Abstract
Plastid-specific ribosomal proteins (PSRPs) have been proposed to play roles in the light-dependent regulation of chloroplast translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A- and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation. We find that the presence of PSRP1/pY lowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool.
Highlights
Organelle originated through engulfment of a photosynthetic unicellular prokaryote by a eukaryotic host cell, and the subsequent integration of the two genomes through a process of gene transfers from the chloroplast to the nuclear genome
Binding of Spinach PSRP1 to the E. coli Ribosome—The mature form of spinach PSRP1 was cloned into pET32b; the construct introduces an N-terminal Trx fusion linked to the protein via a six-histidine (6ϫHis) affinity tag
SDS-PAGE and Western blotting against the 6ϫ histidine (6ϫHis) tag indicated that Trx-PSRP1 migrated with 70S ribosomes
Summary
70S1⁄7PSRP1 Complexes—The gene encoding the mature PSRP1 was amplified from cDNA template and cloned into pET32b, introducing N-terminal thioredoxin (Trx) and 6ϫ histidine (6ϫHis) tags onto the mature PSRP1. Binding assays and sucrose gradients were performed as previously described [18]. Dissociation assays were performed using tightcouple E. coli 70S ribosomes that were incubated in the presence or absence of protein factors in Buffer. E. coli 70S ribosomes (0.4 M) were incubated with 10ϫ molar excess of protein factors indicated for each reaction in binding buffer (20 mM Hepes-KOH, pH 7.6, 8.2 mM MgCl2, 80 mM NH4Cl, 4 mM -mercaptoethanol) for 20 min at 37 °C in a total volume of 150 l, before being loaded on a 5–30% sucrose gradient in the same buffer and centrifuged in a SW40 rotor at 19,000 rpm for 16.5 h at 4 °C. Visualization and interpretation of the map, and docking of crystallographic structures, were performed using SPIDER, IRIS Explorer (Numerical Algorithms Group, Inc., Downers Grove, IL), O [24], and Ribbons [25]
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