Structure of the Chloroplast Ribosome: Novel Domains for Translation Regulation

Citation: Manuell AL, Quispe J, Mayfield SP ( Structure of the Chloroplast Ribosome: Novel Domains for Translation Regulation Andrea L. Manuell 0 1 Joel Quispe 0 1 Stephen P. Mayfield 0 1 0 Academic Editor: Gregory A. Petsko, Brandeis University , United States of America 1 1 Department of Cell Biology, The Scripps Research Institute, La Jolla, California, United States of America, 2 The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, United States of America, 3 National Resource for Automated Molecular Microscopy, The Scripps Research Institute , La Jolla, California , United States of America 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 A . 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. - The chloroplast of plants and algae is believed to have originated from the endosymbiosis of an ancient photosynthetic bacteria into a eukaryotic host. Remnants of that ancient bacteria remain in the modern chloroplast, as it maintains a circular genome and transcription and translation machinery similar to that of prokaryotes [1,2]. Chloroplasts are responsible for photosynthetic energy production in plants and algae, and have recently been targeted as a platform for production of recombinant therapeutic proteins, making the understanding of translation in this organelle essential [3]. Approximately 60 proteins are translated in the plastid, a small fraction of the total proteins functioning in this organelle. The majority of chloroplast proteins are encoded by the nuclear genome and post-translationally imported into the plastid [4]. Coordinate expression from the nuclear and plastid genomes is required for development in photosynthetic organisms, and is achieved in chloroplasts primarily through regulation of translation [5,6]. Translation of many chloroplast genes is also regulated in response to light, and to maintain stoichiometric accumulation of multiprotein-complex subunits [7,8]. All of this regulation involves a host of protein translation factors, and the formation of RNAprotein complexes on chloroplast mRNA 59 untranslated regions (59 UTRs) [913]. Some of these protein factors are specific to individual mRNAs, whereas others serve classes of messages. Due to the bacterial ancestry of the organelle, translation in the chloroplast has been considered bacterial-type translation, and many of the requisite bacterial-type translation factors can be identified in chloroplasts, although not all of these are exact homologs of the bacterial proteins [14]. Translation regulation in the chloroplast is more complex than in bacteria, and this complexity requires additional RNA and protein components not found in prokaryotic systems (reviewed in [5,15]). A number of protein factors have been identified as essential components of chloroplast translation, although how these factors interact with an mRNA to facilitate chloroplast translation is not known. Chloroplast messages also experience pausing during their translation, which has been implicated in maintaining the proper stoichiometry of gene expression from polycistronic mRNAs, as well as in cotranslational membrane insertion or cofactor association [16,17]. mRNA secondary structures or rare codon usage are often suggested as the cause of pausing during elongation; however, for mRNAs studied in chloroplasts (particularly psbA and atpA), these alone are insufficient to account for the pause sites. RNA elements identified as regulatory components in the translation of chloroplast messages are primarily located in the 59 UTR. These elements include Shine-Dalgarno (S-D) sequences, stem-loop structures, and A/U rich elements [10,1820]. Nearly all bacterial mRNAs use base pairing between a S-D sequence located in the 59 UTR of the mRNA Translation of mRNA into protein is the main step for the regulation of gene expression in the chloroplast, the photosynthetic organelle of plant cells. Translation is conducted by the ribosome, a large macromolecular machine composed of RNA and protein. Studies have shown that the composition of the chloroplast ribosome is similar to that of bacterial ribosomes, but also that chloroplast ribosomes contain a number of unique proteins. We present the three-dimensional structure of the chloroplast ribosome, as calculated using cryo-electron microscopy and single-particle reconstruction. Chloroplast-unique structures are clearly visible on our ribosome map, and expand upon a basic bacterial ribosome-like core. The role of these chloroplast-unique ribosomal proteins in regulating translation of chloroplast mRNAs, including light-regulated translation, is suggested by the location of these structures on the ribosome. Biochemical data confirm a predicted function in chloroplast translation for some of the unique proteins. Our model for translation in the chloroplast incorporates decades of biochemical and genetic studies with the structure presented here, and should help guide future studies to understand the molecular mechanisms of translation regulation in the chloroplast. and a complementary sequence located near the 39 end of the 16S rRNA [21]. Base pairing between these sequences is essential for bacterial translation initiation, and bacterial S-D elements are located 7 6 2 nucleotides (nt) upstream of the initiator AUG to allow for a simple physical positioning of the initiator AUG in the P-site of the ribosome [21,22]. In plastids, only some mRNAs have recognizable S-D sequences, and these are found over a large range of the 59 UTR, some up to 100 nt upstream of the start site AUG [23,24]. This diverse positioning of S-D elements precludes a simple physical posit (...truncated)