Structure of a hibernating 100S ribosome reveals an inactive conformation of the ribosomal protein S1

Abstract

To survive under conditions of stress, such as nutrient deprivation, bacterial 70S ribosomes dimerize to form hibernating 100S particles 1 . In g-proteobacteria, such as Escherichia coli, 100S formation requires the ribosome modulation factor (RMF) and the hibernation promoting factor (HPF) 2-4 . Although structures of E. coli 100S particles have been reported 5,6 , the low resolution (18-38 Å) prevented the mechanism of ribosome inactivation and dimerization to be fully elucidated. Here we present single particle cryo-electron microscopy structures of hibernating 70S and 100S particles isolated from stationary phase E. coli cells at 3.0-7.9 Å resolution, respectively. Preferred orientation bias for the complete 100S particle was overcome using tilting during data collection. The structures reveal the binding sites for HPF and RMF as well as the unexpected presence of deacylated E-site tRNA and ribosomal protein S1 in the 100S particle. HPF interacts with the anticodon-stemloop of the E-tRNA and occludes the binding site for the mRNA as well as A-and Psite tRNAs. RMF stabilizes a compact conformation of S1, which together sequester the anti-Shine-Dalgarno (SD) sequence of the 16S ribosomal RNA (rRNA), thereby inhibiting translation initiation. At the dimerization interface, S1 and S2 form intersubunit bridges with S3 and S4 and the C-terminus of S2 probes the mRNA entrance channel of the symmetry related particle, thus suggesting that only translationally inactive ribosomes are prone to dimerization. The back-to-back 100S dimerization mediated by HPF and RMF is distinct from that observed previously in Gram-positive bacteria 7-10 and reveals a unique function for S1 in ribosome dimerization and inactivation, rather than its canonical role in facilitating translation initiation.bioRxiv Page 3 of 39The hibernation state mediated by RMF and HPF is not only important for bacterial survival during stationary phase, but also under stress conditions, such as osmotic, heat and acid stress 1 . Since RMF and HPF are present in many clinically important bacterial pathogens, such E. coli, Salmonella typhimurium, Yersinia pestis and Pseudomonas aeruginosa 11 , the hibernation pathway represents a attractive target for development of novel antimicrobial agents. While structures of hibernating 100S particles exist, the low resolution (18-38 Å) precluded identification of the RMF and HPF binding sites 5,6 .Structures of E. coli HPF in complex with 70S ribosomes revealed a binding site overlapping with the A-and P-sites 12,13 , whereas E. coli RMF has only been visualized on a 70S ribosome from T. thermophilus 13 , a bacterial species that does not even contain the gene encoding RMF. Therefore, we set out to determine the structure of a physiologically relevant 100S particle isolated from nutrient deprived bacteria.For structural analysis, E. coli 100S (Ec100S) particles were isolated by sucrose density gradient centrifugation of cellular lysates prepared from stationary phase cells of the E. coli BW25112DyfiA strain, which lacks...

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An energy charge sensor for balancing RNA polymerase recycling and hibernation

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