Synthesis Of Large Subunit Research Articles

SnapShot: Eukaryotic ribosome biogenesis II

Ribosome production is essential for cell growth. Approximately 200 assembly factors drive this complicated pathway that starts in the nucleolus and ends in the cytoplasm. A large number of structural snapshots of the pre-60S pathway have revealed the principles behind large subunit synthesis. To view this SnapShot, open or download the PDF.

Ribulose-1,5-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 Is Required for Holoenzyme Assembly in Maize

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for Rubisco accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process.

A mechanism for light-induced translation of the rbcL mRNA encoding the large subunit of ribulose-1,5-bisphosphate carboxylase in barley chloroplasts.

Translational regulation plays a key role in light-induced expression of photosynthesis-related genes at various levels in chloroplasts. We here present the results suggesting a mechanism for light-induced translation of the rbcL mRNA encoding the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase (Rubisco). When 8-day-old dark-grown barley seedlings that have low plastid translation activity were illuminated for 16 h, a dramatic increase in synthesis of large subunit of Rubisco and global activation of plastid protein synthesis occurred. While an increase in polysome-associated rbcL mRNA was observed upon illumination for 16 h, the abundance of translation initiation complexes bound to rbcL mRNA remained constant, indicating that translation elongation might be controlled during this dark-to-light transition. Toeprinting of soluble rbcL polysomes after in organello plastid translation showed that ribosomes of rbcL translation initiation complexes could read-out into elongating ribosomes in illuminated plastids whereas in dark-grown plastids, read-out of ribosomes of translation initiation complexes was inhibited. Moreover, new rounds of translation initiation could also occur in illuminated plastids, but not in dark-grown plastids. These results suggest that translation initiation complexes for rbcL are normally formed in the dark, but the transition step of translation initiation complexes entering the elongation phase of protein synthesis and/or the elongation step might be inhibited, and this inhibition seems to be released upon illumination. The release of such a translational block upon illumination may contribute to light-activated translation of the rbcL mRNA.

C4 Gene Expression in Photosynthetic and Nonphotosynthetic Leaf Regions of Amaranthus tricolor.

Throughout most of its growth and development, Amaranthus tricolor produces fully green leaves. However, near the onset of flowering, unique leaves emerge that consist of three distinct color regions: green apices, yellow middle regions, and red basal regions. The green apices are identical to fully green leaves in terms of pigment composition, photosynthetic function, and C4 gene expression. The yellow and red regions possess greatly reduced levels of chlorophyll and they lack photosynthetic activity. The absence of photosynthetic capacity in the nongreen leaf regions was associated with three distinct alterations in C4 gene expression. First, there was a reduction in the translation of C4 polypeptides, and in the yellow regions synthesis of the ribulose-1,5-bisphosphate carboxylase small subunit occurred in the absence of large subunit synthesis. Second, there was a reduction in the relative transcription rates of two plastid-encoded photosynthetic genes, rbcL and psbA. Third, there was a loss of bundle-sheath cell-specific accumulation of the rbcL and RbcS mRNAs (but not the polypeptides, which remained bundle-sheath-specific). This study indicates that alterations in photosynthetic activity or developmental processes responsible for the loss of activity can influence C4 gene expression at multiple regulatory levels.

Differential regulation of chloroplast gene expression in Chlamydomonas reinhardtii during photoacclimation: light stress transiently suppresses synthesis of the Rubisco LSU protein while enhancing synthesis of the PS II D1 protein.

Transfer of Chlamydomonas reinhardtii cells grown photoautotrophically in low light to higher light intensities has a dramatic transient effect on the differential expression of the two major chloroplast encoded photosynthetic proteins. Synthesis of the D1 protein of Photosystem II increases more than 10-fold during the first six hours in high light (HL), whereas synthesis of the large subunit (LSU) of Rubisco drops dramatically within 15 min and only gradually resumes at about 6 h. Synthesis of the chloroplast-encoded ATP synthase beta subunit, the nuclear-encoded Rubisco small subunit and the nuclear-encoded beta-tubulin is not noticeably affected. Up regulation of psbA mRNA translation accounts for a substantial fraction of the increased D1 synthesis, since accumulation of psbA mRNA increases 4.2- and 6.3-fold less than D1 synthesis at 6 and 18 h in HL. Down-regulation of LSU synthesis is not correlated with a reduction in the steady-state level of the rbcL transcript. Primer extension mapping of the 5' ends of the rbcL mRNAs reveals transcripts with start points located at -93 and -168 relative to the first translated ATG. Transfer of low light (LL)-grown cells to HL temporarily decreases the ratio of the -93 to -168 transcripts, but this ratio normalizes after 6 h in HL, coincident with the recovery in the synthesis of LSU. These several distinct effects of temporary light stress were correlated with a rapid, sustained increase in the reduction state of QA, a transient decline in photosynthetic efficiency, a less rapid drop in total chlorophyll content and a delay in cell division.

Photosynthesis, Dark Respiration and Protein Synthesis of Rice Leaves at Low Temperature. Analysis of Ribulose-1,5-Bisphosphate Carboxylase.

The effects of low temperature on photosynthesis, dark respiration and protein synthesis in developing fourth-leaf blades of rice (Oryza sativa L.) seedlings were investigated. Photosynthesis and respiration were measured with an oxygen electrode system, and protein synthesis was analyzed by SDS-polyacrylamide gel electrophoresis, fluorography and immunoprecipitation of ribulose-1, 5-bisphosphate carboxylase (RuBPC) after in vivo labeling of leaf proteins with [35S]methionine. When the rice seedlings were grown at a low temperature of 15°C for 10 days, the photosynthetic rate was severely inhibited, whereas the dark respiration rate of the stressed plants was even higher than the control plants grown at 25°C. The de novo synthesis of total proteins was also inhibited by low temperature, and the distribution of synthesized proteins drastically changed with growth and incubation temperatures, especially in the proteins related to photosynthesis. An immunological analysis of RuBPC revealed that synthesis of the RuBPC proteins was suppressed more severely than the other soluble proteins at low temperature, and that the synthesis of large subunits of RuBPC was more sensitive to the temperature treatments than that of small subunits. These results indicate that the synthesis of RuBPC proteins, especially in the part associated with chloroplasts, is specifically susceptible to low temperature, and that this may be one of the causes of retarded photosynthesis and the poor development of leaves on rice plants grown at low temperatures.

Synthesis and Assembly of Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase in Chloroplast Extracts

We have developed a new system for the in vitro synthesis of large subunits and their assembly into ribulose bisphosphate carboxylase oxygenase (Rubisco) holoenzyme in extracts of higher plant chloroplasts. This differs from previously described Rubisco assembly systems because the translation of the large subunits occurs in chloroplast extracts as opposed to isolated intact chloroplasts, and the subsequent assembly of large subunits into holoenzyme is completely dependent upon added small subunits. Amino acid incorporation in this system displayed the characteristics previously reported for chloroplast-based translation systems. Incorporation was sensitive to chloramphenicol or RNase but resistant to cycloheximide, required magnesium, and was stimulated by nucleotides. The primary product of this system was the large subunit of Rubisco. However, several lower molecular weight polypeptides were formed. These were structurally related to the Rubisco large subunit. The initiation inhibitor aurintricarboxylic acid (ATA) decreased the amount of lower molecular weight products accumulated. The accumulation of completed large subunits was only marginally reduced in the presence of ATA. The incorporation of newly synthesized large subunits into Rubisco holoenzyme occurred under conditions previously identified as optimal for the assembly of in organello-synthesized large subunits and required the addition of purified small subunits.

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO

Higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) cannot reassociate after dissociation, and its subunits do not assemble into active RuBisCO when synthesized in Escherichia coli. Newly synthesized subunits of RuBisCO are associated with a high molecular weight binding protein complex in pea chloroplasts. The immediate donor for large subunits which assemble into RuBisCO is a low molecular weight complex which may be derived from the high molecular weight binding protein complex. When the high molecular weight binding protein complex is diluted, it tends to dissociate, forming low molecular weight complexes. When the large subunit-binding protein complexes were examined after in organello protein synthesis, it was found that the low molecular weight complexes were more abundant when protein synthesis was carried out under hypotonic conditions. This increase in the assembly competent population of low molecular weight large subunit complexes can account for the increased amount of in vitro RuBisCO assembly which occurs under these conditions. The data indicate that the assembly of large subunits into RuBisCO is a function of the aggregation state of the large subunit binding protein complex during protein synthesis. This implies that the binding protein exerts its effects during or shortly after large subunit synthesis.

Catalysis by cyanobacterial ribulose-bisphosphate carboxylase large subunits in the complete absence of small subunits.

An expression plasmid incorporating the structural gene for the large subunit of a cyanobacterial ribulose-bisphosphate carboxylase, but not the gene for its complementary small subunit, directs the synthesis of large subunits in Escherichia coli. This provides a means for obtaining a preparation of large subunits completely devoid of small subunits, which is not otherwise achievable. In extracts, these large subunits were found predominantly in the form of octamers, but intersubunit interactions were weaker than in the holoenzyme, which contains eight small subunits as well as eight large subunits, and tended to be broken by procedures which separated octamers from lower oligomers and monomers. However, partial purification by anion-exchange chromatography was possible. The large subunits recognized the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate, thus enabling measurement of catalytic site concentrations, but the binding was much weaker than to the holoenzyme. E. coli-produced large subunits catalyzed carboxylation with a kcat of 1% of that of the holoenzyme and the substrate affinities were 3- to 5-fold weaker. They also assembled with heterologous small subunits isolated from spinach ribulose-P2 carboxylase with a 100-fold increase in catalytic activity under standard assay conditions. Since catalysis can proceed in their absence, the small subunits cannot be directly involved in the catalytic chemistry. Their stimulative influence upon catalysis must be exerted by conformational means.

Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts

The incorporation of newly synthesized large subunits into ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in pea chloroplast extracts occurs at the expense of intermediate forms of the large subunit which are complexed with a binding protein. Most subunits of this binding protein are found in dodecameric complexes in chloroplast extracts. Addition of small subunits to these extracts results in approximately 40 to 60% increased incorporation of newly made large subunits into RuBisCO at low or zero concentrations of ATP, but is without significant effect at high concentrations of ATP, a condition in which the dodecameric binding protein complex is dissociated into subunits. Overall, these data support the assumption that the incorporation of large subunits into RuBisCO in chloroplast extracts reflects de novo assembly rather than ;mere' exchange of subunits. The in vitro assembly of large subunits into RuBisCO is a function of the conditions under which the large subunits are synthesized in organello. When the large subunits are made in chloroplasts suspended in 188 millimolar sorbitol, they are approximately 2- to 3-fold better able to assemble into RuBisCO when subsequently incubated in vitro than when they are synthesized in chloroplasts suspended in 375 millimolar sorbitol. This observation indicates that mere synthesis of large subunits is not sufficient to confer maximal assembly competence on large subunits.

Co-expression of both the maize large and wheat small subunit genes of ribulose-bisphosphate carboxylase in Escherichia coli.

A cDNA clone for the precursor form of the small subunit of wheat ribulose-bisphosphate carboxylase has been modified to allow the expression in Escherichia coli of a mature form of small subunit that lacks the transit peptide. Synthesis of the protein is controlled by a lac promoter, and translation is initiated from a lacZ ribosome binding site, giving rise to a small subunit with several beta-galactosidase amino acids fused to its N-terminus. A plasmid has been constructed that enables both wheat small subunits and maize large subunits to be synthesized in the bacterial cell, but using different promoters to allow independent expression of the rbcS and rbcL genes. When the small subunit is synthesized in the absence of the large subunit, it is found in the soluble fraction but the polypeptide is unstable and has a half-life of less than 15 min. Its size on sucrose gradients indicates a monomeric or dimeric form. When large subunit synthesis is induced in cells containing the small subunit, both subunits are found predominantly in the insoluble fraction and are fully stable for more than 120 min, suggesting that aggregation of the subunits may occur. The two subunits do not assemble together to form an active holoenzyme in vivo, even when nascent large subunits ware synthesized in a pool of mature small subunits. This indicates that other factors may be required to mediate the assembly of the higher plant enzyme.

Effects of specific inhibitors on the coordination of the concentrations of ribulose-bisphosphate-carboxylase subunits and their corresponding mRNAs in the alga Chlorogonium

Investigations were carried out on the effects of inhibitors of transcription and translation on the concentrations of the subunits of the plastid enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) and their corresponding mRNAs in the unicellular green alga Chlorogonium elongatum Dangeard. The light-induced increase of nuclear-coded small-subunit mRNA was strongly inhibited by α-amanitin while the increase of plastid-coded large-subunit mRNA was only weakly affected: Consequently, the mRNAs of the two subunits were present in very different proportions. Nevertheless, the light-induced increase of both subunits was strongly reduced by α-amanitin to the same degree, and hence the ratio of their concentrations was not affected compared with the untreated control cells. The effect of cycloheximide on the subunit mRNAs was similar to but weaker than that of α-amanitin. Again the increases in the subunit levels were strongly inhibited to the same degree. By contrast, rifampicin and chloramphenicol inhibited the light-induced increase of large-subunit mRNA more strongly than that of small-subunit mRNA, but the differences were less distinct than those caused by α-amanitin and cycloheximide. Again, the increase in both subunits was inhibited almost to the same extent. These results - especially those of the α-amanitin experiments - clearly show that the fine coordination of the RuBPCase subunits occurs posttranscriptionally at the level of translation and-or degradation. This conclusion was confirmed by pulse-chase experiments. Inhibition of the synthesis of the large subunits by chloramphenicol resulted - as also found by other authors-in a degradation of excess small subunits in the plastid. On the other hand, inhibition of the concentration of small subunits caused a proportionate reduction in the synthesis of large subunits, but no rapid degradation of large subunits could be detected. Therefore, the fine coordination of both subunits of RuBPCase is achieved by the degradation of an excess of small subunits, while the level of large subunits is adapted to the small subunit concentration, probably by adjustment of translation of the large-subunit mRNA. Furthermore, our experiments with α-amanitin and cycloheximide allow us to conclude that in the blue-light induction of large-subunit mRNA in the plastid the nucleocytoplasmic compartment is not directly involved.

Control of gene expression during higher plant chloroplast biogenesis. Protein synthesis and transcript levels of psbA, psaA-psaB, and rbcL in dark-grown and illuminated barley seedlings.

Etioplasts of 4.5-day-old dark-grown barley synthesize and accumulate most of the membrane and nearly all the soluble polypeptides of mature chloroplasts of light-grown seedlings. Etioplasts do not synthesize a limited set of chloroplast-encoded polypeptides which are major constituents of chloroplast thylakoid membranes: two chlorophyll apoproteins of photosystem I (68 and 65 kDa), two chlorophyll apoproteins of photosystem II (47 and 43 kDa), and a 32-kDa polypeptide which has now been identified as the psbA gene product. Throughout development in the dark, etioplasts were unable to synthesize the chlorophyll apoproteins of photosystem I and II or the psbA gene product despite the presence of significant transcript levels for psbA and psaA-psaB (encode for photosystem I chlorophyll apoproteins). Light was not required for the synthesis of ribulose bisphosphate carboxylase large subunit with the highest rate of large subunit synthesis occurring in young dark-grown seedlings. Illumination of 4.5-day-old dark-grown barley rapidly induced the synthesis of the chlorophyll apoproteins and the psbA gene product at a time when transcript levels for psbA and psaA-psaB did not increase appreciably. Therefore, during the early stages of light-induced development the synthesis of the chlorophyll apoproteins of photosystem I and psbA gene product is regulated at the translational level. With continued chloroplast development in the light, the synthesis of the chlorophyll apoproteins of photosystem I and II decline rapidly as does the synthesis of the large subunit of ribulose bisphosphate carboxylase. The decline in polypeptide synthesis correlated with a decline in rbcL and psaA-psaB transcript levels and a light-dependent decline in plastid rRNA content. In contrast, synthesis of the psbA gene product was maintained throughout light-induced chloroplast development which correlated with the maintenance of psbA transcript levels. However, light is not strictly required for psbA transcript accumulation since psbA transcript levels increased slightly with continued plastid development in dark-grown seedlings.

Synthesis of large subunit of ribulosebisphosphate carboxylase by thylakoid-bound polyribosomes from spinach chloroplasts

Intact chloroplasts were isolated from developing first leaves of spinach. The chloroplasts were broken and separated into an extensively washed membrane (thylakoid) fraction and a soluble (stroma) fraction. The membrane fraction contained polyribosomes with properties similar to those of thylakoid-bound polyribosomes of other organisms. The distribution of mRNA for large-subunit ribulosebisphosphate carboxylase (LS) was determined by translating RNA from chloroplasts, thylakoids, and stroma in a wheat germ cell-free translation system. LS translation product was identified by immunoprecipitation with antibody to LS from spinach, electrophoresis of the immunoprecipitated product, and fluorography. At least 44% of translatable chloroplast LS-mRNA was in the washed thylakoid fraction. Thylakoid-bound LS-mRNA was in polyribosomes since LS was produced by thylakoids in an Escherichia coli cell-free translation system under conditions where initiation did not take place. Our results demonstrate that membrane-bound polyribosomes can synthesize the stroma-localized polypeptide LS, and suggest that the thylakoids may be an important site of its synthesis.

Amplification of maize ribulose bisphosphate carboxylase large subunit synthesis in E. coli by transcriptional fusion with the lambda N operon.

The maize chloroplast gene coding for the large subunit of ribulose bisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) has been placed under the transcriptional control of the bacteriophage lambda promoter PL, by fusion with the lambda N operon located on a multicopy plasmid. Transcription from PL was repressed at 32 degrees C by the presence in the E. coli chromosome of a cIts gene that specifies a temperature-sensitive repressor. After inactivation of the repressor at 45 degrees C unmoderated transcription of the chloroplast gene occurred from the PL promoter. Translation was probably initiated from a chloroplast Shine-Dalgarno sequence located five nucleotides from the N-terminal methionine initiation codon to yield a polypeptide the same size as that synthesised in maize. This direct translation results in a level of expression of the chloroplast gene corresponding to approximately 2% of the total E. coli cell protein as ribulose bisphosphate carboxylase large subunits. Transcriptional fusions with the lambda N operon should provide a generally applicable, simple method for the amplification and regulation of chloroplast gene expression in E. coli.

Expression in E. coli of maize and wheat chloroplast genes for large subunit of ribulose bisphosphate carboxylase.

Synthesis of the large subunit polypeptide of ribulose bisphosphate carboxylase can be detected in Escherichia coli cells containing the chloroplast genes from maize and wheat. The chloroplast DNA sequences contain a 'promoter' that, in Escherichia coli, initiates transcription and translation of the genes. This synthesis of large subunits has been substantially increased using the powerful lambda promoter, PL, to drive transcription of the maize gene.

Protein synthesis in chloroplasts IX. Assembly of newly-synthesized large subunits into ribulose bishopshate carboxylase in isolated intact pea chloroplasts

Isolated pea ( Pisum sativum) chloroplasts incorporate [ 35S]methionine into the large subunit of the chloroplast enzyme ribulose bisphosphate carboxylase. When chloroplasts are incubated in a medium containing KCl as osmoticum, newly-synthesised large subunits are not incorporated into the holoenzyme but can be separated from pre-existing enzyme by gel electrophoresis under non-denaturing conditions. Furthermore, newly-synthesised large subunits are not precipitated by antibodies which precipitate pre-existing holoenzyme and large subunit prepared from holoenzyme. When chloroplasts are incubated in a medium containing sorbitol as osmoticum, some of the newly-synthesised large subunits comigrate with holoenzyme on both 3% and 5% polyacrylamide non-denaturing gels. Such comigrating large subunits are precipitated by antibodies raised against the holoenzyme. These results indicate assembly of large subunits into ribulose bisphosphate carboxylase in the sorbitol medium. Time course experiments indicate that there is a time-lag of several minutes between the onset of synthesis of large subunits and the onset of assembly. Newly-synthesised large subunits which do not comigrate with holoenzyme on both 3% and 5% polyacrylamide non-denaturing gels are associated with a protein of subunit molecular weight 60 000. This protein may be specifically combined with newly-synthesised large subunits, and the resulting aggregate be involved in the assembly of complete molecules of ribulose bisphosphate carboxylase.

Identification and Properties of the Messenger RNA Activity in Chlamydomonas reinhardi Coding for the Large Subunit of d-ribulose-1,5-bisphosphate Carboxylase

Properties of the mRNA coding for the large subunit of ribulose-1,5-bisphosphate carboxylase from Chlamydomonas reinhardi were determined. Large subunit synthesis, directed by RNA from partially purified whole cell extracts, was detected by specific immunoprecipitation of polypeptide products synthesized in a heterologous translation system derived from Escherichia coli. Large subunit synthesis showed sharp RNA concentration dependence in an E. coli translation system, and at optimal RNA concentrations, immunoprecipitable large subunit synthesis accounted for 2% of the total incorporation. Large subunit messenger activity sedimented at 12 to 14S on nondenaturing sucrose gradients and did not bind to oligo(dT)-cellulose suggesting the mRNA is not polyadenylated. The immunoprecipitable products translated in vitro are not complete polypeptide chains, but are smaller peptides identifiable as large subunit fragments by tryptic fingerprint analysis. No immunoprecipitable product was obtained when similar RNA fractions were tested in a wheat germ translation system.

Identification and Precipitation of the Polyribosomes in Chlamydomonas reinhardi Involved in the Synthesis of the Large Subunit of d-ribulose-1,5-bisphosphate Carboxylase

The size classes of polyribosomes involved in the synthesis of ribulose-1,5-bisphosphate carboxylase large subunit were determined by binding radioiodinated specific antibodies to polyribosomal preparations from Chlamydomonas reinhardi. Antibodies specific to the denatured large subunit and to the native enzyme bound primarily to small polyribosomes (N = two to five ribosomes). The binding of antibodies to small polyribosomes was unexpected since the large subunit is a large polypeptide (molecular weight 55,000) coded for by a corresponding large mRNA (12-14S). Control experiments showed that this unexpected pattern of antibody binding was not a result of messenger RNA degradation, "run-off" of ribosomes from polyribosomes, or adventitious binding of the completed enzyme to a selected class of polyribosomes. In addition, polyribosomes bearing nascent large subunit chains have been immunoprecipitated from small polyribosome fractions. A large RNA species that can direct the synthesis of large subunit in vitro was extracted from small polyribosomes.