Biology subjects

Beckmann, R.

Publications and source records attributed to Beckmann, R..

2 recordsLinked to original sources

bioRxiv2018

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

To survive under conditions of stress, such as nutrient deprivation, bacterial 70S ribosomes dimerize to form hibernating 100S particles1. In {gamma}-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 reported5,6, the low resolution (18-38 [A]) 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 [A] 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-stem-loop of the E-tRNA and occludes the binding site for the mRNA as well as A- and P-site 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 bacteria7-10 and reveals a unique function for S1 in ribosome dimerization and inactivation, rather than its canonical role in facilitating translation initiation.

molecular biology↗

bioRxiv2018

The Folding Pathway of an Ig Domain is Conserved On and Off the Ribosome

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-b immunoglobulin domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force-dependence of escape from arrest, accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations - which also reproduce experiments on mutant forms of I27 - show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.\n\nSignificance StatementMost proteins need to fold into a specific three-dimensional structure in order to function. The mechanism by which isolated proteins fold has been thoroughly studied by experiment and theory. However, in the cell proteins do not fold in isolation, but are synthesized as linear chains by the ribosome during translation. It is therefore natural to ask at which point during synthesis proteins fold, and whether this differs from the folding of isolated protein molecules. By studying folding of a well characterized protein domain, titin I27, stalled at different points during translation, we show that it already folds in the mouth of the ribosome exit tunnel, and that the mechanism is almost identical to that of the isolated protein.

biophysics↗