence of the gene, corresponding to the position mapped by earlier workers. The larger transcript, 1.86 Kb in length, has not been previously described and originates from a site on the gene which is 302 nucleotides upstream from the coding sequence. The increased size of the largerbcL mRNA transcript from maize, as compared to the transcripts from spinach and tobacco, results from the presence of a 130 nucleotide insert between the two maizerbcL gene mRNA start sites. The DNA sequence adjacent to the start site for the large mRNA transcript is shown to have greater than 90% homology to the DNA sequences adjacent to the mRNA start sites for the spinach and tobaccorbcL gene. Discounting the presence of the insert in the maizerbcL gene, the nucleotides upstream from the coding sequence in the maize, spinach and tobaccorbcL genes, all share approximately the same amount of homology to each other. This homology, along with other evidence, suggests that the large mRNA species are the primary transcripts and that the smaller RNA species are the result of post-transcriptional processing. The finding that spinach also contains two different mRNA transcripts for therbcL gene is consistent with this model.

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

Cited by 24 publications

References 42 publications

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.

Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

Characterization of the mRNA transcripts of the maize, ribulose-1,5-bisphosphate carboxylase, large subunit gene

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