In vivo analysis of the overlapping functions of DnaK and trigger factor

Abstract

Trigger factor (TF) is a ribosome-bound protein that combines catalysis of peptidyl-prolyl isomerization and chaperone-like activities in Escherichia coli. TF was shown to cooperate with the DnaK (Hsp70) chaperone machinery in the folding of newly synthesized proteins, and the double deletion of the corresponding genes (tig and dnaK) exhibited synthetic lethality. We used a detailed genetic approach to characterize various aspects of this functional cooperation in vivo. Surprisingly, we showed that under specific growth conditions, one can delete both dnaK and tig, indicating that bacterial survival can be maintained in the absence of these two major cytosolic chaperones. The strain lacking both DnaK and TF exhibits a very narrow temperature range of growth and a high level of aggregated proteins when compared to either of the single mutants. We found that, in the absence of DnaK, both the N-terminal ribosome-binding domain and the C-terminal domain of unknown function are essential for TF chaperone activity. In contrast, the central PPIase domain is dispensable. Taken together, our data indicate that under certain conditions, folding of newly synthesized proteins in E. coli is not totally dependent on an interaction with either TF and/or DnaK, and suggest that additional chaperones may be involved in this essential process.

Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action

“…Furthermore, it appears that the conformational change of the extended signature motif plays a key role in facilitating TFa docking on the 50S subunit. Consistently, a recent biochemical report has indicated that mutations and even deletion of the TF signature motif exert only a mild effect on TF function, compared with the complete deletion of the N-terminal domain (22). As association with the ribosome is a prerequisite for TF chaperone activity (23), the interactions of helix A2 with L29 and the C terminus of L23 seem to contribute significantly for TF binding.…”

SecB is a bona fide generalized chaperone in Escherichia coli

“…Recently, we reported that of many other known chaperone and protease genes tested, only GroEL͞GroES overproduction could provide enough assistance to support growth of our ⌬tig ⌬dnaKdnaJ strain as well as prevent aggregation under these conditions (9). Therefore, it is conceivable that TF, DnaK, GroEL, and SecB may compete for the same pool of both secretory and cytosolic proteins, although with a considerable advantage for TF, being more abundant and localized near the ribosome exit channel (28).…”

“…To answer this question, we took advantage of a previously described mutation in secB (resulting in the E77K amino acid substitution in the SecB protein), which Fresh transformants were then grown for 24 h at 20°C in LB-ampicillin, serially diluted, and spotted on LB-ampicillin agar plates without (Ϫ) or with (ϩ) 2 mM IPTG inducer at the indicated temperatures. Note that TF overproduction is toxic in the ⌬tig ⌬dnaKdnaJ triple mutant, as described (9).…”

“…However, it was recently shown that E. coli can tolerate the deletion of both chaperones at temperatures Ͻ30°C (9,10). In contrast to their respective isogenic single mutants, the strains lacking both DnaK͞DnaJ and TF exhibit a very narrow range of growth and, in addition, accumulate high levels of aggregated proteins at the permissive temperatures.…”

Reconstitution of a Mycobacterium tuberculosis proteostasis network highlights essential cofactor interactions with chaperone DnaK

“…Transposon mutagenesis analysis predicts that dnaK is also essential for growth in Mtb (14,15). In contrast, DnaK is nonessential in other bacteria studied, such as Escherichia coli, where its functions are redundant with those of trigger factor (encoded by tig), such that dnaK and tig form a synthetic lethal pair (16)(17)(18). In mycobacteria, the repressor HspR negatively regulates the heat shock response and is encoded in the same operon as dnaK (Fig.…”