rbcL Transcripts Research Articles

A methyl jasmonate-induced shift in the length of the 5' untranslated region impairs translation of the plastid rbcL transcript in barley.

The plant growth substance (-)-jasmonic acid methyl ester (methyl jasmonate, JaMe) affects plastid gene expression at the protein and mRNA levels when applied exogenously to detached leaf segments of Hordeum vulgare L. cv. Salome. Translation of the large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (LSU, rbcL gene products) and of the 65 and 68 kDa proteins of photosystem I (psaA and psaB gene products, respectively) ceased, whereas synthesis of the 32 kDa photosystem II protein (D1, psbA gene product) continued in JaMe-treated leaf tissues. These changes were not caused by corresponding alterations in transcript abundances. The loss of LSU protein synthesis, occurring within 24 h of JaMe treatment, correlated with a decline in the in vitro-translatable rbcL mRNA, but contrasted with an almost constant transcript level. The 5' ends of the rbcL transcripts shifted from '-59' in freshly harvested or water-treated leaves to '-94' in JaMe-treated leaf tissues. Transcripts ending at these positions presumably arise from alternative processing of the primary transcript ending at position '-316'. The '-94' transcript contains, within the 5' untranslated region, a 35-base motif with remarkable complementarity to the extreme 3' terminal part of the 16S rRNA, involved in intramolecular base pairing within the ribosome and can associate with 30S but not 70S complexes in organello, suggesting that intermolecular base pairing impairs translation initiation, probably by competing for ribosome binding at the Shine-Dalgarno sequence. In contrast, transcripts ending at '-59' lack the 5' terminal 'extra' sequence and are active in terms of translation initiation.

Plastid Genes Encoding the Transcription/Translation Apparatus Are Differentially Transcribed Early in Barley (Hordeum vulgare) Chloroplast Development (Evidence for Selective Stabilization of psbA mRNA).

Chloroplast genomes encode rRNAs, tRNAs, and proteins involved in transcription, translation, and photosynthesis. The expression of 15 plastid genes representing each of these functions was quantitated during chloroplast development in barley (Hordeum vulgare). The transcription of all plastid genes increased during the initial phase of chloroplast development and then declined during chloroplast maturation. RNAs corresponding to rpoB- rpoC1-rpoC2, which encode subunits of a plastid RNA polymerase, and rps16, which encodes a ribosomal protein, reached maximal abundance early in chloroplast development prior to genes encoding subunits of the photosynthetic apparatus (rbcL, atpB, psaA, petB). Transcription of rpoB as well as 16S rRNA, trnfM-trnG, and trnK was high early in chloroplast development and declined 10-fold relative to rbcL transcription during chloroplast maturation. RNA hybridizing to psbA and psbD, genes encoding reaction center proteins of photosystem II, was differentially maintained in mature chloroplasts of illuminated barley. Differential accumulation of psbD mRNA relative to rbcL mRNA was due to light-stimulated transcription of psbD. In contrast, enhanced levels of psbA mRNA in mature chloroplasts were due primarily to selective stabilization of the psbA mRNA. These data document dynamic modulation of plastid gene transcription and mRNA stability during barley chloroplast development.

Regulation of Light-Induced Chloroplast Transcription and Translation in Eight-Day-Old Dark-Grown Barley Seedlings

Plastid transcription and translation are light-activated in 8-day-old dark-grown barley (Hordeum vulgare L.) seedlings. Pretreatment of dark-grown seedlings with cycloheximide (inhibitor of cytoplasmic protein synthesis) abolished the activation of rbcL, psbA, and psaA-B transcription by light. In contrast, inhibition of plastid protein synthesis by chloramphenicol stimulated light-activated transcription of rbcL, psbA, and psaA-B. Light-induced transcription of the plastid genome occurred normally in the chlorophyll-deficient mutant xan-J(64). These results suggest that although the light-induced activation of plastid transcription is modulated by cytoplasmic and organellar protein synthesis, transcriptional activation is not dependent on the absorption of light by protochlorophyllide or the attainment of photosynthetic competence. In addition, plastid translation increased dramatically when 8-day-old dark-grown seedlings were illuminated and activation was dependent on cytoplasmic protein synthesis. Blockage of light-activated plastid transcription by Tagetin treatment (inhibitor of plastid RNA polymerase) did not attenuate the activation of plastid translation by light. These results suggest that while light simultaneously activates plastid transcription and translation, the rapid burst in plastid protein synthesis is due mainly to cytoplasmic-derived changes that regulate the rate of translation of pre-existing mRNAs.

Analysis of chloroplast promoters using bidirectional transcription vectors

Spinach chloroplast RNA polymerase has been shown to efficiently terminate transcription at the threonine attenuator (thra) from Escherichia coli. In this study, efficient transcription termination by the chloroplast RNA polymerase was observed at a second prokaryotic terminator, the histidine attenuator (hisa) from Salmonella typhimurium. Termination occurred regardless of the orientation of either attenuator. In higher-plant chloroplast DNA, the genes for the beta subunit of the ATPase (atpB) and the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) are adjacent and divergently transcribed. Bidirectional transcription vectors, containing the histidine and threonine terminators, were constructed to analyze the divergently oriented atpB and rbcL promotors. One plasmid construction, pRTT7, contained two tandem copies of the threonine attenuator (pRTT7). Two additional constructs, pRHT1 and pRHT2, each contained oppositely oriented copies of thra and hisa. A DNA fragment containing the rbcL and atpB promoters was inserted between the two terminators present in the pRTT7, pRHT1, and pRHT2 plasmids. Transcription of these recombinant DNAs by spinach chloroplast RNA polymerase resulted in discretely sized rbcL and atpB transcripts. In addition, these bidirectional transcription vectors were used to identify previously uncharacterized chloroplast promoters.

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Expression in Second Leaves of Maize Seedlings from Low and High Yield Populations

Ribulose-1,5-bisphosphate carboxylase oxygenase (EC 4.1.1.39) (Rubisco) activity, Rubisco-protein, and Rubisco large and small subunit gene (rbcL and rbcS) transcripts were measured at seven stages of development in the second leaf of maize (Zea mays L.) seedlings belonging to low and high yield populations. During the three early stages of development, when the leaf has not yet expanded, it was determined that increments in Rubisco-activity were caused by increases in Rubisco-protein and its mRNAs. Afterward, the rbcS level decreased sharply down to nondetectable levels at the seventh stage, when the leaf was at the beginning of senescence. As a contrast, rbcL transcript decreased slowly and Rubisco-protein accumulated up to the fifth stage, when the leaf reached its maximum expansion. A slight decrease in Rubisco-protein was then observed. These results suggest that at early stages of development Rubisco-activity and Rubisco-protein are regulated mainly at the transcriptional level. At the later phase the regulation seems to be at other biochemical levels. Neither Rubisco activity nor Rubisco-protein showed correlation with yield for both maize populations at this stage of development. Slightly higher levels of both transcripts were observed in the high yield population.

Stem-loop structures at the 3′ end of tobacco Rubisco large subunit mRNA

The are two inverted repeat nucleotide (nt) sequences, each capable of forming a stem-loop structure ( sls) at the 3′ end of the tobacco Rubisco large subunit mRNA ( rbcL). The smaller sls is followed by a larger sls. The in vivo functions of the 3′ sls of the rbcL mRNA were characterized using the Escherichia coli system. S1 mapping of the rbcL transcripts synthesized in E. coli revealed that the 3′ end of a major transcript in the bacterial cell is almost identical to the 3′ end of authentic chloroplast (cp) rbcL mRNA. This native 3′ end is located 4 nt downstream from the larger sls for the cp mRNA and 6 nt for the bacterial transcript, respectively. Deletion expriements show that the larger sls is essential for producing the native 3′ end of rbcL mRNA in E. coli. The sls do not function an an efficient transcription terminator but can stabilize upstream mRNA segments in vivo.

Transcriptional interaction between the promoters of the maize chloroplast genes which encode the β subunit of ATP synthase and the large subunit of ribulose 1,5-bisphosphate carboxylase

The genes encoding the beta subunit of ATP synthase and the large subunit of ribulose 1,5-bisphosphate carboxylase are located on opposite strands of the maize chloroplast genome. Their transcription start sites are separated by a 159 bp sequence that includes the promoters for both genes. The effects of deleting or modifying one of the two promoters on transcription from the adjacent, unaltered promoter were assessed in vitro using maize chloroplast extracts to transcribe cloned maize DNA templates. When the atpB promoter was disrupted by an 8 bp insertion, rbcL transcription was not altered. When the rbcL promoter was disrupted by a 2 bp insertion, atpB transcription decreased, whereas when the rbcL promoter region was deleted, atpB transcription increased. Activity of the atpB promoter was also reduced when the + 2 bp-rbcL promoter template was transcribed in vitro by Escherichia coli RNA polymerase. The changes in atpB transcriptional efficiency were only seen when the atpB and rbcL promoters were closely spaced on the same template molecule. These results established that the atpB and rbcL promoters interact in vitro in a cis and spacing dependent manner. The interaction may have physiological relevance in vivo.

Light-regulated translation of chloroplast proteins. I. Transcripts of psaA-psaB, psbA, and rbcL are associated with polysomes in dark-grown and illuminated barley seedlings.

We have previously observed (Klein, R. R., and J. E. Mullet, 1986, J. Biol. Chem. 261:11138-11145) that translation of two 65-70-kD chlorophyll a-apoproteins of Photosystem I (gene products of psaA and psaB) and a 32-kD quinone-binding protein of Photosystem II (gene product of psbA) was not detected in plastids of dark-grown barley seedlings even though transcripts for these proteins were present. In the present study it was found that nearly all of the psaA-psaB transcripts in plastids of dark-grown plants were associated with membrane-bound polysomes. Membrane-associated polysomes from plastids of dark-grown plants synthesized the 65-70-kD chlorophyll a-apoproteins at low levels when added to a homologous in vitro translation extract capable of translation elongation. However, when etioplast membranes were disrupted with detergent, in vitro synthesis of the 65-70-kD chlorophyll a-apoproteins increased to levels observed with polysomes of plastids from illuminated plants. These results suggest that synthesis of the chlorophyll a-apoproteins of Photosystem I is arrested on membrane-bound polysomes at the level of polypeptide chain elongation. In addition to the selective activation of chlorophyll a-apoprotein translation, illumination also caused an increase in chloroplast polysomes (membrane-associated and stromal) and induced a recruitment of psbA and rbcL transcripts into chloroplast polysomes. These results indicate that in conjunction with the selective activation of chlorophyll a-apoprotein elongation, illumination also caused a general stimulation of chloroplast translation initiation.

The Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene. II. The spliced mRNA and its product.

The polypeptide product of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase from Euglena gracilis based on the DNA sequence of the chloroplast-encoded gene is described. The large subunit polypeptide of 475 codons is co-linear with the homologous polypeptides from other chloroplasts and cyanobacteria. The amino acid sequence is 92% homologous to that of Chlamydomonas, 84% homologous to spinach, 82% homologous to maize, and 80% homologous to that of the cyanobacterium Anabaena variabilis. Known functional domains of the protein are coded by the larger exons of the gene. Introns in the gene generally occur at coding sequences specifying hydrophilic, presumably surface exposed, regions of the polypeptide. The location of some of the introns may reflect a separation of functional domains. The 5'- and 3'-ends of the rbcL transcript were determined by primer extension sequencing using reverse transcriptase and S1 nuclease protection, respectively. The transcribed but untranslated sequences are quite distinct from those from other rbcL loci.

Antitermination is required for readthrough transcription of the maize rbcL gene by a bacteriophage promoter in Escherichia coli.

Sequences upstream of the 5' end of the rbcL gene in maize chloroplast fragment Bam 9, have a polar effect on the expression of rbcL from an external upstream bacteriophage PL promoter in Escherichia coli. This polarity can be suppressed by the bacteriophage transcription antitermination protein N or Q. The requirement for transcription antitermination is abolished if DNA upstream of rbcL is removed by a deletion. We have also investigated the ability of RNA polymerase initiating transcription at PL in the presence of N to transcribe through the normal rbcL transcription terminator and into sequences beyond. RNA polymerase initiating at PL can traverse rbcL and its 5' and 3'-flanking regions in the presence of N.

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

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.

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

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.

Control of psbA gene expression: in mature Spirodela chloroplasts light regulation of 32-kd protein synthesis is independent of transcript level

Spirodela oligorrhiza plants were used to study direct effects of light on plastid gene expression uncoupled from sequential chloroplast-developmental processes. Specific transcript levels were analysed using chloroplast, gene-fragment probes. Protein synthesis in vivo and in vitro was also measured. In tissue with mature chloroplasts, light/dark regimes had no substantial effect on transcript levels of the psbA gene coding for the 32-kd protein. However, under the same conditions, synthesis of the protein itself was dramatically affected. We conclude that in mature chloroplasts synthesis of 32-kd protein is regulated mainly at a translational level. Transcript levels of the rbcL gene, coding for the large subunit of ribulosebisphosphate carboxylase-oxygenase, were somewhat sensitive to light/dark effects, although not to the same degree as was synthesis of the protein. We conclude that translational events and transcript levels are involved in regulating synthesis of this polypeptide. Carbon deprivation in the dark reduced psbA and rbcL transcript levels appreciably below those found in non-starved, dark-grown tissue. This suggests that starvation and dark effects must be experimentally separated from each other for valid conclusions to be drawn about light/dark regulation of chloroplast gene expression.

Multiple transcripts for higher plantrbcL andatpB genes and localization of the transcription initiation site of therbcL gene

We have compared therbcL andatpB transcription units from spinach, maize, and pea. In most cases multiple transcripts were found for a given chloroplast gene. The 5' termini of these transcripts were determined by S1 nuclease protection and primer extension analyses. TherbcL transcripts have 5' termini 178-179 and 64 nucleotides (spinach), 300 and 59-63 nucleotides (maize), and 178 and 65 nucleotides (pea) upstream from their respective protein coding regions. TheatpB transcripts have 5' termini (453-454, 272-273, 179, and 99 nucleotides (spinach), 298-302 nucleotides (maize), and 351-355 nucleotides (pea) upstream from their respective protein coding regions. The intergenic distance between therbcL andatpB genes is relatively constant (152 to 157 base pairs) among the three chloroplast genomes. In spinach, maize, and pea, the 80 base pairs surrounding the 5' end of therbcL gene (±40 base pairs) have 85% sequence homology. Similarly, the 60 base pairs preceding theatpB gene have 48% sequence homology. Both genes have '-10' and '-35' regions that resemble the prokaryotic consensus promoter sequence. The larger, but not smaller,rbcL transcripts from spinach and pea can be labeled with alpha-(32)P-GTP by guanylyltransferase. These data suggest that DNA sequences 178-179 (spinach), 300 (maize), and 178 (pea) base pairs before therbcL protein coding regions represent sites of transcription initiation. The sequences 59-65 base pairs before therbcL protein coding regions may correspond to sites of RNA cleavage.

Anin vitrosystem for accurate transcription Initiation of chloroplast protein genes

We have developed an homologous in vitro system from spinach chloroplasts that correctly initiates transcription of the plastid genes for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) and the beta subunit of the plastid ATPase (atpB). The transcriptionally active extracts from spinach chloroplasts require circular DNA templates for specific initiation. The RNA polymerase activity is insensitive to rifampicin. The extent of transcription in vitro is a function of the extract:template ratio. The efficiency of the rbcL transcription in vitro is more than one transcript per one hundred templates per hour.