A primary role for release factor 3 in quality control during translation elongation in Escherichia coli

Abstract

Release factor 3 (RF3) is a GTPase found in a broad range of bacteria where it is thought to play a critical "recycling" role in translation by facilitating the removal of class 1 release factors (RF1 and RF2) from the ribosome following peptide release. More recently, RF3 was shown in vitro to stimulate a retrospective editing reaction on the bacterial ribosome wherein peptides carrying mistakes are prematurely terminated during protein synthesis. Here, we examine the role of RF3 in the bacterial cell and show that the deletion of this gene sensitizes cells to other perturbations that reduce the overall fidelity of protein synthesis. We further document substantial effects on mRNA stability and protein expression using reporter systems, native mRNAs and proteins. We conclude that RF3 plays a primary role in vivo in specifying the fidelity of protein synthesis thus impacting overall protein quantity and quality.

Figure 1

A model for the action of RF3 in controlling nonsense suppression. During protein synthesis, stop codons are typically recognized by class I release factors, leading to termination (a). Occasionally, stop codons are misread by near-cognate tRNAs, leading to a mismatched pairing between the P-site codon and the anticodon of the peptidyl-tRNA. Under normal conditions, this ribosomal complex would be recognized by the post PT QC machinery (RF1/2 and RF3), leading to premature termination of protein synthesis (b). In the absence of RF3, recognition of the mismatched complex is compromised leading to QC evasion and hence elevated levels of nonsense suppression (c).

Figure 2

Loss of RF3 appears to make cells sensitive to perturbations that reduce the accuracy of protein synthesis. (A) A spotting assay of a dilution series showing that deletion of the prfC gene confers streptomycin-sensitivity. WT refers to the BW25113 strain, whereas ΔprfC refers to the JW5873 strain. (B) Growth curves of the indicated strains showing that the deletion of the prfC gene results in growth defects in an error-prone strain. WT refers to the Xac strain, rpsL refers to the hyperaccurate US157 strain and rpsD refers to the error-prone UD131 strain. See also Figures S1 and S2.

Figure 3

Post PT QC takes place in vivo and depends on RF3. (A) Schematic of reporter dimers (Rep-Dim) used for following premature termination as a function of asparagine starvation. (B) An autoradiograph of an SDS-PAGE gel of an S30 in vitro translation assay demonstrating that premature termination takes place during bona fide elongation and is stimulated by the addition of RF3. The ratio of the monomer to the dimer is indicated at the bottom of the gel. (C) An autoradiograph of a Tris-Tricine gel used to follow the status of in vivo pulse-labeled reporters. Constructs were transformed into the asparagine auxotroph JK463 (labeled WT), its ΔprfC derivative HZ001 (labeled ΔprfC), and a derivative of the latter carrying a copy of the prfC gene on a plasmid (labeled pBADprfC). The cells were pulse-labeled with [³⁵S]-methionine in the presence and absence of asparagine, and protein products affinity purified using the N-terminal HA-tag. (D) 2D-PAGE analysis of the prematurely terminated product from Rep-DimN indicates the substitution of a positively-charged amino acid (most likely lysine) in the product. fN^N represents a correctly decoded full-length product, fN^K represents a full-length product with a lysine substitution, tN^X represents a monomer-length product with no additional amino acids, tN^K indicates a monomer-length product with one lysine residue added, and tN^K* indicates monomer-length products containing additional positively charged residues. (E) Quantitative analysis of the 2D-gel in D. The fractional radioactivity corresponding to the indicated spot is plotted. (F) A coomassie-stained SDS-PAGE gel used to follow the purification of the indicated reporter protein products for mass-spec analysis. Ni-NTA lane denotes proteins that bound to the resin, while TEV-cleavage denotes the flow-through over Ni-NTA resin after TEV-cleavage of the His-tag. (G) MALDI-TOF mass spectra of the indicated sample in the presence or absence of asparagine. In the presence of asparagine, the Rep-DimN sample generated an m/z M⁺ peak (labeled fN^N(+))as well as a corresponding M²⁺ peak (labeled fN^N(2+)). In the absence of asparagine, a collection of peaks corresponding to masses slightly greater than the fN^N(2+) and stalled M⁺ monomer peak (tN^X(+)) were additionally observed. In contrast, the Rep-DimX sample generated almost identical spectra in the presence and absence of asparagine, with one prominent m/z peak (fX^X(+)). See also Figure S3.

Figure 4

Protein and mRNA levels are impacted by RF3 in a fidelity-dependent manner. (A) Western blot used to assess the expression of Rep-DimK in the indicated strains (WT, rpsL and rpsD here refer to the Xac, hyperaccurate US157 and error-prone UD131 strains, respectively). (B) Northern blot of the same samples as in A used to assess the level of the reporter transcripts in the different strains. (C) Northern blot used to determine the stability of mRNAs by measuring the levels of transcripts following the inhibition of transcription initiation using rifampicin (Rif). The half-lives, indicated at the bottom of the blots, were determined by plotting the relative amount of the transcript to the 23S rRNA and were fit to a single-exponential decay function. See also Figures S4 and S5.

Figure 5

Deletion of RF3 increases ribosome occupancy of the mRNAs in an error-prone background. (A) Polysome profile of the error-prone rpsD strain (UD131) and its prfC deletion derivative. (B) Northern blot of the same fractions follows the distribution of two endogenous transcripts, ompA and lrpA, across the gradient. (C) Western blot of fractions from the sucrose-density gradient in A used to follow the distribution of RF1 and RF2 across the gradient. See also Figure S6.

Figure 6

Deletion of RF3 leads to elevated levels of frameshifting. (A) Schematic of the reporter used to assess the levels of frameshifting with relevant elements labeled. The myc-tag is in the −1 frame, and as a result is only expressed when a +1 frameshift occurs. (B) Western blot using antibodies against the N-terminal His-tag and the out of frame C-terminal myc-tag. In all strains, deletion of prfC is accompanied by an increase in expression of the dimer. Conversely, over-expression of RF3 is accompanied by a decrease in expression of the dimer. See also Figure S7.

Figure 7

Schematic of the proposed model for RF3 action in maintaining high-fidelity protein synthesis. Under conditions where the accuracy of protein synthesis is relatively high, few mistakes occur during protein synthesis and translation continues until canonical termination takes place. Under conditions (such as starvation) where the accuracy of protein synthesis is compromised, more mistakes occur during protein synthesis, leading to the generation of mismatched complexes, to iterated miscoding, eventually resulting in recognition of the aberrant complexes by class I RFs and RF3.