Structure of the chloroplast ribosome: novel domains for translation regulation

Gene expression in chloroplasts is controlled primarily through the regulation of translation. This regulation allows coordinate expression between the plastid and nuclear genomes, and is responsive to environmental conditions. Despite common ancestry with bacterial translation, chloroplast translation is more complex and involves positive regulatory mRNA elements and a host of requisite protein translation factors that do not have counterparts in bacteria. Previous proteomic analyses of the chloroplast ribosome identified a significant number of chloroplast-unique ribosomal proteins that expand upon a basic bacterial 70S-like composition. In this study, cryo-electron microscopy and single-particle reconstruction were used to calculate the structure of the chloroplast ribosome to a resolution of 15.5 Å. Chloroplast-unique proteins are visualized as novel structural additions to a basic bacterial ribosome core. These structures are located at optimal positions on the chloroplast ribosome for interaction with mRNAs during translation initiation. Visualization of these chloroplast-unique structures on the ribosome, combined with mRNA cross-linking, allows us to propose a model for translation initiation in chloroplasts in which chloroplast-unique ribosomal proteins interact with plastid-specific translation factors and RNA elements to facilitate regulated translation of chloroplast mRNAs.     

植物叶绿体基因组基因表达调控的研究

叶绿体基因的表达在许多方面与原核基因表达相似,所以最早的理论认为叶绿体基因的表达与原核相似,是在转录起始水平上的调控,进一步的研究认为叶绿体基因表达调控是在不同水平上进行的如：转录水平的调节、转录后调节与修饰、翻译和翻译后修饰等。     

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.     

Light-activated translation of chloroplast mRNAs

The integrated regulation of mRNA stability, processing and translation facilitates the expression of several chloroplast genes, particularly in response to changes in illumination. Nuclear and chloroplast-encoded factors that mediate the expression of specific chloroplast messages have been characterized from green algae and plants. Recent studies suggest that the chloroplast might have recruited eukaryotic proteins, which are usually found in the cytoplasm or the endoplasmic reticulum, to couple the level of photosynthetic activity to gene expression via translational activation. Consequently, elements required for translational initiation of chloroplast messages differ from their prokaryotic ancestors. These results suggest that chloroplast translational regulation is a hybrid between prokaryotic and eukaryotic systems.     

Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5' untranslated region

Translational regulation is a key modulator of gene expression in chloroplasts of higher plants and algae. Genetic analysis has shown that translation of chloroplast mRNAs requires nuclear-encoded factors that interact with chloroplastic mRNAs in a message-specific manner. Using site-specific mutations of the chloroplastic psbA mRNA, we show that RNA elements contained within the 5' untranslated region of the mRNA are required for translation. One of these elements is a Shine- Dalgarno consensus sequence, which is necessary for ribosome association and psbA translation. A second element required for high levels of psbA translation is located adjacent to and upstream of the Shine-Dalgarno sequence, and maps to the location on the RNA previously identified as the site of message-specific protein binding. This second element appears to act as a translational attenuator that must be overcome to activate translation. Mutations that affect the secondary structure of these RNA elements greatly reduce the level of psbA translation, suggesting that secondary structure of these RNA elements plays a role in psbA translation. These data suggest a mechanism for translational activation of the chloroplast psbA mRNA in which an RNA element containing the ribosome-binding site is bound by message- specific RNA binding proteins allowing for increased ribosome association and translation initiation. These elements may be involved in the light-regulated translation of the psbA mRNA.     

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.     

Translation in plants-rules and exceptions

Translation processes in plants are very similar to those in other eukaryotic organisms and can in general be explained with the scanning model. Particularly among plant viruses, unconventional mRNAs are frequent, which use modulated translation processes for their expression: leaky scanning, translational stop codon readthrough or frameshifting, and transactivation by virus-encoded proteins are used to translate polycistronic mRNAs; leader and trailer sequences confer (cap-independent) efficient ribosome binding, usually in an end-dependent mechanism, but true internal ribosome entry may occur as well; in a ribosome shunt, sequences within an RNA can be bypassed by scanning ribosomes. Translation in plant cells is regulated under conditions of stress and during development, but the underlying molecular mechanisms have not yet been determined. Only a small number of plant mRNAs, whose structure suggests that they might require some unusual translation mechanisms, have been described.     

Translational control of growth factor and proto-oncogene expression

Control of translation is now understood to be one of the major regulatory events in eukaryotic gene expression. Moreover there is evidence which suggests that aberrant expression of growth-related genes by translational mechanisms makes a significant contribution to cell transformation. However, the mechanisms which regulate translation of specific growth-related mRNAs have yet to be fully elucidated. The majority of these mRNAs have long 5' untranslated regions (UTRs) and three features which are important in translational control have been identified, namely (i) structured regions which inhibit the scanning mechanisms of translation, (ii) regulatory upstream open reading frames and (iii) internal ribosome entry segments which are capable of initiating cap-independent translation. In this review the translational regulation of specific mRNAs encoding growth factors and proto-oncogenes by these three mechanisms will be discussed, together with examples of altered translational regulation in neoplasia.     

Cis-acting elements and trans-acting factors for accurate translation of chloroplast psbA mRNAs: development of an in vitro translation system from tobacco chloroplasts.

Translational regulation is an important step of gene expression in chloroplasts. To analyze biochemical mechanisms of translational regulation unique to higher plant chloroplasts, an in vitro translation system has been developed from tobacco chloroplasts. Conditions for chloroplast extraction and the in vitro translation reaction have been optimized with a tobacco psbA-lacZ fusion mRNA. The in vitro system supports accurate translation of a variety of chloroplasts mRNAs. Using a series of mutant psbA mRNAs, we showed that three elements within the 5'-untranslated region of the mRNA are required for translation. Two of them are complementary to the 3'-terminus of chloroplast 16S rRNA (termed RBS1 and RBS2) and the other is an AU-rich sequence (UAAAUAAA) located between RBS1 and RBS2 and is termed the AU box. mRNA competition experiments using the in vitro translation reaction and gel mobility shift assays revealed the existence of a trans-acting factor(s) for translation and its possible interaction with the AU box. We propose a model for the initiation of psbA translation whereby RBS1 and RBS2 bind cooperatively to the 3'-end of 16S rRNA resulting in looping out of the AU box, which facilitates the interaction of a trans-acting factor(s).