Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons - PubMed (original) (raw)

Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons

K Ito et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Prokaryotic translational release factors, RF1 and RF2, catalyze polypeptide release at UAG/UAA and UGA/UAA stop codons, respectively. In this study, we isolated a bacterial RF2 mutant (RF2*) containing an E167K substitution that restored the growth of a temperature-sensitive RF1 strain of Escherichia coli and the viability of a chromosomal RF1/RF2 double knockout. In both in vivo and in vitro polypeptide termination assays, RF2* catalyzed UAG/UAA termination, as does RF1, as well as UGA termination, showing that RF2* acquired omnipotent release activity. This result suggests that the E167K mutation abolished the putative third-base discriminator function of RF2. These findings are interpreted as indicating that prokaryotic and eukaryotic release factors share the same anticodon moiety and that only one omnipotent release factor is sufficient for bacterial growth, similar to the eukaryotic single omnipotent factor.

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Figures

Figure 1

Bacterial RF2 mutant that substitutes for RF1 function. (A) Preference in stop codon recognition by RFs and rationale of RF2* selection. The plasmid-bearing RF2 gene was mutagenized in vitro, and the RF2* allele was isolated among temperature-resistant survivors of the Ts RF1 (prfA1) strain on transformation as described in Materials and Methods. Shaded triangles mean the loss of function by Ts or knockout mutations. It is assumed that RF1 and RF2 encode “second- and third-base discriminator” functions, respectively, and this paper provides genetic evidence for the existence of the third-base discriminator function in RF2. (B) Amino acid sequence around the mutation site (E167K) of RF2* and effects of relevant mutations on RF function. Substitutions generated by site-directed mutagenesis by using designed primers coding for the substitutions are indicated below the wild-type sequence of RF1 and RF2. These altered protein genes were cloned into plasmid pSUIQ and transformed and expressed in the Ts RF1 (prfA1) and RF2 (prfB286) strains, and the transformant growth was examined at 42°C on LB agar plates containing 1 mM IPTG (complementation assay). “normal” means that the mutation did not reduce or alter the activity to complement the relevant Ts allele, and “loss of function” means that the mutation abolished the activity.

Figure 2

Replacement of RF1 and RF2 function with RF2* by chromosomal gene disruption. (A) Disruption of RF1 (prfA) and RF2 (prfB) genes by KmR and CmR cassettes, respectively, on the E. coli chromosome. RF2* was cloned in plasmid pSUIQT so as to be expressed by the addition of IPTG. (B) Viability of pSUIQT-RF2* transformants of RF1 knockout (ΔA, _prfA_∷KmR), RF2 knockout (ΔB, _prfB_∷CmR), and RF1/RF2 double knockout (ΔAB, _prfA_∷KmR _prfB_∷CmR) strains. Cells were grown at 37°C on LB agar plates containing different IPTG concentrations as indicated. Cell viability can be judged by single colony formability, not by appearance of a cell zone (caused by cell density effect carrying over some IPTG) at the top of the cell suspension streak. (C) Western immunoblot analysis of the level of RF2* required for suppression of prfA and/or prfB knockout strains. Equal amounts (10 μg of bulk protein) of cell lysates were analyzed by SDS/PAGE and subjected to immunoblot staining with anti-RF1 (Top) and anti-RF2 (Bottom) antibodies. Lanes: 1, E. coli W3110 (nontransformant control); 2, pSUIQT-RF2* transformant of _prfA_∷KmR disruptant (0.04 mM IPTG); 3, pSUIQT-RF2* transformant of _prfB_∷CmR disruptant (0.01 mM IPTG); and 4, pSUIQT-RF2* transformant of _prfA_∷KmR _prfB_∷CmR double disruptant (0.04 mM IPTG). Lanes 2–4 represent RF2* synthesized with the minimal concentrations of IPTG sufficient to restore viability of prfA and/or prfB knockout strains. Relative intensities of immunoblots compared with that of lane 1 are shown in parentheses.

Figure 3

Polypeptide release activity of RF2* at a UAG codon. (A) in vitro fMet release assay with histidine-tagged RF2 and RF2*. f[3H]Met release from the [f[3H]Met-tRNAf⋅AUG⋅ribosome] complex on addition of RFs and terminator triplets was determined (21). Reactions contained 20 μM UAG (Left), UAA (Center), and UGA (Right), as well as equal molar amounts (50 pmol) of RF proteins. The relative fMet-release activity of RF2* to RF1 at UAG is approximately one-fourth or one-fifth under these experimental conditions (data not shown). (B) The 3A′ reporter gene construct (pAB96) for UAG readthrough assay (15, 16). (C) Influence on UAG readthrough of expression of various RF constructs. Readthrough (RT) values, i.e., molar amounts of 3A′ domain protein (translation readthrough) relative to 2A′ domain protein (translation termination), were measured as described (13, 15, 16). Bars: 1, pSUIQ (vector control); 2, pSUIQT-RF1; 3, pSUIQT-RF2; 4, pSUIQT-RF2*. Experiments were performed independently at least five times in A and C, and the values are expressed with SDs.

Figure 4

Comparison of the amino acid sequences of prokaryotic RF1 and RF2. The similarity alignments of RFs were accomplished as described (7). Relevant sequences around positions 157 (general enhancer allele; in shade) and 167 (omnipotent allele; in black) are shown.

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Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons - PubMed (original) (raw)