Question

1. Positive regulation of translation is used to control protein synthesis in chloroplasts after light stimulation. Please describe the translational activation of chloroplast mRNA. What is the translational activator responding to light intensity? And how does light activate this translational activator?

Answer 1

Chloroplasts are the characteristic organelle of plant cells. They host numerous essential metabolic pathways including photosynthesis, which makes chloroplasts the primary source of chemical energy on earth. All chloroplasts are likely derived from a single ancient photosynthetic cyanobacterium that was engulfed by a mitochondrial eukaryotic cell more than a billion years ago. During subsequent host-endosymbiont coevolution, the genome of the endosymbiont shrank significantly

Chloroplast gene expression is primarily regulated at the post-transcriptional level, where a variety of complex mechanism has evolved to govern the interaction between the chloroplast and nuclear genomes. These rely on different nuclear-encoded proteins that act on chloroplast mRNAs, including RNA polymerase subunits, RNA stability factors, and translational activators. Such control mechanisms must be viewed in the light of the evolutionary heritage of the chloroplast, as an independent unicellular organism. Although some features of prokaryotic gene expression still occur in chloroplasts, their multiple genetic mechanisms for synthesizing RNA and protein are new and appear to have arisen to provide the flexibility of responding to the developmental or environmental state of the plant.

It has been shown that in both chloroplasts and mitochondria, following components of translation apparatus are found : (i) ribosomes specific to organelle, (ii) tRNAs specific to organelle, (iii) other factors for translation (little is known about these factors). Some of these components may be synthesized outside these organelles and then transported into organelles.

It has been shown that the translation apparatus in chloroplasts and mitochondria differ from that in cytoplasm in eukaryotes in the following respects, (i) Ribosomes in these organelles are smaller in size (the 70S) than those in cytoplasm (80S), (ii) The tRNAs are specific and differ, the number of tRNAs in mitochondria being 22 as against 55 in cytoplasm, (iii) Initiation of translation takes place by formyl-methionyl tRNA both in chloroplasts and mitochondria, although no formylation takes place in cytoplasm, (iv) Translation in chloroplasts and mitochondria can be inhibited by chloramphenicol, as in bacteria since the 70S ribosomes are sensitive to chloramphenicol and not to cycloheximide; on the other hand, the translation in cytoplasm is inhibited by cycloheximide, since 80S ribosomes are sensitive to cycloheximide. There are other antibiotics like spectinomycin, lincomycin, and erythromycin which also inhibit translation in bacteria and in all organelles of eukaryotes. These antibiotics help to stop the translation preferentially either (i) in the cytoplasm (by cycloheximide) thus permitting protein Synthesis only in chloroplasts or mitochondria or (ii) in organelles (by chloramphenicol) thus permitting protein synthesis only in the cytoplasm

Studies on the synthesis of 'chlorophyll-binding proteins', which play a major role in the primary reactions of photosynthesis have also provided new information about chloroplast translation and its regulation. In many plant species, in the absence of light the mRNA of chlorophyll-binding proteins, including those encoded by chloroplast genome, remain associated with thylakoid bound polysomes but are not translated. Transfer of seedlings to light induces synthesis of proteins, although no increase in transcription has been observed. This suggests that post-transcriptional processes play a key role in light-induced chloroplast gene expression.

It has also been shown that the polycistronic mRNA encoded by the psbB operon in maize, need not be cleaved into tri-, di- or monocistronic forms for successful translation (psbB operon = psbB + psbH + petB + petD). This suggests that plastid ribosomes can bind directly to internal initiation regions for initiation of translation as in prokaryotes. However, RNA processing does take place in chloroplasts and mitochondria, and the transcripts may differ in their translatability.

There are also nuclear genes, whose products are essential for the translation of specific genes. Several of these nuclear-encoded factors interact with the 5' untranslated region of chloroplast massages, but little is known about the identity and precise function of these factors. These products may be translational activators of the type described in Regulation of Gene Expression 1. Operon Circuits in Bacteria and other Prokaryotes and Regulation of Gene Expression 3. A Variety of Mechanisms in Eukaryotes for prokaryotic and eukaryotic genes respectively.

Protein synthesis in isolated chloroplasts of pea was studied by R.J. Ellis of U.K. The proteins which could be synthesized in isolated chloroplasts included (i) large subunit of Fraction I protein, (ii) five unidentified proteins of the internal lamellar system, and (iii) two or three unidentified polypeptides of the envelope. These are only a few of a large number of proteins found in chloroplasts. It has also been demonstrated that while the large subunit of Fraction I protein is synthesized under the influence of chloroplast DNA the small subunit is synthesized in the cytoplasm under the influence of nuclear DNA. The small subunit is then transported to the chloroplast where it associates with large subunit to give rise to Fraction I protein

Genetic analysis has revealed a set of nuclear-encoded factors that regulate chloroplast mRNA translation by interacting with the 5' leaders of chloroplastic mRNAs. We have identified and isolated proteins that bind specifically to the 5' leader of the chloroplastic psbA mRNA, encoding the photosystem II reaction center protein D1. Binding of these proteins protects a 36 base RNA fragment containing a stem-loop located upstream of the ribosome binding site. Binding of these proteins to the psbA mRNA correlates with the level of translation of psbA mRNA observed in light- and dark-grown wild type cells and in a mutant that lacks D1 synthesis in the dark. The accumulation of at least one of these psbA mRNA-binding proteins is dependent upon chloroplast development, while its mRNA-binding activity appears to be light modulated in developed chloroplasts. These nuclear-encoded proteins are prime candidates for regulators of chloroplast protein synthesis and may play an important role in coordinating nuclear-chloroplast gene expression as well as provide a mechanism for regulating chloroplast gene expression during development in higher plants.