Authentic precursors to ribosomal subunits accumulate in Escherichia coli in the absence of functional DnaK chaperone

Abstract

SummaryEscherichia coli dnaK -ts mutants are defective in the late stages of ribosome biogenesis at high temperature. Here, we show that the 21S, 32S and 45S ribosomal particles that accumulate in the dnaK756 -ts mutant at 44 ∞ ∞ ∞ ∞ C contain unprocessed forms of their 16S and 23S rRNAs (partially processed in the case of 45S particles). Their 5S rRNA stoichiometry and ribosomal protein composition are typical of the genuine ribosomal precursors found in a wild-type ( dnaK + + + + ) strain. Despite the lack of a functional DnaK, a very slow maturation of these 21S, 32S and 45S particles to structurally and functionally normal 30S and 50S ribosomal subunits still occurs at high temperature. This conversion is accompanied by the processing of p16S and p23S rRNAs to their mature forms. We conclude that: (i) 21S, 32S and 45S particles are not dead-end particles, but true precursors to active ribosomes (21S particles are converted to 30S subunits, and 32S and 45S to 50S subunits); (ii) DnaK is not absolutely necessary for ribosome biogenesis, but accelerates the late steps of this process considerably at high temperature; and (iii) 23S rRNA processing depends on the stage reached in the stepwise assembly of the 50S subunit, not directly on DnaK.

The CRM domain: An RNA binding module derived from an ancient ribosome-associated protein

“…3B). Incompletely processed 23S rRNA, a hallmark of intermediates in the assembly of 50S ribosomal subunits (Srivastava and Schlessinger 1988;Hage and Alix 2004), was enriched in the YhbY peak fractions (Fig. 3B).…”

“…To distinguish between these possibilities we took advantage of the fact that 50S subunits that have participated in translation harbor mature 23S rRNA, whereas pre-50S subunits are enriched in incompletely processed 23S rRNA (Srivastava and Schlessinger 1988;Hage and Alix 2004). Ribonuclease-protection ( Fig.…”

Ribosome Biogenesis and the Translation Process inEscherichia coli

“…It stabilizes the hydrophobic regions of extended polypeptide segments in an ATP-dependent manner upon heat shock induction (25). At 45°C, temperature-sensitive DnaK mutants give rise to the accumulation of 30S and 50S precursor particles, implying that DnaK is involved in ribosome biogenesis at high temperatures (3,59). However, the involvement of the protein at temperatures below 30°C is more controversial.…”

Time course of large ribosomal subunit assembly in E. coli cells overexpressing a helicase inactive DbpA protein

“…In order to fulfill the cell's demand for new proteins and to avoid wasting valuable cellular energy, ribosome assembly needs to occur rapidly and accurately. To ensure this, cells use a number of different protein and RNA maturation factors (El Hage et al 2001;Charollais et al 2003Charollais et al , 2004El Hage and Alix 2004;Jain 2008). One class of RNA maturation factors used by the cell during ribosome assembly is DEAD-box RNA helicases (Cordin et al 2006;Linder 2006;Pyle 2008;Pan and Russell 2010;Shajani et al 2011;Henn et al 2012;Putnam and Jankowsky 2013).…”