Chloroplast elongation factor Tu: Evidence that it is the product of a chloroplast gene in Euglena

The chloroplast protein synthesizing factor responsible for the binding of aminoacyl-tRNA to ribosomes (EF-Tu_chl) has been identified in extracts of Euglena gracilis. This factor is present in low levels when Euglena is grown in the dark and can be induced more than 10-fold when the organism is exposed to light. The induction of the chloroplast EF-Tu by light is inhibited by streptomycin, an inhibitor of protein synthesis on chloroplast ribosomes, indicating that protein synthesis within the chloroplast itself is required for the induction of this factor. The induction of the chloroplast EF-Tu by light is also inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. The effect of cycloheximide probably results from the inhibition of chloroplast ribosome synthesis which requires the synthesis of many proteins by the cytoplasmic translational system. Chloroplast EF-Tu cannot be induced by light in an aplastidic mutant (strain W₃BUL) of Euglena which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-Tu resides in the chloroplast genome and that this protein is synthesized within the organelle itself.     

Processing of the psbA 5′ Untranslated Region in Chlamydomonas reinhardtii Depends upon Factors Mediating Ribosome Association

The 5′ untranslated region of the chloroplast psbA mRNA, encoding the D1 protein, is processed in Chlamydomonas reinhardtii. Processing occurs just upstream of a consensus Shine-Dalgarno sequence and results in the removal of 54 nucleotides from the 5′ terminus, including a stem-loop element identified previously as an important structure for D1 expression. Examination of this processing event in C. reinhardtii strains containing mutations within the chloroplast or nuclear genomes that block psbA translation reveals a correlation between processing and ribosome association. Mutations within the 5′ untranslated region of the psbA mRNA that disrupt the Shine-Dalgarno sequence, acting as a ribosome binding site, preclude translation and prevent mRNA processing. Similarly, nuclear mutations that specifically affect synthesis of the D1 protein specifically affect processing of the psbA mRNA. In vitro, loss of the stem-loop element does not prohibit the binding of a message-specific protein complex required for translational activation of psbA upon illumination. These results are consistent with a hierarchical maturation pathway for chloroplast messages, mediated by nuclear-encoded factors, that integrates mRNA processing, message stability, ribosome association, and translation.     

Construction of a chloroplast DNA library from rye (Secale cereale), a cereal plant of temperature-inducible chloroplast ribosome deficiency.

The chloroplast genomes of flowering plants are circular DNA molecules, 120 to 160 kilobase pairs long, encoding the rRNA, all tRNAs, and 21 r-proteins of the chloroplast translational apparatus as well as key protein components of the photosynthetic and carbon reduction cycle reactions. In this paper we describe some characteristics of the rye chloroplast (plastid) genome and the construction and characterization of a clone library of 93% of its DNA in a plasmid and a cosmid vector. The size of rye chloroplast DNA is estimated at 135 kbp, similar to that for wheat and rice but slightly smaller than the estimate for maize (139 kbp). Chloroplast ribosome deficiency is induced in rye seedlings by germination and growth at 32 degrees-34 degrees C; therefore these clones would be useful for analyzing the regulation of chloroplast ribosome synthesis in higher plants, a process that requires coordinate expression of genes located in the nucleus and the chloroplast.     

Synthesis of two proteins in chloroplasts and mRNA distribution between thylakoids and stroma during the cell cycle of Chlamydomonas reinhardii

Chloroplasts contain thylakoid-bound and free ribosomes and polysomes. Whether binding of polysomes plays an immediate role in the regulation of chloroplast protein synthesis is not yet clear. In the present work, variations of protein synthesis and of mRNA content were measured not in greening, but in fully differentiated chloroplasts during the cell cycle of synchronized cultures of Chlamydomonas reinhardii. At different times of the vegetative cell cycle, the RNA was extracted from free and thylakoid-bound chloroplast polysomes and the partition of mRNAs between stroma and thylakoids was measured for two proteins, i.e. the 32-kDa herbicide-binding membrane protein and the soluble large subunit of the ribulose-1,5-bisphosphate carboxylase. At the same time the rates of synthesis of these two proteins were also determined. At 2 h after the onset of light, the content of both mRNAs in chloroplasts had doubled and 75-90% of each of these mRNAs were found to be bound to the thylakoids. The rate of protein synthesis, however, increased 10-fold, but reached its maximum only after about 6 h in the light. The differences in the time courses, in the stimulation of the rate of protein synthesis, and in the mRNA-binding to thylakoids point to a translational regulation of protein synthesis. Furthermore, since a very high proportion of polysomes were bound to thylakoids, containing mRNA for both a membrane and a soluble protein, this light-induced binding of polysomes to thylakoids seems to be an essential, but not the only, prerequisite for protein synthesis in chloroplasts.     

Translation of heterologous mRNAs by chloroplast stroma components

In vitro translation of exogenous mRNAs has been difficult to achieve using chloroplast components. An in vitro protein synthesis system is described, based on a pea ( Pisum sativum L, cv. Spring) chloroplast stroma 30000 g supernatant, which was capable of translating polyuridylie acid and MS2 phage RNA into corresponding proteins. The kinetics of label incorporation and fluorograms of unique translation products are presented. The unusual steps which may have contributed to successful translation include the following: the removal of exogenous unlabeled amino acids from the incubation mixture, ultrafiltration of the stromal supernatant and the removal of thylakoid-bound polyribosomes. The system could be further developed to study translational control of gene expression in the chloroplasts.     

Molecular Factors Affecting the Accumulation of Recombinant Proteins in the <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> Chloroplast

In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis, and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase, and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced when compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions this could be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well when compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.     

Identification of the chloroplast adenosine-to-inosine tRNA editing enzyme

Plastids (chloroplasts) of higher plants exhibit two types of conversional RNA editing: cytidine-to-uridine editing in mRNAs and adenosine-to-inosine editing in at least one plastid genome-encoded tRNA, the tRNA-Arg(ACG). The enzymes catalyzing RNA editing reactions in plastids are unknown. Here we report the identification of the A-to-I tRNA editing enzyme from chloroplasts of the model plant Arabidopsis thaliana. The protein (AtTadA) has an unusual structure in that it harbors a large N-terminal domain of >1000 amino acids, which is not required for catalytic activity. The C-terminal region of the protein displays sequence similarity to tadA, the tRNA adenosine deaminase from Escherichia coli. We show that AtTadA is imported into chloroplasts in vivo and demonstrate that the in vitro translated protein triggers A-to-I editing in the anticodon of the plastid tRNA-Arg(ACG). Suppression of AtTadA gene expression in transgenic Arabidopsis plants by RNAi results in reduced A-to-I editing in the chloroplast tRNA-Arg(ACG). The RNAi lines display a mild growth phenotype, presumably due to reduced chloroplast translational efficiency upon limited availability of edited tRNA-Arg(ACG).     

Repressible chloroplast gene expression in Chlamydomonas: A new tool for the study of the photosynthetic apparatus

A repressible/inducible chloroplast gene expression system has been used to conditionally inhibit chloroplast protein synthesis in the unicellular alga Chlamydomonas reinhardtii. This system allows one to follow the fate of photosystem II and photosystem I and their antennae upon cessation of chloroplast translation. The main results are that the levels of the PSI core proteins decrease at a slower rate than those of PSII. Amongst the light-harvesting complexes, the decrease of CP26 proceeds at the same rate as for the PSII core proteins whereas it is significantly slower for CP29, and for the antenna complexes of PSI this rate is comprised between that of CP26 and CP29. In marked contrast, the components of trimeric LHCII, the major PSII antenna, persist for several days upon inhibition of chloroplast translation. This system offers new possibilities for investigating the biosynthesis and turnover of individual photosynthetic complexes in the thylakoid membranes. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

A nuclear gene in maize required for the translation of the chloroplast atpB/E mRNA.

To elucidate mechanisms that regulate chloroplast translation in land plants, we sought nuclear mutations in maize that disrupt the translation of subsets of chloroplast mRNAs. Evidence is presented for a nuclear gene whose function is required for the translation of the chloroplast atpB/E mRNA. A mutation in atp1 results in a failure to accumulate the chloroplast ATP synthase complex due to reduced synthesis of the AtpB subunit. This decrease in AtpB synthesis does not result from a change in atpB mRNA structure or abundance. Instead, the atpB mRNA is associated with abnormally few ribosomes in atp1-1 mutants, indicating that atp1 function is required during translation initiation or early in elongation. Previously, only one nuclear gene that is required for the translation of specific chloroplast mRNAs had been identified in a land plant. Thus, atp1 will be a useful tool for dissecting mechanisms of translational control in chloroplasts.