Chloroplast translation regulation

Chloroplast gene expression is primarily controlled during the translation of plastid mRNAs. Translation is regulated in response to a variety of biotic and abiotic factors, and requires a coordinate expression with the nuclear genome. The translational apparatus of chloroplasts is related to that of bacteria, but has adopted novel mechanisms in order to execute the specific roles that this organelle performs within a eukaryotic cell. Accordingly, plastid ribosomes contain a number of chloroplast-unique proteins and domains that may function in translational regulation. Chloroplast translation regulation involves cis-acting RNA elements (located in the mRNA 5′ UTR) as well as a set of corresponding trans-acting protein factors. While regulation of chloroplast translation is primarily controlled at the initiation steps through these RNA-protein interactions, elongation steps are also targets for modulating chloroplast gene expression. Translation of chloroplast mRNAs is regulated in response to light, and the molecular mechanisms underlying this response involve changes in the redox state of key elements related to the photosynthetic electron chain, fluctuations of the ADP/ATP ratio and the generation of a proton gradient. Photosynthetic complexes also experience assembly-related autoinhibition of translation to coordinate the expression of different subunits of the same complex. Finally, the localization of all these molecular events among the different chloroplast subcompartments appear to be a crucial component of the regulatory mechanisms of chloroplast gene expression.     

植物叶绿体分裂的分子机制

叶绿体是植物细胞内一种重要的细胞器．它不仅是光合作用的场所,还是其它多种中间代谢的场所．叶绿体起源于蓝细菌,与其原核祖先类似,通过二分裂方式进行增殖．最近的研究表明,叶绿体的分裂装置包含原核起源和真核起源的蛋白质,它们在叶绿体的内膜内侧和外膜外侧协同作用以完成叶绿体的分裂．在过去十几年里,包括丝状温度敏感蛋白Z（FtsZ）、Min系统蛋白、质体分裂蛋白（PDV）和ARC蛋白等在内的多个叶绿体分裂相关组分被分离鉴定．本文简要介绍了叶绿体分裂装置各成员的发现、叶绿体被膜的收缩和叶绿体分裂位点的选择机制．另外,植物发育过程中叶绿体分裂可能受到细胞的控制,但目前对细胞如何调控叶绿体分裂知之甚少．本文对该领域的最新研究进展也进行了综述．     

Euglena gracilis chloroplast elongation factor Tu. Purification and initial characterization

The chloroplast protein synthesis elongation factor Tu (EF-Tuchl) has been purified to near homogeneity from Euglena gracilis. Chromatography of the postribosomal supernatant of light-induced Euglena on DEAE-Sephadex reveals two forms of EF-Tuchl. Further purification has shown that one species consists of a complex between EF-Tuchl and a factor that stimulates its activity. The other species consists of free EF-TUchl. The factor has been purified from both chromatographic forms by taking advantage of the molecular weight shift that occurs upon disruption of the complex between EF-Tuchl and the stimulatory factor. EF-Tuchl consists of a single polypeptide chain with a molecular weight of about 50,000. EF-Tuchl is as active on Escherichia coli ribosomes as it is on its homologous ribosomes but displays no detectable activity on eukaryotic cytoplasmic ribosomes. It is stimulated in polymerization by E. coli EF-Ts and will form a complex with the prokaryotic factor that can be isolated by gel filtration chromatography. Like E. coli EF-Tu, it is sensitive to modification by N-ethylmaleimide and is inhibited by the antibiotic kirromycin. Thus, the chloroplast factor has many features that reflect the close relationship between prokaryotic and chloroplast translational systems.     

毕赤酵母表达动物传染病病原基因的研究进展

毕赤酵母表达系统是一种新的真核基因表达系统,由于其有许多突出的优点,如它既有原核生物的特点,又有真核生物的特性,可以对目的蛋白质进行糖基化、二硫键形成等翻译后修饰等特点,越来越受到分子生物学界的重视。已有多种重组蛋白在该系统成功表达,其中许多已被广泛应用于临床诊断治疗或科研工作中。总结了毕赤酵母的表达系统的组成及其在表达动物传染病病原方面的主要研究进展。     

Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba)

Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced. Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic sequences and relatively similar to the GAPDHs of thermophilic bacteria. These results are compatible with the hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin. They are in puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to the eukaryotic enzymes [Eur. J. Biochem. 150, 61-66 (1985)].     

Stability and degradation of mRNA

Differential mRNA stability plays an important role in the regulation of gene expression. Several recent advances have helped to define the general pathways by which mRNA is degraded in prokaryotic cells, although many details remain to be elucidated. Much less is known about the pathways of degradation in eukaryotic cells, but recent studies on specific systems have highlighted both differences from and similarities to prokaryotic pathways.     

Conservation of structure and cold-regulation of RNA-binding proteins in cyanobacteria: probable convergent evolution with eukaryotic glycine-rich RNA-binding proteins

The rbp gene family of the cyanobacterium Anabaena variabilis strain M3 consists of eight members that encode small RNA-binding proteins containing a single RNA recognition motif (RRM). Similar genes are found in the genomes of Synechocystis sp. PCC6803, Helicobacter pylori and Treponema pallidum, but are absent from the other completely sequenced prokaryotic genomes. The expression of the rbp genes of Anabaena is induced by low temperature, with the exception of the rbpD gene. We found four stretches of conserved sequences in the 5'-untranslated region of the cyanobacterial rbp genes that are known to be induced by low temperature. The cold-regulated Rbp proteins contain a short C-terminal glycine-rich domain. In this respect, these proteins are similar to plant and mammalian glycine-rich RNA-binding proteins (GRPs), which also contain a single RRM domain with a C-terminal glycine-rich domain and are highly expressed at low temperature. Detailed phylogenetic analysis showed, however, that the cyanobacterial Rbp proteins and the eukaryotic GRPs do not belong to a single lineage, but that the glycine-rich domains are likely to have been added independently. The cold-regulation of both types of proteins is also likely to have evolved independently. Furthermore, the chloroplast RNA-binding proteins are not likely to have originated from the Rbp proteins of endosymbiont cyanobacterium, but are supposed to have diverged from the GRPs. These results suggest that the cyanobacterial Rbp proteins and the eukaryotic GRPs are similar in both structure and regulation, but that this apparent similarity has resulted from convergent evolution.     

The Shine-Dalgarno-like sequence is a negative regulatory element for translation of tobacco chloroplast rps2 mRNA: an additional mechanism for translational control in chloroplasts

Most prokaryotic mRNAs contain within the 5' untranslated region (UTR), a Shine-Dalgarno (SD) sequence, which is complementary to the 3' end of 16S rRNA and serves as a major determinant for correct translational initiation. The tobacco chloroplast rps2 mRNA possesses an SD-like sequence (GGAG) at a proper position (positions -8 to -5 from the start codon). Using an in vitro translation system from isolated tobacco chloroplasts, the role of this sequence in translation was examined. Unexpectedly, the mutation of the SD-like element resulted in a large increase in translation. Internal and external deletions within the 5' UTR revealed that the region from -20 to -5 was involved in the negative regulation of translation. Scanning mutagenesis assays confirmed the above result. Competition assays suggested the existence of a trans-acting factor(s) involved in translational regulation. In this study, we discuss a possible mechanism for the negative regulation of rps2 mRNA translation.     

Why chloroplasts and mitochondria contain genomes

Chloroplasts and mitochondria originated as bacterial symbionts. The larger, host cells acquired genetic information from their prokaryotic guests by lateral gene transfer. The prokaryotically-derived genes of the eukaryotic cell nucleus now function to encode the great majority of chloroplast and mitochondrial proteins, as well as many proteins of the nucleus and cytosol. Genes are copied and moved between cellular compartments with relative ease, and there is no established obstacle to successful import of any protein precursor from the cytosol. Yet chloroplasts and mitochondria have not abdicated all genes and gene expression to the nucleus and to cytosolic translation. What, then, do chloroplast- and mitochondrially-encoded proteins have in common that confers a selective advantage on the cytoplasmic location of their genes? The proposal advanced here is that co-location of chloroplast and mitochondrial genes with their gene products is required for rapid and direct regulatory coupling. Redox control of gene expression is suggested as the common feature of those chloroplast and mitochondrial proteins that are encoded in situ. Recent evidence is consistent with this hypothesis, and its underlying assumptions and predictions are described.     

The evolution of chloroplast RNA editing

RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.     

The mRNA of Human Cytoplasmic Arginyl-tRNA Synthetase Recruits Prokaryotic Ribosomes Independently

There are two isoforms of cytoplasmic arginyl-tRNA synthetase (hcArgRS) in human cells. The long form is a component of the multiple aminoacyl-tRNA synthetase complex, and the other is an N-terminal truncated form (ΔNhcArgRS), free in the cytoplasm. It has been shown that the two forms of ArgRS arise from alternative translational initiation in a single mRNA. The short form is produced from the initiation at a downstream, in-frame AUG start codon. Interestingly, our data suggest that the alternative translational initiation of hcArgRS mRNA also takes place in Escherichia coli transformants. When the gene encoding full-length hcArgRS was overexpressed in E. coli, two forms of hcArgRS were observed. The N-terminal sequencing experiment identified that the short form was identical to the ΔNhcArgRS in human cytoplasm. By constructing a bicistronic system, our data support that the mRNA encoding the N-terminal extension of hcArgRS has the capacity of independently recruiting E. coli ribosomes. Furthermore, two critical elements for recruiting prokaryotic ribosomes were identified, the “AGGA” core of the Shine-Dalgarno sequence and the “A-rich” sequence located just proximal to the alternative in-frame initiation site. Although the mechanisms of prokaryotic and eukaryotic translational initiation are distinct, they share some common features. The ability of the hcArgRS mRNA to recruit the prokaryotic ribosome may provide clues for shedding light on the mechanism of alternative translational initiation of hcArgRS mRNA in eukaryotic cells.     

Phylogenetic Origin of the Chloroplast*†

SYNOPSIS. The 16S ribosomal RNA of the chloroplast of Euglena gracilis strain Z has been characterized in terms of its 2-dimensional electrophoretic “fingerprint” (T1 ribonuclease). Over 100 spots were resolved on the “fingerprint” and each spot was characterized as to which RNA oligonucleotide fragment(s) it contained. When compared to similar analyses of prokaryotic 16S rRNAs and eukaryotic cytoplasmic 18S rRNAs, the chloroplast 16S rRNA was a typically prokaryotic RNA, but bore little if any relationship to eukaryotic 18S rRNAs. Therefore, the cistrons for chloroplast 16S rRNA are related to the equivalent prokaryotic cistrons, but, apparently, are not related to the equivalent eukaryotic cistrons. Among the organisms available for comparison, the Euglena chloroplast 16S rRNA appears most closely related to the 16S rRNA of the eukaryote, Porphyridium cruentum (a red alga), and at least distantly related to the 16S rRNAs of the blue-green algae and perhaps also to the bacilli.     

Glycerolipid synthesis in Chlorella kessleri 11h. I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C16 and C18 acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [14C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C18-sn-2-C16 (C18/C16) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C18-sn-2-C18 (C18/C18) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C18/C18 species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C18/C18 species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

Remote Origins of Tail‐Anchored Proteins

C‐tail‐anchored (TA) proteins constitute a heterogeneous group of membrane proteins that are inserted into membranes by unique post‐translational mechanisms and that play key roles within cells. During recent years, bioinformatic screens on eukaryotic genomes have helped to obtain comprehensive pictures of the number, intracellular distribution and functions of TA proteins, but similar screens had not yet been carried out on prokaryotic cells. Here, we report the results of a bioinformatic screen of the genomes of two bacteria and one archeon. We find that all three of these prokaryotes contain TA proteins in proportions approaching those found in eukaryotic cells, indicating that this protein group is present in all three domains of life. Although some of our hits correspond to proteins of unknown function, others are enzymes with hydrophobic substrates or have functions carried out at the inner face of the cytoplasmic membrane. To generate hypotheses on the insertion mechanisms of prokaryotic TA proteins, we compared the sequences of the prokaryotic and eukaryotic versions of Asna1/Trc40/GET3, a cytosolic ATPase that plays a key role in TA protein post‐translational delivery to membranes in eukaryotic cells. We found that hydrophobic residues involved in TA binding by the eukaryotic chaperone (Mateja et al., Nature 2009;461:361–366) are generally replaced with equally hydrophobic amino acids in the archeal homologue (ArsA), whereas this is not the case for the bacterial protein. Thus, eukaryotes may have inherited the GET3 targeting pathway from our archeal ancestor, while the bacterial homologue may be exclusively dedicated to heavy metal resistance.