Chloroplasts, the photosynthetic factories of green algae or land plants, derive from a cyanobacterial endosymbiont engulfed by a primitive eukaryotic cell 1.5-billion years ago. Similarly, mitochondria, from which most eukaryotes derive energy through aerobic respiration, originate from an α-proteobacterium captured about 2-billion years ago. Over time, the genomes of these organelles have lost most of their coding capacity; they encode only 50-200 and 3-67 proteins for chloroplast and mitochondria, respectively, instead of thousands for free living bacteria. Thus, the chloroplast from the green alga Chlamydomonas reinhardtii only codes for part of its own transcription and translation machineries, for a subset of the subunits of each of the photosynthetic complexes, and for a few other proteins. Yet, organelles contain as many different proteins as free-living bacteria, showing that most of these proteins are nuclear-encoded. Still, gene expression in organelles retains a pronounced prokaryotic character because most of the involved genes are actually ancestral prokaryotic genes that have been transferred to the nucleus. For instance, in C. reinhardtii chloroplast, transcription is achieved by an RNA polymerase (α2ββ'σ) similar to that of E. coli, and the resulting mRNAs are translated by 70S ribosomes that resemble those of bacteria. Also, chloroplast mRNAs are degraded by endo- or exonucleases that are orthologs of those existing in E. coli or B. subtilis. Although poorly documented, the degradation pathways are logically thought to resemble those prevailing in bacteria.
A major difference between gene expression in bacteria and in organelles is the existence of unique regulatory mechanisms that allow the eukaryotic cell to control the activity of its ancient symbiont. For instance, chloroplast gene expression is under the hierarchical control of nucleus-encoded factors that control the translation or stability of mRNAs in a gene specific manner. In contrast, the Shine-Dalgarno sequence (SD), which is critical for translation initiation in bacteria, has been lost in more than 70% of the chloroplast genes.
Task 1 of DYNAMO aims at comparing gene expression in organelles and in model free-living prokaryotes. One project involves the characterization of the mRNA degradation machinery from the C. reinhardtii chloroplast. Another important research direction consists in assessing the similarities and differences between post-transcriptional gene regulation in bacteria and in chloroplast.
RNases, from bacteria to chloroplast.
RNase J is an important ribonuclease conserved over a large fraction of the bacterial world as well as in chloroplasts. The Bacillus subtilis enzyme, which carries the first 5’->3’ exonucleolytic activity ever identified in a bacterium, was discovered and characterised in the teams of Harald Putzer and Ciaran Condon (FRE3630). The upper left part of Figure 1 shows the structure of the B. subtilis enzyme. Upper, right : detail of the active site complexed with the nucleotide UMP. This image suggests how the enzyme might work as a 5’->3’ exonuclease. Lower : sequence comparison between RNase J from B. subtilis and chloroplast. The latter enzyme is currently being characterized through a collaborative effort between the labs of Harald Putzer (FRE3630) and Francis-André Wollman (UMR7141). Particularly involved in this project is the PhD student Anna Liponska (lower right) and the Postdoctoral researcher Loreto Suay, both of whom are supported by DYNAMO.
YacP, a putative RNase of unknown function, is conserved in bacilli, cyanobacteria, and in higher plants, where it is predicted to be localised to chloroplasts (Figure 2, lower left). The chromosomal context of The B. subtilis yacP gene is surrounded by genes involved in translation (Figure 2, upper), leading us to suspect that YacP is involved in the maturation or turnover of an RNA involved in the process of translation. The structure of the enzyme has been solved by Jérémie Piton, a postdoc from the team of Ciaran Condon (FRE3630) (Figure 2, lower middle). Further studies on YacP enzymes of various origins are underway in the Condon lab, by Magali Leroy (Figure 2, lower right), a Post-doctoral researcher supported by DYNAMO. The aim is to identify the substrate(s) of this orphan RNase, and to characterize the basis of its specificity.
Post-transcriptional regulation of gene expression in bacteria and chloroplast.
Expression from chloroplast mRNAs is regulated by specific nuclear factors, called “M” and “T”, that are present in limiting amounts and are required for the stability (M factors) or translatability (T factors) of individual mRNAs (Figure 3). M factors are thought to behave as barricades against uncharacterized 5’->3’ exonucleases (RNase J?), whereas T factors would favour translation by melting unfavourable mRNA structures around the start codon or by directly binding chloroplast ribosomes. Current projects aim at better characterising the mechanism of action of M and T factors by reproducing their activity in the model bacteria E. coli and B. subtilis, where translational activation or protection against 5’->3’ exonucleases (in B. subtilis only) are well documented. This collaborative project between the labs FRE3630 and UMR7141 has been started by Agata Staszak, an Erasmus student supported by DYNAMO; a second Erasmus position is currently available along the same lines.