Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions

Abstract

Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.

Structure of the Bacillus subtilis hibernating 100S ribosome reveals the basis for 70S dimerization

“…Aligning the E. coli SHPF-70S structure (Polikanov et al, 2012) to the 70S-A ribosome in the Bs100S based on the 16S rRNA revealed the expected similarity in their binding positions ( Fig 2C). As noted previously for E. coli YfiA and HPF (Vila-Sanjurjo et al, 2004;Polikanov et al, 2012) and for the NTD of the LHPF from Spinach chloroplast (Sharma et al, 2007(Sharma et al, , 2010Graf et al, 2016;Bieri et al, 2017), the binding position of BsHPF-NTD overlaps with the mRNA and anticodon-stem loop regions of tRNAs bound in the ribosomal A-and P-sites ( Fig 2D), thus explaining the observed inhibitory effect by LHPFs when added to in vitro translation assays (Ueta et al, 2013;Basu & Yap, 2016).…”

“…Here we present a cryo-EM structure of the B. subtilis 100S particle (Bs100S) revealing the binding site for the BsHPF (also referred to as YvyD). The BsHPF-NTD binds in a position overlapping the mRNA, A-and P-tRNAs, analogous to YfiA, SHPF, and the NTD of the LHPF from spinach chloroplasts (Vila-Sanjurjo et al, 2004;Sharma et al, 2007Sharma et al, , 2010Polikanov et al, 2012;Graf et al, 2016;Bieri et al, 2017), indicating how LHPFs inhibit translation (Ueta et al, 2013;Basu & Yap, 2016). Unexpectedly, we observe that the BsHPF-CTD forms a homodimer with the CTD of the BsHPF from the second 70S ribosome, thus providing a structural basis for LHPF-mediated 100S formation.…”

“…The BsHPF-NTD binds in a position overlapping the mRNA, A-and P-tRNAs, analogous to YfiA, SHPF, and the NTD of the LHPF from spinach chloroplasts (Vila-Sanjurjo et al, 2004;Sharma et al, 2007Sharma et al, , 2010Polikanov et al, 2012;Graf et al, 2016;Bieri et al, 2017), indicating how LHPFs inhibit translation (Ueta et al, 2013;Basu & Yap, 2016). The BsHPF-NTD binds in a position overlapping the mRNA, A-and P-tRNAs, analogous to YfiA, SHPF, and the NTD of the LHPF from spinach chloroplasts (Vila-Sanjurjo et al, 2004;Sharma et al, 2007Sharma et al, , 2010Polikanov et al, 2012;Graf et al, 2016;Bieri et al, 2017), indicating how LHPFs inhibit translation (Ueta et al, 2013;Basu & Yap, 2016).…”

Functional Analysis of PSRP1, the Chloroplast Homolog of a Cyanobacterial Ribosome Hibernation Factor

“…Extracts of leaves harvested at midday or at the end of the night were fractionated by sedimentation through sucrose gradients under conditions that resolved monosomes and free ribosomal subunits in the middle of the gradient ( Figure 2). Prior data show that PSRP1 binds 30S ribosomal subunits and 70S ribosomes, and suggest that it could not bind ribosomes in the process of translation [6,[8][9][10]15]. Therefore, we did not aim to maintain polysome integrity in these assays.…”

“…The PSRP1 sequence places it in the "long Hibernation Promoting Factor" (l-HPF) subclass, members of which generally promote the formation of inactive 100S ribosome dimers [8]. Structural and biochemical data show, however, that PSRP1 binds and inactivates 70S ribosomes in a manner analogous to Rai1/pY [6][7][8][9][10][11]. PSRP1 binds the intersubunit space of chloroplast ribosomes, preventing access of tRNAs to the A and P sites [9,10].…”