Ribosome Dimerization Protects the Small Subunit

Abstract

When nutrients become scarce, bacteria can enter an extended state of quiescence. A major challenge of this state is how to preserve ribosomes for the return to favorable conditions. Here, we show that the ribosome dimerization protein hibernation-promoting factor (HPF) functions to protect essential ribosomal proteins. Ribosomes isolated from strains lacking HPF (Δhpf) or encoding a mutant allele of HPF that binds the ribosome but does not mediate dimerization were substantially depleted of the small subunit proteins S2 and S3. Strikingly, these proteins are located directly at the ribosome dimer interface. We used single-particle cryo-electron microscopy (cryo-EM) to further characterize these ribosomes and observed that a high percentage of ribosomes were missing S2, S3, or both. These data support a model in which the ribosome dimerization activity of HPF evolved to protect labile proteins that are essential for ribosome function. HPF is almost universally conserved in bacteria, and HPF deletions in diverse species exhibit decreased viability during starvation. Our data provide mechanistic insight into this phenotype and establish a mechanism for how HPF protects ribosomes during quiescence. IMPORTANCE The formation of ribosome dimers during periods of dormancy is widespread among bacteria. Dimerization is typically mediated by a single protein, hibernation-promoting factor (HPF). Bacteria lacking HPF exhibit strong defects in viability and pathogenesis and, in some species, extreme loss of rRNA. The mechanistic basis of these phenotypes has not been determined. Here, we report that HPF from the Gram-positive bacterium Bacillus subtilis preserves ribosomes by preventing the loss of essential ribosomal proteins at the dimer interface. This protection may explain phenotypes associated with the loss of HPF, since ribosome protection would aid survival during nutrient limitation and impart a strong selective advantage when the bacterial cell rapidly reinitiates growth in the presence of sufficient nutrients.

The hibernating 100S complex is a target of ribosome-recycling factor and elongation factor G in Staphylococcus aureus

“…The recycling of PoTc, reactivation of the silent 100S ribosomes, and prevention of ribosome degradation are cost-saving activities because ribosome biogenesis is the most energyconsuming cellular process. A recent study offered the first clue that 100S dimers are disassembled and converted into active ribosomes in a reconstituted cell-free translation system consisting solely of purified components, including RRF and EF-G, but not the HflX (30).…”

“…The significance of 100S ribosomes during logarithmic growth is unclear, although they are thought to function as storage sites to preserve unused ribosomes (e.g., posttermination recycled ribosomes) from degradation (3)(4)(5)29). In fact, 70S dimerization is strongly linked to the protection of ribosomes and the maintenance of active translation pools (3,23,30).…”

Analyzing Ribosome Remodeling in Health and Disease

“…The combination of mass spectrometry with structural biology has been used to elucidate several facets of the ribosome complex subunits, including ribosome assembly in bacteria [139,144,145] and the effect of dimerization on ribosomes when nutrients are scarce. [146] In addition, ribosome remodeling has been found to occur in the bacterial ribosome through the use of MS and X-ray crystallography. [147] This paper emphasized the need to use multiple complementary approaches for quantifying ribosome remodeling and briefly reviewed sources of systematic biases as well as approaches for mitigating their influence.…”

Bacterial Spore mRNA – What’s Up With That?

“…Moreover, spore ribosomes appeared to be translationally active, in the sense that when extracted, they were capable of synthesizing polyphenylalanine from a polyuracil template in cell-free reaction mixtures at comparable rates to ribosomes purified from log-phase vegetative cells (Chambon et al, 1968;Deutscher et al, 1968). Whether ribosome hibernation factors, such as the Hpf protein, have a role in spores in preserving the structural integrity of ribosomes during dormancy, analogous to that observed in stationary phase vegetative cells (Feaga et al, 2020), perhaps facilitating rapid resumption of activity upon germination, has not been established. Regardless, and most importantly, the thinking about spores until ~2006 was that they did not have "functional" mRNA.…”