Late steps of ribosome assembly in E. coli are sensitive to a severe heat stress but are assisted by the HSP70 chaperone machine

Abstract

The late stages of 30S and 50S ribosomal subunits biogenesis have been studied in a wild-type (wt) strain of Escherichia coli (MC4100) subjected to a severe heat stress (45–46°C). The 32S and 45S ribosomal particles (precursors to 50S subunits) and 21S ribosomal particles (precursors to 30S subunits) accumulate under these conditions. They are authentic precursors, not degraded or dead-end particles. The 21S particles are shown, by way of a modified 3′5′ RACE procedure, to contain 16S rRNA unprocessed, or processed at its 5′ end, and not at the 3′ end. This implies that maturation of 16S rRNA is ordered and starts at its 5′-terminus, and that the 3′-terminus is trimmed at a later step. This observation is not limited to heat stress conditions, but it also can be verified in bacteria growing at a normal temperature (30°C), supporting the idea that this is the general pathway. Assembly defects at very high temperature are partially compensated by plasmid-driven overexpression of the DnaK/DnaJ chaperones. The ribosome assembly pattern in wt bacteria under a severe heat stress is therefore reminiscent of that observed at lower temperatures in E. coli mutants lacking the chaperones DnaK or DnaJ.

Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process

“…Second, intermediates from the Δ rimM strain show underrepresented levels for all secondary and tertiary binding proteins from the 3′-domain. Interestingly, the Δ rimM intermediates are to some extent similar in protein composition to a previously identified in vivo 21S intermediates (44,53), but differ from the in vitro RI intermediates (54). The close resemblance of the Δ rimM intermediates to the naturally populated in vivo 21S intermediates suggests that they represent an early stage during the assembly of the 3′-domain, likely the entry stage.…”

“…Identification of the two sets of 16S rRNA precursors by a previously established 5′3′-rapid amplification of complementary DNA ends (RACE) technique (44) reveals that a majority of these precursors are unprocessed at both the 5′- and 3′-ends (Supplementary Figure S1). The protein gel analysis shows that some ribosomal proteins, e.g.…”

“…Nevertheless, if we view the in vivo assembly as a multi-branched process, the seemingly function redundancy among assembly factors is merely a sign of altered contribution of different, inter-connected assembly pathways. As shown in Figure 6C, the late-stage assembly in vivo starts with an in vivo 21S intermediate (44,53) and proceeds along a highly efficient pathway in the presence of all assembly factors. Assembly factors come in play at different time points to assist certain kinetically disfavored assembly events.…”

The toxin GraT inhibits ribosome biogenesis

“…The DnaK–DnaJ chaperone system has been shown to contribute to ribosome biogenesis, although a specific mechanism has not been determined (Al Refaii and Alix, ; René and Alix, ). To test if the transposon insertions into dnaK had also suppressed the GraT‐caused ribosome defects, we examined the ribosome profiles.…”

Inhibition of staphyloxanthin biosynthesis in Staphylococcus aureus by rhodomyrtone, a novel antibiotic candidate

“…The results indicated that rhodomyrtone inhibited the synthesis of DnaK protein in the treated pathogens (Sianglum et al, 2011). DnaK protein is associated with a wide variety of cellular processes (Hu et al, 2006;Kurt et al, 2006;Singh et al, 2007;Al Refaii & Alix, 2009;René & Alix, 2011), and deletion of dnaK in S. aureus demonstrated an increase in susceptibility to oxidative stress conditions and reduced carotenoid production (Singh et al, 2007). Sigma factor s B was significantly downregulated in rhodomyrtone-treated S. aureus cells (Sianglum et al, 2012).…”