Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins

Abstract

Protein synthesis in the chloroplast is carried out by chloroplast ribosomes (chloro-ribosome) and regulated in a light-dependent manner. Chloroplast or plastid ribosomal proteins (PRPs) generally are larger than their bacterial counterparts, and chloro-ribosomes contain additional plastid-specific ribosomal proteins (PSRPs); however, it is unclear to what extent these proteins play structural or regulatory roles during translation. We have obtained a threedimensional cryo-EM map of the spinach 70S chloro-ribosome, revealing the overall structural organization to be similar to bacterial ribosomes. Fitting of the conserved portions of the x-ray crystallographic structure of the bacterial 70S ribosome into our cryo-EM map of the chloro-ribosome reveals the positions of PRP extensions and the locations of the PSRPs. Surprisingly, PSRP1 binds in the decoding region of the small (30S) ribosomal subunit, in a manner that would preclude the binding of messenger and transfer RNAs to the ribosome, suggesting that PSRP1 is a translation factor rather than a ribosomal protein. PSRP2 and PSRP3 appear to structurally compensate for missing segments of the 16S rRNA within the 30S subunit, whereas PSRP4 occupies a position buried within the head of the 30S subunit. One of the two PSRPs in the large (50S) ribosomal subunit lies near the tRNA exit site. Furthermore, we find a mass of density corresponding to chlororibosome recycling factor; domain II of this factor appears to interact with the flexible C-terminal domain of PSRP1. Our study provides evolutionary insights into the structural and functional roles that the PSRPs play during protein synthesis in chloroplasts.chloroplast protein synthesis ͉ cryo-EM structure ͉ ribosomal RNA ͉ plastid ribosome recycling factor ͉ protein synthesis C ellular organelles, like mitochondria and chloroplasts, possess their own genome and components of gene-expression system, including ribosomes (for reviews, see refs. 1 and 2). The chloroplast (or plastid) ribosome (henceforth referred to as the ''chlororibosome'') is specialized in that it is involved in the biosynthesis of only the small number of proteins encoded by the chloroplast genome. The complete genome sequence of the spinach (Spinacea oleracea) chloroplast identified a total of 146 genes, of which 98 encode protein products, and 45 represent structural RNAs (3). The majority of the chloroplast-encoded proteins is targeted to the thylakoid membranes and include components of the ATP synthase, cytochrome b/f, and especially photosystem I and II complexes (3). In addition, chloro-ribosomes translate NADH dehydrogenase, the large subunit of RuBisCO, RNA polymerase subunits, and a distinct subset of ribosomal proteins: 12 from the small (30S) subunit and 8 from the large (50S) subunit, and the rest are nuclear-encoded and imported into the chloroplast.The origin of chloroplasts is understood to be through an early endosymbiotic event between a photosynthetic prokaryotic ancestor related to cyanobacteria and a eukaryotic cell (4). In fa...

PSRP1 Is Not a Ribosomal Protein, but a Ribosome-binding Factor That Is Recycled by the Ribosome-recycling Factor (RRF) and Elongation Factor G (EF-G)

“…3c). Indeed, the binding position of PSRP1 reported here on an E. coli ribosome, as well as in the homologous chloroplast ribosome (26), is incompatible with translation. Furthermore, the similarity in binding sites of PSRP1 and pY (17) on the ribosome, together with their high sequence homology suggests a functional similarity between the two proteins, leading us to suggest that PSRP1 is a pY-like stress response factor, rather than a ribosomal protein.…”

“…In the chloroplast ribosome, RRF has been found to directly contact PSRP1 (26). This led us to hypothesize that perhaps RRF, EF-G, and IF3 play a role in recycling PSRP1 from the ribosome after the stress conditions are relieved.…”

Rpl33, a Nonessential Plastid-Encoded Ribosomal Protein in Tobacco, Is Required under Cold Stress Conditions

“…In tobacco (Nicotiana tabacum) and most other higher plants, 12 of the 25 proteins in the small ribosomal subunit (30S) and nine of the 33 proteins in the large ribosomal subunit (50S) are encoded by plastid genes. In addition to these classical ribosomal proteins, plastids also possess a small set of ribosome-associated proteins that are not found in bacterial 70S ribosomes and therefore are referred to as plastid-specific ribosomal proteins Manuell et al, 2007;Sharma et al, 2007).…”

Nonessential Plastid-Encoded Ribosomal Proteins in Tobacco: A Developmental Role for Plastid Translation and Implications for Reductive Genome Evolution

“…Plastid ribosomes also contain a small number of proteins that are not found in bacterial ribosomes, the plastid-specific ribosomal proteins. They are encoded by nuclear genes, and their functions in protein biosynthesis and/or ribosome assembly are still largely unknown Manuell et al, 2007;Sharma et al, 2007).…”