YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes

Abstract

In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl-tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.

Figure 1.

YaeJ can hydrolyze peptidyl–tRNA on stalled ribosomes in vitro. (A) Schematic drawing of the wild-type template (stop) and non-stop templates (non-stop-1, -2 and -3), and the templates into which a tandem repeat of four rare Arg codons and four and eight major Leu codons were inserted (R4L4-Stop and R4L8-Stop, respectively), which were used in the in vitro translation system. The box indicates the open reading frame of the crp gene, with the DNA sequences of the engineered C-terminal region. Stop codon is indicated by an asterisk. (B) In vitro translation of the non-stop template (non-stop) with YaeJ that was expressed using the in vitro translation system. The reaction mixture containing YaeJ was directly added to the solution in which a preliminary 1-h in vitro translation reaction had been performed using the non-stop-1 template. The resulting mixture, incubated for another 10 min, was analyzed by NuPAGE. The gel was visualized using a laser-based fluorescent gel scanner. The final concentration of YaeJ is shown in each lane. (C) In vitro translation of non-stop template (non-stop) and wild-type template (stop) with a purified recombinant His-tagged YaeJ protein. His-tagged YaeJ protein was added to the solution in which a 1-h in vitro translation reaction has been performed using the non-stop-1 (left panel) and stop (right panel) templates at different concentrations. The final concentration of His-tagged YaeJ is shown in each lane. (D) In vitro translation of R4L4-Stop and R4L8-Stop templates with YaeJ that was expressed using the in vitro translation system. The reaction mixture containing YaeJ was directly added to the solution in which a 1-h in vitro translation reaction had been performed using each template. The final concentration of YaeJ was 5 μM.

Figure 2.

Mutation of the GGQ motif of YaeJ affected PTH activity. Wild-type YaeJ and four YaeJ mutants in which the GGQ residues were changed to GAQ, GGE, GAE or VAQ were expressed using the in vitro translation system. The in vitro translation reaction mixture containing a YaeJ mutant was directly added to another solution in which a preliminarily 1-h in vitro translation reaction had been performed using the non-stop-1 template. The final concentration of YaeJ and the mutants was 5 μM.

Figure 3.

YaeJ protein is always associated with ribosomes in vivo. Localization of YaeJ in log phase cells of wild-type strain (MG1655) (A), polysomes (B) and in stationary phase cells (C). After separation through 5–20% and 10–40% sucrose gradients for (A) or (C) and (B), respectively, fractions were analyzed by western blotting using an anti-YaeJ antibody.

Figure 4.

YaeJ plays no role in ribosome assembly or maturation. The ribosome profiles between wild-type and ΔyaeJ strains in log phase (A), polysomes (B) and stationary phase (C), were examined by sucrose density gradient centrifugation.

Figure 5.

C-terminal extension of YaeJ is required for PTH activity and ribosomal binding. (A) Sequence alignment of the C-terminal extension of YaeJ proteins from various bacteria. The C-terminal extensions that appeared unstructured in solution were determined according to the solution structure of the human YaeJ homolog, ICT1 (PDB code, 1J26) (21). White letters with a black background show basic amino acid residues. Bold letters indicate highly conserved residues. The numbering shown corresponds to the E. coli YaeJ protein. (B) Loss of PTH activity by truncation of the C-terminal extension of YaeJ. Wild-type YaeJ (1–140) and the three C-terminal truncation mutants (1–130, 1–119 and 1–100) were expressed using the in vitro translation system. Each of the mutant-containing samples was added to the translation reaction mixture using the non-stop-1 template. The final concentration of YaeJ and the mutants was 5 μM. (C) Ribosome association with His-tagged YaeJ and the C-terminal truncation mutant (1–130). His-tagged proteins were overexpressed in E. coli, and each was then added to the lysates of ΔyaeJ strain. After separation through a 5–20% sucrose gradient, fractions were analyzed by western blotting using an anti-His₆ antibody.