The Heat Shock Protein YbeY Is Required for Optimal Activity of the 30S Ribosomal Subunit

Abstract

The highly conserved bacterial ybeY gene is a heat shock gene whose function is not fully understood. Previously, we showed that the YbeY protein is involved in protein synthesis, as Escherichia coli mutants with ybeY deleted exhibit severe translational defects in vivo. Here we show that the in vitro activity of the translation machinery of ybeY deletion mutants is significantly lower than that of the wild type. We also show that the lower efficiency of the translation machinery is due to impaired 30S small ribosomal subunits.Many heat shock proteins are chaperones and proteases that constitute the protein quality control system (4,5,13,18). Recent studies demonstrated that beyond protein quality control, the heat shock response includes proteins implemented in the translation machinery (16,17), such as FtsJ (2, 3) and Hsp15 (11).FtsJ catalyzes methylation of U2552 in 23S rRNA (3). This modification occurs during the final steps of 50S biogenesis and is important for the structural stability of the 50S subunit (2). ftsJ deletion mutants accumulate ribosomal subunits at the expense of polysomes (2). Consequently, crude ribosome extracts prepared from ftsJ deletion mutants are far less active than wild-type preparations (3). Hsp15 recognizes and binds with high affinity to the aberrant state of the 50S subunit in complex with peptidyl tRNA positioned at the A site (10), which is more frequent at high temperatures (10). It has been proposed that Hsp15 participates in releasing the bound peptide and thereby helps recycle the 50S subunit (8, 10). Thus, heat shock proteins play a significant role both in the biogenesis of ribosomes and in the translation process.YbeY is a 17-kDa heat shock protein, highly conserved among bacteria, that belongs to the UPF0054 family of metaldependent hydrolases, suggesting that it may have a potential hydrolytic function (14,21). In Aquifex aeolicus, analysis of YbeY structure homology showed similarity to eukaryotic extracellular proteinases such as collagenase and gelatinase. However, in vitro experiments could not detect collagenase, gelatinase, or other hydrolase activity in YbeY (14).Recently, we showed that ybeY deletion mutants exhibit severe translational defects manifested by a very low level of polysomes and accumulation of free ribosomes and ribosomal subunits, indicating that most ribosomes in the cell are not engaged in translation. This translational defect intensifies at elevated temperatures (42°C) and results in growth arrest (17).Here we present in vitro studies indicating that the activity of the translation machinery prepared from ybeY deletion mutants is lower than in the wild type. In addition, we show that this lower activity stems specifically from a defective 30S ribosomal subunit. MATERIALS AND METHODSPurification of ribosomes and S100 subunits. Ribosomes and S100 subunits were prepared essentially as described previously (1). Cultures of Escherichia coli MG1655 and its ⌬ybeY derivative were grown in LB broth (Difco) medium at 37°C to an optical density at 60...

Conserved Bacterial RNase YbeY Plays Key Roles in 70S Ribosome Quality Control and 16S rRNA Maturation

“…The Δ ybeY mutant is reported to have both defective 30S and 50S subunits (Davies et al, 2010; Rasouly et al, 2010). To determine whether this unique 70S ribosome quality control is mediated specifically by either the defective 30S or 50S subunit, we purified subunits from wild type and the Δ ybeY mutant.…”

Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

“…Association of Era with membranes versus association with RNA appears to be competitive (33). It should be added that the third gene of the operon, ygfG , encodes a protein homologous to E. coli YbeY, which is also thought to be involved in regulating ribosomes (77,78) and is structurally similar to certain eukaryotic metalloproteases (107). These observations suggest that UDPK plays a role in a stress-induced pathway that modulates ribosome function.…”

The Escherichia coli Translation-Associated Heat Shock Protein YbeY Is Involved in rRNA Transcription Antitermination

“…Previous studies indicated that YbeY is important for translation and its absence results in production of impaired 30 S ribosomal subunits because of abnormal maturation of rRNA [26]–[28]. Recently [29] YbeY was shown to be a metallo endoribonuclease and with several functions including rRNA maturation and 70 S ribosome quality control.…”