Nascentome analysis uncovers futile protein synthesis in Escherichia coli

Abstract

Although co-translational biological processes attract much attention, no general and easy method has been available to detect cellular nascent polypeptide chains, which we propose to call collectively a "nascentome." We developed a method to selectively detect polypeptide portions of cellular polypeptidyl-tRNAs and used it to study the generality of the quality control reactions that rescue dead-end translation complexes. To detect nascent polypeptides, having their growing ends covalently attached to a tRNA, cellular extracts are separated by SDS-PAGE in two dimensions, first with the peptidyl-tRNA ester bonds preserved and subsequently after their in-gel cleavage. Pulse-labeled nascent polypeptides of Escherichia coli form a characteristic line below the main diagonal line, because each of them had contained a tRNA of nearly uniform size in the first-dimension electrophoresis but not in the second-dimension. The detection of nascent polypeptides, separately from any translation-completed polypeptides or degradation products thereof, allows us to follow their fates to gain deeper insights into protein biogenesis and quality control pathways. It was revealed that polypeptidyl-tRNAs were significantly stabilized in E. coli upon dysfunction of the tmRNA-ArfA ribosome-rescuing system, whose function had only been studied previously using model constructs. Our results suggest that E. coli cells are intrinsically producing aberrant translation products, which are normally eliminated by the ribosome-rescuing mechanisms.

Figure 1. Non-uniform stability of polypeptidyl-tRNA ester bonds.

(A) In vitro translation was directed by a truncated proOmpA_1-143 (Met1 to Thr143) message that was followed by a codon for one of 20 amino acids . Reaction was allowed at 37°C for 30 min in the presence of [³⁵S]methionine. Macromolecules were precipitated with trichloroacetic acid (final concentration, 5%) and the precipitates were washed twice with acetone and dissolved in 2 x Laemmli sample buffer (see Materials and Methods for the composition) that had been treated with RNASecure (Ambion), at room temperature followed by incubation at 37°C for 5 min. Samples were then divided into 3 portions. The first sample was untreated. The second and the third samples were mixed with an equal volume of 0.4 M Tris-base and incubated at 70°C for 15 and 30 min, respectively. They were separated by NuPAGE to visualize ³⁵S-labeled proOmpA_1-143-X-tRNA bands with phosphor imaging. Numbers indicate intensities of the bands, after incubation with Tris-base, relative (%) to that of the untreated sample. (B) Stability of proOmpA_1-143-Val-tRNA and proOmpA_1-143-His-tRNA was examined as described in (A) except that protein precipitates were dissolved in 1 x sample buffer, to which 1/10 volume of either 2 M Na₂CO₃ or 2 N NaOH was added and incubated at 37°C or 70°C for 15 min as indicated. (C) Stability of proOmpA_1-143-Val-tRNA and proOmpA_1-143-His-tRNA was examined as described in (A) except that samples were mixed with an equal volume of either 0.4 M, 1.0 M or 2.0 M Tirs-base and incubated at 80°C for 20 min as indicated.

Figure 2. Detection of the E. coli nascentome by pulse-chase labeling and two-dimensional separation.

(A and C) E. coli strain W3110 was grown at 20°C (A) or 37°C (C) and pulse-labeled for 0.5 min with [³⁵S]methionine and chased with unlabeled methionine for the indicated lengths of time. Samples were prepared as described in Materials and Methods and subjected to separation by the nascentome two-dimensional electrophoresis. Radioactive materials were visualized by phosphor imaging. The upper (main diagonal) line represents translation-completed polypeptide and the lower line (nascent line) represents polypeptide portions of polypeptidyl-tRNAs originally present in the cell. The value of %NL (proportion of radioactivities on the nascent line in the total radioactivities displayed on the gel) is shown in each gel panel. (B) A sample identical to that of zero-chase in (A) was prepared without using RNASecure and incubated with 0.1 mg/ml of RNase A at 30°C for 20 min before electrophoresis.

Figure 3. Stabilization of aberrant polypeptidyl-tRNAs upon dysfunction of the tmRNA-ArfA ribosome-rescuing system.

(A) CH111 (ΔssrAΔarfA/pBAD24-ssrA⁺) cells grown at 37°C in the presence of arabinose were harvested by centrifugation, washed and resuspended in the medium containing arabinose (open circles) or glucose (solid circles). Growth curves were recorded by absorbance measurement at 660 nm. (B) Portions of the same cultures as shown in (A) were sampled for pulse-labeling and nascentome two-dimensional separation. Cells in the presence of arabinose (Ara) as well as those at indicated time points after the arabinose to glucose (Glu) medium change were pulse-labeled for 0.5 min followed by chase for 1.5 min. Radioactive samples were prepared and separated in two-dimensions as described in Figure 2. The %NL value is given in each gel panel. (C) CH111 cells at 110 min after arabinose to glucose medium change were pulse-labeled for 0.5 min and chased for the indicated lengths of time for electrophoretic visualization of radioactive polypeptidyl-tRNAs. The %NL value is given in each gel panel.