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)

Manjuli R Sharma,Alexandra Dönhöfer,Chandana Barat,Viter Marquez,Partha P Datta,Paola Fucini,Daniel N Wilson,Rajendra K Agrawal

doi:10.1074/jbc.m109.062299

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

EXPERIMENTAL PROCEDURES

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]

RESULTS

Factor Rather Than a Ribosomal

DISCUSSION