# Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock
## 3 Discussion  
Until now, in vivo characterization of ribosome precursors has mainly been done by deleting or depleting ribosome assembly factors to force the accumulation of precursor particles. However, such mutations dramatically affect cellular metabolism and growth, resulting in significant feedback regulations which then affect ribosome synthesis. Here we took advantage of the increase in precursor content which occurs when cells are affected by heat shock. Indeed, the DnaK molecular chaperone induced upon heat shock to promote ATP-dependent refolding or degradation of damaged proteins is also necessary for the later steps of ribosome assembly. Under heat shock, its quantity is limited, since it is titrated by unfolded proteins, and this results in a transient reduction in the amounts of this chaperone that can be devoted to ribosome assembly, resulting in the temporary accumulation of ribosomal precursors [22]. However, this does not compromise ribosome synthesis because these precursors slowly mature into 30S ribosomal subunits, suggesting that they are authentic precursors. Indeed, similar amounts of ribosomes are neosynthesized at both 30 and 42 \({}^{\circ}\)C, which is unsurprising since the growth rates at these two non-optimal growth temperatures are nearly identical. We constructed strains with tags on certain ribosomal  
Figure 6: Structural comparison of 21S and 30S ribosomal subunits. (**A**) Front (top) and back (bottom) views of the 21S from this study, with the density corresponding to the rRNA in grey, and the ribosomal proteins highlighted in gold. Note that the density of uS7 is poorly defined. (**B**) Same views, but here of an atomic model of the _E. coli_ 30S ribosomal subunit (PDB 4V4Q) filtered to adjust the resolution to match the 21S map. The ribosomal proteins uS2 and bS21, missing in our 21S structure, are light blue and red, while the uS7 is blue. (**C**) Same, but for the 30S + RsgA cryo-EM map (EMD-3663), with the RsgA maturation factor in blue. proteins. These chromosomal tags had no effect on growth at any temperature, nor did they affect antibiotic susceptibility or in vitro translation, so these tags do not affect bacterial metabolism or ribosomal activity. Even if we cannot exclude the idea that heat stress could affect ribosome biogenesis by changing the transcription of rRNA and r-protein operons, the effect of heat stress on transcription has already been analyzed in many studies, which have concluded that rRNA operon transcription is not affected at 45 \({}^{\circ}\)C [35; 36; 37; 38]. We analyzed ribosome biogenesis data (Supplementary Figure S4) from a transcriptomic study of the effects of heat stress on the MG1655 strain grown in the same medium and under identical heat constraints as studied here [37]. There, the expression of ribosomal protein genes was reduced immediately after stress but was maintained at high levels, which is in accordance with the reduced growth rates known to occur after a shift to high temperatures [39]. This is particularly true for proteins that are poorly represented in the 21S subunit, whose reduction in 21S is not correlated with a similar reduction in their synthesis. These data suggest that the synthesis of ribosomal components is not dramatically affected by growth at very high temperatures, so the accumulation of precursors is instead due to a defect in ribosome assembly. When cells are grown at 45 \({}^{\circ}\)C, expressions of the KsgA, RimM, RsgA, RbfA, and RimP maturation factors follow the same pattern as that of ribosomal proteins: there is an immediate decrease after stress, but their expressions remain high (Supplementary Figure S4). This is not surprising, since many of these genes are part of operons that also encode for ribosomal proteins. This suggests that the moderate reduction of these ribosome assembly factors does not cause any drastic effects. From our data, we saw structural defects in the head of 21S particles.