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1.  Evolution and Diversification of the Organellar Release Factor Family 
Molecular Biology and Evolution  2012;29(11):3497-3512.
Translation termination is accomplished by proteins of the Class I release factor family (RF) that recognize stop codons and catalyze the ribosomal release of the newly synthesized peptide. Bacteria have two canonical RFs: RF1 recognizes UAA and UAG, RF2 recognizes UAA and UGA. Despite that these two release factor proteins are sufficient for de facto translation termination, the eukaryotic organellar RF protein family, which has evolved from bacterial release factors, has expanded considerably, comprising multiple subfamilies, most of which have not been functionally characterized or formally classified. Here, we integrate multiple sources of information to analyze the remarkable differentiation of the RF family among organelles. We document the origin, phylogenetic distribution and sequence structure features of the mitochondrial and plastidial release factors: mtRF1a, mtRF1, mtRF2a, mtRF2b, mtRF2c, ICT1, C12orf65, pRF1, and pRF2, and review published relevant experimental data. The canonical release factors (mtRF1a, mtRF2a, pRF1, and pRF2) and ICT1 are derived from bacterial ancestors, whereas the others have resulted from gene duplications of another release factor. These new RF family members have all lost one or more specific motifs relevant for bona fide release factor function but are mostly targeted to the same organelle as their ancestor. We also characterize the subset of canonical release factor proteins that bear nonclassical PxT/SPF tripeptide motifs and provide a molecular-model-based rationale for their retained ability to recognize stop codons. Finally, we analyze the coevolution of canonical RFs with the organellar genetic code. Although the RF presence in an organelle and its stop codon usage tend to coevolve, we find three taxa that encode an RF2 without using UGA stop codons, and one reverse scenario, where mamiellales green algae use UGA stop codons in their mitochondria without having a mitochondrial type RF2. For the latter, we put forward a “stop-codon reinvention” hypothesis that involves the retargeting of the plastid release factor to the mitochondrion.
PMCID: PMC3472500  PMID: 22688947
release factor; translation termination; mitochondrion; plastid; evolution; genetic code
2.  mtRF1a Is a Human Mitochondrial Translation Release Factor Decoding the Major Termination Codons UAA and UAG 
Molecular Cell  2007;27(5):745-757.
Human mitochondria contain their own genome, encoding 13 polypeptides that are synthesized within the organelle. The molecular processes that govern and facilitate this mitochondrial translation remain unclear. Many key factors have yet to be characterized—for example, those required for translation termination. All other systems have two classes of release factors that either promote codon-specific hydrolysis of peptidyl-tRNA (class I) or lack specificity but stimulate the dissociation of class I factors from the ribosome (class II). One human mitochondrial protein has been previously identified in silico as a putative member of the class I release factors. Although we could not confirm the function of this factor, we report the identification of a different mitochondrial protein, mtRF1a, that is capable in vitro and in vivo of terminating translation at UAA/UAG codons. Further, mtRF1a depletion in HeLa cells led to compromised growth in galactose and increased production of reactive oxygen species.
PMCID: PMC1976341  PMID: 17803939
3.  A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome 
The EMBO Journal  2010;29(6):1116-1125.
Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes.
PMCID: PMC2845271  PMID: 20186120
mitoribosomes; peptidyl-tRNA hydrolase; translation release factor
4.  Regulation of mtrF Expression in Neisseria gonorrhoeae and Its Role in High-Level Antimicrobial Resistance 
Journal of Bacteriology  2005;187(11):3713-3720.
The obligate human pathogen Neisseria gonorrhoeae uses the MtrC-MtrD-MtrE efflux pump to resist structurally diverse hydrophobic antimicrobial agents (HAs), some of which bathe mucosal surfaces that become infected during transmission of gonococci. Constitutive high-level HA resistance occurs by the loss of a repressor (MtrR) that negatively controls transcription of the mtrCDE operon. This high-level HA resistance also requires the product of the mtrF gene, which is located downstream and transcriptionally divergent from mtrCDE. MtrF is a putative inner membrane protein, but its role in HA resistance mediated by the MtrC-MtrD-MtrE efflux pump remains to be determined. High-level HA resistance can also be mediated through an induction process that requires enhanced transcription of mtrCDE when gonococci are grown in the presence of a sublethal concentration of Triton X-100. We now report that inactivation of mtrF results in a significant reduction in the induction of HA resistance and that the expression of mtrF is enhanced when gonococci are grown under inducing conditions. However, no effect was observed on the induction of mtrCDE expression in an MtrF-negative strain. The expression of mtrF was repressed by MtrR, the major repressor of mtrCDE expression. In addition to MtrR, another repressor (MpeR) can downregulate the expression of mtrF. Repression of mtrF by MtrR and MpeR was additive, demonstrating that the repressive effects mediated by these regulators are independent processes.
PMCID: PMC1112036  PMID: 15901695
5.  Two ABC Transporter Operons and the Antimicrobial Resistance Gene mtrF Are pilT Responsive in Neisseria gonorrhoeae▿  
Journal of Bacteriology  2007;189(14):5399-5402.
Retraction of type IV pili is mediated by PilT. We show that loss of pilT function leads to upregulation of mtrF (multiple transferable resistance) and two operons encoding putative ABC transporters in Neisseria gonorrhoeae MS11. This effect occurs indirectly through the transcriptional regulator FarR, which until now has been shown to regulate only farAB. l-Glutamine can reverse pilT downregulation of the ABC transporter operons and mtrF.
PMCID: PMC1951848  PMID: 17496077
6.  Unusual base pairing during the decoding of a stop codon by the ribosome 
Nature  2013;500(7460):107-110.
During normal translation, binding of a release factor to one of the three stop codons (UGA, UAA or UAG) results in termination of protein synthesis. However, modification of the initial uridine to a pseudouridine (Ψ) allows efficient recognition and read-through of these stop codons by a transfer RNA (tRNA), although it requires formation of two normally forbidden purine-purine base pairs1. We have determined the crystal structure at 3.1 Å resolution of the 30S ribosomal subunit in complex with the anticodon stem loop of tRNASer bound to the ΨAG stop codon in the A site. The ΨA base pair at the first position is accompanied by the formation of purine-purine base pairs at the second and third positions of the codon, which display an unusual Watson-Crick/Hoogsteen geometry. The structure shows a previously unsuspected ability of the ribosomal decoding center to accommodate non-canonical base pairs.
PMCID: PMC3732562  PMID: 23812587
7.  Common and specific amino acid residues in the prokaryotic polypeptide release factors RF1 and RF2: possible functional implications 
Nucleic Acids Research  2005;33(16):5226-5234.
Termination of protein synthesis is promoted in ribosomes by proper stop codon discrimination by class 1 polypeptide release factors (RFs). A large set of prokaryotic RFs differing in stop codon specificity, RF1 for UAG and UAA, and RF2 for UGA and UAA, was analyzed by means of a recently developed computational method allowing identification of the specificity-determining positions (SDPs) in families composed of proteins with similar but not identical function. Fifteen SDPs were identified within the RF1/2 superdomain II/IV known to be implicated in stop codon decoding. Three of these SDPs had particularly high scores. Five residues invariant for RF1 and RF2 [invariant amino acid residues (IRs)] were spatially clustered with the highest-scoring SDPs that in turn were located in two zones within the SDP/IR area. Zone 1 (domain II) included PxT and SPF motifs identified earlier by others as ‘discriminator tripeptides’. We suggest that IRs in this zone take part in the recognition of U, the first base of all stop codons. Zone 2 (domain IV) possessed two SDPs with the highest scores not identified earlier. Presumably, they also take part in stop codon binding and discrimination. Elucidation of potential functional role(s) of the newly identified SDP/IR zones requires further experiments.
PMCID: PMC1214553  PMID: 16162810
8.  Lack of peptide-release activity responding to codon UGA in Mycoplasma capricolum. 
Nucleic Acids Research  1993;21(6):1335-1338.
In Mycoplasma capricolum, a relative of Gram-positive eubacteria with a high genomic AT-content (75%), codon UGA is assigned to tryptophan instead of termination signal. Thus, in this bacterium the release factor 2 (RF-2), that recognizes UAA and UGA termination codons in eubacteria such as Escherichia coli and Bacillus subtilis, would be either specific to UAA or deleted. To test this, we have constructed a cell-free translation system using synthetic mRNA including codon UAA [mRNA(UAA)], UAG [mRNA(UAG)] and UGA [mRNA(UGA)] in-frame. In the absence of tryptophan, the translation of mRNA(UGA) ceased at UGA sites without appreciable release of the synthesized peptides from the ribosomes, whereas with mRNA(UAA) or mRNA(UAG) the bulk of the peptides was released. Upon addition of the E.coli S-100 fraction or B.subtilis S-100 fraction to the translation system, the synthesized peptides with mRNA(UGA) were almost completely released from the ribosomes, presumably because of the presence of RF-2 active to UGA in the added S-100 fraction. These data suggest that RF-2 is deleted or its activity to UGA is strongly weakened in M.capricolum.
PMCID: PMC309316  PMID: 8464722
9.  MpeR Regulates the mtr Efflux Locus in Neisseria gonorrhoeae and Modulates Antimicrobial Resistance by an Iron-Responsive Mechanism 
Previous studies have shown that the MpeR transcriptional regulator produced by Neisseria gonorrhoeae represses the expression of mtrF, which encodes a putative inner membrane protein (MtrF). MtrF works as an accessory protein with the Mtr efflux pump, helping gonococci to resist high levels of diverse hydrophobic antimicrobials. Regulation of mpeR has been reported to occur by an iron-dependent mechanism involving Fur (ferric uptake regulator). Collectively, these observations suggest the presence of an interconnected regulatory system in gonococci that modulates the expression of efflux pump protein-encoding genes in an iron-responsive manner. Herein, we describe this connection and report that levels of gonococcal resistance to a substrate of the mtrCDE-encoded efflux pump can be modulated by MpeR and the availability of free iron. Using microarray analysis, we found that the mtrR gene, which encodes a direct repressor (MtrR) of mtrCDE, is an MpeR-repressed determinant in the late logarithmic phase of growth when free iron levels would be reduced due to bacterial consumption. This repression was enhanced under conditions of iron limitation and resulted in increased expression of the mtrCDE efflux pump operon. Furthermore, as judged by DNA-binding analysis, MpeR-mediated repression of mtrR was direct. Collectively, our results indicate that both genetic and physiologic parameters (e.g., iron availability) can influence the expression of the mtr efflux system and modulate levels of gonococcal susceptibility to efflux pump substrates.
PMCID: PMC3294918  PMID: 22214775
10.  Distinct eRF3 Requirements Suggest Alternate eRF1 Conformations Mediate Peptide Release During Eukaryotic Translation Termination 
Molecular cell  2008;30(5):599-609.
Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, while variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.
PMCID: PMC2475577  PMID: 18538658
11.  Decoding accuracy in eRF1 mutants and its correlation with pleiotropic quantitative traits in yeast 
Nucleic Acids Research  2010;38(16):5479-5492.
Translation termination in eukaryotes typically requires the decoding of one of three stop codons UAA, UAG or UGA by the eukaryotic release factor eRF1. The molecular mechanisms that allow eRF1 to decode either A or G in the second nucleotide, but to exclude UGG as a stop codon, are currently not well understood. Several models of stop codon recognition have been developed on the basis of evidence from mutagenesis studies, as well as studies on the evolutionary sequence conservation of eRF1. We show here that point mutants of Saccharomyces cerevisiae eRF1 display significant variability in their stop codon read-through phenotypes depending on the background genotype of the strain used, and that evolutionary conservation of amino acids in eRF1 is only a poor indicator of the functional importance of individual residues in translation termination. We further show that many phenotypes associated with eRF1 mutants are quantitatively unlinked with translation termination defects, suggesting that the evolutionary history of eRF1 was shaped by a complex set of molecular functions in addition to translation termination. We reassess current models of stop-codon recognition by eRF1 in the light of these new data.
PMCID: PMC2938225  PMID: 20444877
12.  Histidine 197 in Release Factor 1 is Essential for A Site Binding and Peptide Release 
Biochemistry  2010;49(43):9385-9390.
Class I peptide release factors 1 and 2 (RF1 and RF2) recognize the stop codons in the ribosomal decoding center and catalyze peptidyl-tRNA hydrolysis. High-fidelity stop codon recognition by these release factors is essential for accurate peptide synthesis and ribosome recycling. X-ray crystal structures of RF1 and RF2 bound to the ribosome have identified residues in the mRNA-protein interface that appear critical for stop codon recognition. Especially interesting is a conserved histidine in all bacterial class I release factors that forms a stacking interaction with the second base of the stop codon. Here we analyzed the functional significance of this conserved histidine (197 in E. coli) of RF1 by point mutagenesis to alanine. Equilibrium binding studies and transient-state kinetic analysis have shown that the histidine is essential for binding with high affinity to the ribosome. Furthermore, analysis of the binding data indicates a conformational change within the RF1•ribosome complex that results in a more tightly bound state. The rate of peptidyl-tRNA hydrolysis was also reduced significantly, more than the binding data would suggest, implying a defect in the orientation of the GGQ domain without the histidine residue.
PMCID: PMC2967428  PMID: 20873815
Ribosome; release factor; stop codon; translation; termination
13.  Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1 
eLife  2013;2:e00411.
Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.
eLife digest
Ribosomes are complex molecular machines that join amino acids together to form proteins. The order of amino acids in the protein is specified by a strand of messenger RNA (mRNA), and the process of decoding the mRNA into a string of amino acids is called translation. A ribosome consists of two subunits—one large, one small—that come together at a particular site on the mRNA strand called the translation initiation site. The ribosome then moves along the mRNA—joining together amino acids brought to it by transfer RNA (tRNA)—until it reaches a termination site and releases the protein.
The ribosome has three sites; the first amino acid to be delivered by a tRNA molecule to the ribosome occupies the site in the middle—also called the P site—and the second amino acid is delivered to the A site. Once the first two amino acids have been joined together, the ribosome moves along the mRNA so that the first amino acid now occupies the third site, called the E or exit site, and the second amino acid occupies the P site, leaving the A site vacant. The third amino acid is then delivered to the A site, and the whole process repeats itself until the ribosome reaches the termination site. Proteins called release factors are responsible for terminating the translation process and releasing the translated string of amino acids, which folds to form a protein. In bacteria this task can by performed by two releases factors, known as RF1 and RF2. However, the release factor must itself be released to leave the ribosome free to translate another strand of mRNA.
Pallesen et al. have used cryo-electron microscopy (cryo-EM) to study how a third release factor, RF3, helps to release RF1 from the ribosome in bacteria. In cells, RF3 usually forms a complex with a molecule called GDP, and the cryo-EM studies show that this molecule is released shortly after the RF3•GDP complex enters the ribosome. Once inside the ribosome, RF3 comes into contact with RF1 and with a protein called L12 that is part of the ribosome. A molecule called GTP—which is well known as a source of energy within cells—then binds to RF3, and this causes the shape of the ribosome to change. This change of shape results in the release of RF1 and the formation of a new RF3•GDP complex, which then leaves the ribosome.
Further work is needed to fully understand the role of L12 in these events, but a detailed understanding of the mechanism for terminating the translation of mRNA by the ribosome is coming into view.
PMCID: PMC3677378  PMID: 23755360
Ribosome; Cryo-EM; Structure; RF1; RF3; L7/L12; E. coli
14.  Class I release factors in ciliates with variant genetic codes 
Nucleic Acids Research  2001;29(4):921-927.
In eukaryotes with the universal genetic code a single class I release factor (eRF1) most probably recognizes all stop codons (UAA, UAG and UGA) and is essential for termination of nascent peptide synthesis. It is well established that stop codons have been reassigned to amino acid codons at least three times among ciliates. The codon specificities of ciliate eRF1s must have been modified to accommodate the variant codes. In this study we have amplified, cloned and sequenced eRF1 genes of two hypotrichous ciliates, Oxytricha trifallax (UAA and UAG for Gln) and Euplotes aediculatus (UGA for Cys). We also sequenced/identified three protist and two archaeal class I RF genes to enlarge the database of eRF1/aRF1s with the universal code. Extensive comparisons between universal code eRF1s and those of Oxytricha, Euplotes and Tetrahymena, which represent three lineages that acquired variant codes independently, provide important clues to identify stop codon-binding regions in eRF1. Domain 1 in the five ciliate eRF1s, particulary the TASNIKS heptapeptide and its adjacent region, differs significantly from domain 1 in universal code eRF1s. This observation suggests that domain 1 contains the codon recognition site, but that the mechanism of eRF1 codon recognition may be more complex than proposed by Nakamura et al. or Knight and Landweber.
PMCID: PMC29606  PMID: 11160924
15.  Identification of residues required for stalled-ribosome rescue in the codon-independent release factor YaeJ 
Nucleic Acids Research  2013;42(5):3152-3163.
The YaeJ protein is a codon-independent release factor with peptidyl-tRNA hydrolysis (PTH) activity, and functions as a stalled-ribosome rescue factor in Escherichia coli. To identify residues required for YaeJ function, we performed mutational analysis for in vitro PTH activity towards rescue of ribosomes stalled on a non-stop mRNA, and for ribosome-binding efficiency. We focused on residues conserved among bacterial YaeJ proteins. Additionally, we determined the solution structure of the GGQ domain of YaeJ from E. coli using nuclear magnetic resonance spectroscopy. YaeJ and a human homolog, ICT1, had similar levels of PTH activity, despite various differences in sequence and structure. While no YaeJ-specific residues important for PTH activity occur in the structured GGQ domain, Arg118, Leu119, Lys122, Lys129 and Arg132 in the following C-terminal extension were required for PTH activity. All of these residues are completely conserved among bacteria. The equivalent residues were also found in the C-terminal extension of ICT1, allowing an appropriate sequence alignment between YaeJ and ICT1 proteins from various species. Single amino acid substitutions for each of these residues significantly decreased ribosome-binding efficiency. These biochemical findings provide clues to understanding how YaeJ enters the A-site of stalled ribosomes.
PMCID: PMC3950681  PMID: 24322300
16.  GTP Hydrolysis by eRF3 Facilitates Stop Codon Decoding during Eukaryotic Translation Termination 
Molecular and Cellular Biology  2004;24(17):7769-7778.
Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.
PMCID: PMC506980  PMID: 15314182
17.  RF1 Knockout Allows Ribosomal Incorporation of Unnatural Amino Acids at Multiple Sites 
Nature Chemical Biology  2011;7(11):779-786.
Stop codons have been exploited for genetic incorporation of unnatural amino acids (Uaas) in live cells, but the efficiency is low possibly due to competition from release factors, limiting the power and scope of this technology. Here we show that the reportedly essential release factor 1 can be knocked out from Escherichia coli by fixing release factor 2. The resultant strain JX33 is stable and independent, and reassigns UAG from a stop signal to an amino acid when a UAG-decoding tRNA/synthetase pair is introduced. Uaas were efficiently incorporated at multiple UAG sites in the same gene without translational termination in JX33. We also found that amino acid incorporation at endogenous UAG codons is dependent on RF1 and mRNA context, which explains why E. coli tolerates apparent global suppression of UAG. JX33 affords a unique autonomous host for synthesizing and evolving novel protein functions by enabling Uaa incorporation at multiple sites.
PMCID: PMC3201715  PMID: 21926996
18.  Interactions between peptidyl tRNA hydrolase homologs and the ribosomal release factor Mrf1 in S. pombe mitochondria 
Mitochondrion  2013;13(6):871-880.
Mitochondrial translation synthesizes key subunits of the respiratory complexes. In Schizosaccharomyces pombe, strains lacking Mrf1, the mitochondrial stop codon recognition factor, are viable, suggesting that other factors can play a role in translation termination. S. pombe contains four predicted peptidyl tRNA hydrolases, two of which (Pth3 and Pth4), have a GGQ motif that is conserved in class I release factors. We show that high dosage of Pth4 can compensate for the absence of Mrf1 and loss of Pth4 exacerbates the lack of Mrf1. Also Pth4 is a component of the mitochondrial ribosome, suggesting that it could help recycling stalled ribosomes.
•In S. pombe the peptidyl tRNA hydrolases Pth3 and Pth4 are mitochondrial proteins.•Pth3 and Pth4 are associated with the mitochondrial ribosome and the large subunit.•Deletion of pth4 and mrf1, encoding the mitochondrial release factor, is co-lethal.•Over-expression of pth4 compensates for the deletion of mrf1.•Pth4 can act as a release factor in S. pombe mitochondria.
PMCID: PMC3919214  PMID: 23892058
Fission yeast; Mitochondria; Translation; Peptidyl tRNA hydrolase; Release factor; Mitochondrial ribosome tagging
19.  Development of K562 cell clones expressing β-globin mRNA carrying the β039 thalassaemia mutation for the screening of correctors of stop-codon mutations 
Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30 % of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in β039-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon–anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of β039-thalassaemia. In this context, we started the development of a cellular model of the β039-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the β039-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of β-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the β039-globin mutation causing β-thalassaemia.
PMCID: PMC3582994  PMID: 19216718
aminoglycoside antibiotics; K562 cell; locus control region; nonsense mutation; thalassaemia
20.  Insights into translational termination from the structure of RF2 bound to the ribosome 
Science (New York, N.Y.)  2008;322(5903):953-956.
The termination of protein synthesis occurs through specific recognition of a stop codon in the A site of the ribosome by a release factor (RF), which then catalyzes the hydrolysis of the nascent protein chain from the P-site tRNA. Here we present the crystal structure at 3.5 Å resolution of release factor RF2 in complex with its cognate UGA stop codon in the 70S ribosome. The structure provides insight into how RF2 specifically recognizes the stop codon and suggests a model for the role of a universally conserved GGQ motif in catalysis of peptide release.
PMCID: PMC2642913  PMID: 18988853
21.  Versatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plants 
PLoS ONE  2009;4(10):e7354.
Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA) in an mRNA. However, many viruses (and some cellular mRNAs) contain “stop” codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a “readthrough”, or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context.
We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP.
We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a ‘leaky’ UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV) readthrough context.
PMCID: PMC2754532  PMID: 19816579
22.  Ribosomes containing the C1054-deletion mutation in E. coli 16S rRNA act as suppressors at all three nonsense codons. 
Nucleic Acids Research  1991;19(19):5281-5283.
It was established some time ago that the deletion of base C1054 in E. coli 16S rRNA specifically affects UGA-dependent termination of translation. Based on this observation, a model for the termination event was proposed in which the UGA nonsense codon on the mRNA base-pairs with a complementary motif in 'helix 34' of the 16S rRNA, thus potentially providing a recognition signal for the binding of the release factor. This model has been re-examined here and evidence is presented which demonstrates that ribosomes containing the C1054 delta mutation enhance the activity of suppressors of both UAG and UAA termination codons introduced into the host. The results do not support the nonsense codon-16S rRNA base pairing model, and rather imply a more general involvement of 'helix 34' in the translation termination reactions.
PMCID: PMC328888  PMID: 1923812
23.  Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae 
Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl – dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough.
We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p.
The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.
PMCID: PMC56590  PMID: 11570975
24.  A ribosomal ambiguity mutation in the 530 loop of E. coli 16S rRNA. 
Nucleic Acids Research  1992;20(16):4221-4227.
A series of base substitution and deletion mutations were constructed in the highly conserved 530 stem and loop region of E. coli 16S rRNA involved in binding of tRNA to the ribosomal A site. Base substitution and deletion of G517 produced significant effects on cell growth rate and translational fidelity, permitting readthrough of UGA, UAG and UAA stop codons as well as stimulating +1 and -1 frameshifting in vivo. By contrast, mutations at position 534 had little or no effect on growth rate or translational fidelity. The results demonstrate the importance of G517 in maintaining translational fidelity but do not support a base pairing interaction between G517 and U534.
PMCID: PMC334129  PMID: 1380697
25.  Characterization of mitochondrial mRNAs in codfish reveals unique features compared to mammals 
Current Genetics  2011;57(3):213-222.
Expression and processing of mitochondrial gene transcripts are fundamental to mitochondrial function, but information from early vertebrates like teleost fishes is essentially lacking. We have analyzed mitogenome sequences of ten codfishes (family Gadidae), and provide complete sequences from three new species (Saithe, Pollack and Blue whiting). Characterization of the mitochondrial mRNAs in Saithe and Atlantic cod identified a set of ten poly(A) transcripts, and six UAA stop codons are generated by posttranscriptional polyadenylation. Structural assessment of poly(A) sites is consistent with an RNaseP cleavage activity 5′ of tRNA acceptor-like stems. COI, ND5 and ND6 mRNAs were found to harbor 3′ UTRs with antisense potential extending into neighboring gene regions. While the 3′ UTR of COI mRNA is complementary to the tRNASer (UCN) and highly similar to that detected in human mitochondria, the ND5 and ND6 3′ UTRs appear more heterogenic. Deep sequencing confirms expression of all mitochondrial mRNAs and rRNAs, and provides information about the precise 5′ ends in mature transcripts. Our study supports an overall evolutionary conservation in mitochondrial RNA processing events among vertebrates, but reveals some unique 5′ and 3′ end characteristics in codfish mRNAs with implications to antisense regulation of gene expression.
Electronic supplementary material
The online version of this article (doi:10.1007/s00294-011-0338-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3097352  PMID: 21484258
Antisense regulation; Deep sequencing; Gadidae; Mitochondrial RNA; Mitogenome; Polyadenylation

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