# Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock
## 1 Introduction  
Bacteria have developed a myriad of mechanisms to allow them to grow in hostile environments or under stressful conditions. Because exposure to elevated temperatures causes a constant denaturation of proteins, heat stress has a large impact on bacterial fitness and induces a regulated response. The molecular mechanisms that control this specific cellular stress response are mainly governed at the transcriptional level by the heat-shock sigma factor Rp0H. The activation of Rp0H at high temperatures enables the increased expression of heat shock proteins (HSPs). Many HSPs function as chaperones, which are needed for both the correct folding of neosynthesized proteins and for the renaturation or degradation of denatured proteins [1]. While these proteins play essential roles in cellular metabolism under regular conditions, their synthesis increases dramatically when temperatures are elevated. Among the many macromolecular complexes affected by stress, ribosomes are of major importance. Indeed, their activity is crucial for cell viability, and they can sense the cell's metabolic state through their interactions with cofactors, notably the Rel/RelA enzymes which monitor the aminoacylation status of ribosomal A-site tRNA [2]. In fact, these enzymes are positively regulated by their product, the alarmone nucleotide (p)ppGpp, which is an activator of the \"stringent\" response which in turn affects ribosome synthesis [3; 4; 5]. These various mechanisms are very well described, but ribosome biogenesis under stressful conditions is less understood. Ribosome synthesis begins with the transcription of a large ribosomal RNA (rRNA) precursor molecule that is then cleaved by RNase III into the precursors of the three rRNAs. The assembly process is sequential and cooperative, with a series of RNA processing andconformational changes as well as the hierarchical binding of ribosomal proteins [6; 7]. These events are favored by the intervention of many ribosome assembly factors that regulate the assembly of the large (50S) and small (30S) ribosomal subunits [6; 8]. The 30S late-assembly intermediates that accumulate in _E. coli_ null mutants deleted from the assembly factors _rimM_, _rbFa_, _yjeQ_ and _era_ have already been isolated and well-described [9; 10; 11; 12; 13; 14; 15]. These immature precursors are structurally related, all resembling the mature 30S subunit but with severe distortions at the decoding center which make translation impossible [16]. Each of these precursors are blocked in the later stages of ribosome maturation, which suggests that multiple parallel assembly pathways exist, and that these pathways converge at the end of the assembly process [17]. In the same way, severe heat stress also affects ribosome biogenesis, inducing the accumulation of ribosome precursors. The formation of these precursors after chaperone titration by denatured proteins has been observed both in vitro and in vivo. Indeed, the depletion of DnaK or GroEL chaperones causes the immature ribosomes to accumulate, indicating that these chaperones are most probably involved in ribosome maturation [18; 19]. Accordingly, at temperatures below 30 \({}^{\circ}\)C, _dnaK_ and _dnaJ_ mutants exhibit less growth than the wild type, but with no ribosome assembly defects. At temperatures above 42 \({}^{\circ}\)C, however, ribosome assembly decreases, while both 30S and 50S precursors accumulate [20]. The overproduction of GroEL chaperones can suppress defects in the large 50S subunit assembly, but does not affect the maturation of the 30S precursor [21]. A transient accumulation of this precursor, the 21S particle, was also observed in wild-type strains subjected to severe heat stress [22]. These are genuine precursors that slowly convert into 30S mature subunits, but are produced in very small amounts. Until now, this had unfortunately prevented their isolation as well as the analysis of their composition and structure, thus limiting our understanding of the influence of heat stress on ribosome maturation at the molecular level.