Recognition of the amber UAG stop codon by release factor RF1

We report the crystal structure of a termination complex containing release factor RF1 bound to the 70S ribosome in response to an amber (UAG) codon at 3.6‐Å resolution. The amber codon is recognized in the 30S subunit‐decoding centre directly by conserved elements of domain 2 of RF1, including T186 of the PVT motif. Together with earlier structures, the mechanisms of recognition of all three stop codons by release factors RF1 and RF2 can now be described. Our structure confirms that the backbone amide of Q230 of the universally conserved GGQ motif is positioned to contribute directly to the catalysis of the peptidyl‐tRNA hydrolysis reaction through stabilization of the leaving group and/or transition state. We also observe synthetic‐negative interactions between mutations in the switch loop of RF1 and in helix 69 of 23S rRNA, revealing that these structural features interact functionally in the termination process. These findings are consistent with our proposal that structural rearrangements of RF1 and RF2 are critical to accurate translation termination.     

The signal for a leaky UAG stop codon in several plant viruses includes the two downstream codons

Expression of the RNA replicase domain of tobacco mosaic virus (TMV) and certain protein-coding regions in other plant viruses, is mediated by translational readthrough of a leaky UAG stop codon. It has been proposed that normal tobacco tyrosine tRNAs are able to read the UAG codon of TMV by non-conventional base-pairing but recent findings that stop codons can also be bypassed as a result of extended translocational shifts (tRNA hopping) have encouraged a re-examination. In light of the alternatives, we investigated the sequences flanking the leaky UAG codon using an in vivo assay in which bypass of the stop codon is coupled to the transient expression of beta-glucuronidase (GUS) reporter genes in tobacco protoplasts. Analysis of GUS constructions in which codons flanking the stop were altered allowed definition of the minimal sequence required for read through as UAG-CAA-UUA. The effects of all possible single-base mutations in the codons flanking the stop indicated that 3' contexts of the form CAR-YYA confer leakiness and that the 3' context permits read through of UAA and UGA stop codons as well as UAG. Our studies demonstrate a major role for the 3' context in the read through process and do not support a model in which teh UAG is bypassed exclusively as a result of anticodon-codon interactions. No evidence for tRNA hopping was obtained. The 3' context apparently represents a unique sequence element that affects translation termination.     

The crystal structure of human eukaryotic release factor eRF1--mechanism of stop codon recognition and peptidyl-tRNA hydrolysis

The release factor eRF1 terminates protein biosynthesis by recognizing stop codons at the A site of the ribosome and stimulating peptidyl-tRNA bond hydrolysis at the peptidyl transferase center. The crystal structure of human eRF1 to 2.8 A resolution, combined with mutagenesis analyses of the universal GGQ motif, reveals the molecular mechanism of release factor activity. The overall shape and dimensions of eRF1 resemble a tRNA molecule with domains 1, 2, and 3 of eRF1 corresponding to the anticodon loop, aminoacyl acceptor stem, and T stem of a tRNA molecule, respectively. The position of the essential GGQ motif at an exposed tip of domain 2 suggests that the Gln residue coordinates a water molecule to mediate the hydrolytic activity at the peptidyl transferase center. A conserved groove on domain 1, 80 A from the GGQ motif, is proposed to form the codon recognition site.     

UAG readthrough in mammalian cells: Effect of upstream and downstream stop codon contexts reveal different signals

Background  

Translation termination is mediated through an interaction between the release factors eRF1 and eRF3 and the stop codon within its nucleotide context. Although it is well known that the nucleotide contexts both upstream and downstream of the stop codon, can modulate readthrough, little is known about the mechanisms involved.     

Initiation of translation at a UAG stop codon in the aldolase gene of Plasmodium falciparum.

The gene coding for the key glycolytic enzyme fructose-1,6-diphosphate aldolase of the human malaria parasite Plasmodium falciparum lacks a functional AUG initiation codon for translation. Protein sequences of natural or in vitro translated aldolase include the candidate start methionine residue at internal positions. No additional AUG start codon is found in genomic DNA, cDNA or mRNA sequences. Instead, a UAG chain termination codon is recognized as the start signal of protein synthesis in vivo and in vitro.     

Context effects: translation of UAG codon by suppressor tRNA is affected by the sequence following UAG in the message

The efficiency of various suppressor tRNAs in reading the UAG amber codon has been measured at 42 sites in the lacI gene. Results indicate that: (1) for all suppressors, efficiency is not an a priori value; rather, it is determined at each site by the specific reading context of the suppressed codon; (2) the degree of sensitivity to context effects differs among suppressors. Most affected is amber suppressor supE (su2), whose activity varies over a 20-fold range depending on context; (3) context effects are produced by residues present at the 3' side of the UAG codon. The most important role appears to be played by the base that is immediately adjacent to the codon. When this base is a purine, the amber codon is suppressed more efficiently than when a pyrimidine is in the same position. Superimposed on this initial pattern, the influence of bases further downstream to the UAG triplet can be detected also. The possibility is discussed that context effects are produced by the whole codon following UAG in the message.     

Translational termination in Escherichia coli: three bases following the stop codon crosslink to release factor 2 and affect the decoding efficiency of UGA-containing signals.

The observations that the Escherichia coli release factor 2 (RF2) crosslinks with the base following the stop codon (+4 N), and that the identity of this base strongly influences the decoding efficiency of stop signals, stimulated us to determine whether there was a more extended termination signal for RF2 recognition. Analysis of the 3' contexts of the 1248 genes in the E.coli genome terminating with UGA showed a strong bias for U in the +4 position and a general bias for A and against C in most positions to +10, consistent with the concept of an extended sequence element. Site-directed crosslinking occurred to RF2 from a thio-U sited at the +4, +5 and +6 bases following the UGA stop codon but not beyond (+7 to +10). Varying the +4 to +6 bases modulated the strength of the crosslink from the +1 invariant U to RF2. A strong selection bias for particular bases in the +4 to +6 positions of certain E. coli UGANNN termination sites correlated in some cases with crosslinking efficiency to RF2 and in vivo termination signal strength. These data suggest that RF2 may recognise at least a hexanucleotide UGA-containing sequence and that particular base combinations within this sequence influence termination signal decoding efficiency.     

mtRF1a is a human mitochondrial translation release factor decoding the major termination codons UAA and UAG

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.     

The nucleic acid-binding zinc finger protein of potato virus M is translated by internal initiation as well as by ribosomal frameshifting involving a shifty stop codon and a novel mechanism of P-site slippage.

The genes for the capsid protein CP and the nucleic acid-binding 12K protein (pr12) of potato virus M (PVM) constitute the 3' terminal gene cluster of the PVM RNA genome. Both proteins are presumably translated from a single subgenomic RNA. We have identified two translational strategies operating in pr12 gene expression. Internal initiation at the first and the second AUG codon of the pr12 coding sequence results in the synthesis of the 12K protein. In addition the protein is produced as a CP/12K transframe protein by ribosomal frameshifting. For these studies parts of the CP and pr12 coding sequences including the putative frameshift region were introduced into an internal position of the beta-glucuronidase gene. Mutational analyses in conjunction with in vitro translation experiments identified a homopolymeric string of four adenosine nucleotides which together with a 3' flanking UGA stop codon were required for efficient frameshifting. The signal AAAAUGA is the first frameshift signal with a shifty stop codon to be analyzed in the eukaryotic system. Substitution of the four consecutive adenosine nucleotides by UUUU increased the efficiency of frameshifting, while substitution by GGGG or CCCC dramatically reduced the synthesis of the transframe protein. Also, UAA and UAG could replace the opal stop codon without effect on the frameshifting event, but mutation of UGA to the sense codon UGG inhibited transframe protein formation. These findings suggest that the mechanism of ribosomal frameshifting at the PVM signal is different from the one described by the 'simultaneous slippage' model in that only the string of four adenosine nucleotides represents the slippery sequence involved in a -1 P-site slippage.     

The properties of chimeric picornavirus IRESes show that discrimination between internal translation initiation sites is influenced by the identity of the IRES and not just the context of the AUG codon.

The internal ribosome entry segment (IRES) of picornaviruses consists of approximately 450 nt of 5'-untranslated region, terminating at the 3' end with an approximately 25 nt element consisting of an absolutely conserved UUUC motif followed by a more variable pyrimidine-rich tract and G-poor spacer, and finally an AUG triplet, which is considered to be the actual ribosome entry site. Events following entry at this site differ among picornaviruses: in encephalomyocarditis virus (EMCV) virtually all ribosomes initiate translation at this site (AUG-11); in foot-and-mouth-disease virus (FMDV), one-third of the ribosomes initiate at this AUG (the Lab site), and the rest at the next AUG 84 nt downstream (Lb site); and in poliovirus (PV), the AUG at the 3' end of the IRES (at nt 586 in PV type 1) is considered to be a silent entry site, with all ribosomes initiating translation at the next AUG downstream (nt 743). To investigate what determines this different behavior, chimeras were constructed with a crossover at the conserved UUUC motif: the body of the IRES, the sequences upstream of this UUUC motif, was derived from one species, and the downstream sequences from another. When the body of the FMDV or PV IRESes was replaced by that of EMCV, there was a marked increase in the absolute and relative frequency of initiation at the upstream AUG, the Lab site of FMDV and 586AUG of PV, respectively. In contrast, when the body of the EMCV IRES was replaced by that of PV, initiation occurred with no preference at three AUGs: the normal site (AUG-11), AUG-10 situated 8 nt upstream, and AUG-12, which is 12 nt downstream. Thus although the context of the AUG at the 3' end of the IRES may influence initiation frequency at this site, as was shown by improving the context of 586AUG of PV, the behavior of the ribosome is also highly dependent on the nature of the upstream IRES. Delivery of the ribosome to this AUG in an initiation-competent manner is particularly efficient and accurate with the EMCV IRES.     

Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences.

In a previous report, we described the presence, in pituitary tissue, of an alternatively processed species of bovine growth hormone mRNA from which the last intron (intron D) has not been removed by splicing (R. K. Hampson and F. M. Rottman, Proc. Natl. Acad. Sci. USA 84:2673-2677, 1987). Using transient expression of the bovine growth hormone gene in Cos I cells, we observed that splicing of intron D was affected by sequences within the downstream exon (exon 5). Deletion of a 115-base-pair FspI-PvuII restriction fragment in exon 5 beginning 73 base pairs downstream of the intron 4-exon 5 junction resulted in cytoplasmic bovine growth hormone mRNA, more than 95% of which retained intron D. This contrasted with less than 5% of the growth hormone mRNA retaining intron D observed with expression of the unaltered gene. Insertion of a 10-base-pair inverted repeat sequence, CTTCCGGAAG, which was located in the middle of this deleted segment, partially reversed this pattern, resulting in cytosolic mRNA from which intron D was predominantly removed. More detailed deletion analysis of this region indicated that multiple sequence elements within the exon 5, in addition to the 10-base-pair inverted repeat sequence, are capable of influencing splicing of intron D. The effect of these exon sequences on splicing of bovine growth hormone precursor mRNA appeared to be specific for the growth hormone intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. Deletions in exon 5 which resulted in marked alterations in splicing of growth hormone intron D had no effect on splicing when exon 5 of bovine growth hormone was placed downstream of the heterologous bovine prolactin intron D. The results of this study suggest a unique interaction between sequences located near the center of exon 5 and splicing of the adjacent intron D.     

Adenine and guanine recognition of stop codon is mediated by different N domain conformations of translation termination factor eRF1

Positioning of release factor eRF1 toward adenines and the ribose-phosphate backbone of the UAAA stop signal in the ribosomal decoding site was studied using messenger RNA (mRNA) analogs containing stop signal UAA/UAAA and a photoactivatable cross-linker at definite locations. The human eRF1 peptides cross-linked to these analogs were identified. Cross-linkers on the adenines at the 2nd, 3rd or 4th position modified eRF1 near the conserved YxCxxxF loop (positions 125-131 in the N domain), but cross-linker at the 4th position mainly modified the tripeptide 26-AAR-28. This tripeptide cross-linked also with derivatized 3'-phosphate of UAA, while the same cross-linker at the 3'-phosphate of UAAA modified both the 26-28 and 67-73 fragments. A comparison of the results with those obtained earlier with mRNA analogs bearing a similar cross-linker at the guanines indicates that positioning of eRF1 toward adenines and guanines of stop signals in the 80S termination complex is different. Molecular modeling of eRF1 in the 80S termination complex showed that eRF1 fragments neighboring guanines and adenines of stop signals are compatible with different N domain conformations of eRF1. These conformations vary by positioning of stop signal purines toward the universally conserved dipeptide 31-GT-32, which neighbors guanines but is oriented more distantly from adenines.     

Accumulation of a mRNA decay intermediate by ribosomal pausing at a stop codon.

A RNA fragment which is protected from degradation by ribosome pausing at a stop codon has been identified in growing Escherichia coli. The fragment is 261 nt long and corresponds to the 3'-end of the mRNA expressed from a semi-synthetic model gene. The 5'-end of the RNA fragment, denoted rpRNA (ribosomal pause RNA), is located 13 bases upstream of the stop codon. In vivo decay of the complete mRNA and accumulation of rpRNA are dependent on the nature of the stop codon and its codon context. The data indicate that the rpRNA fragment arises from interrupted decay of the S3A'mRNA in the 5'-->m3'direction, in connection with a ribosomal pause at the stop codon. RF-2 decoding of UGA is less efficient than RF-1 decoding of UAG in identical codon contexts, as judged from rpRNA steady-state levels. The half-life of UGA-containing rpRNAs is at least 5 min, indicating that ribosomal pausing can be a major factor in stabilising downstream regions of messenger RNAs.     

Evidence that UGA is read as a tryptophan codon rather than as a stop codon by Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma gallisepticum.

Molecular cloning and sequencing showed that Mycoplasma gallisepticum, like Mycoplasma capricolum, contains both tRNA(UCA) and tRNA(CCA) genes, while Mycoplasma pneumoniae and Mycoplasma genitalium each appear to have only a tRNA(UCA) gene. Therefore, these mycoplasma species contain a tRNA with the anticodon UCA that can translate both UGA and UGG codons.     

Structure and dynamics in solution of the stop codon decoding N-terminal domain of the human polypeptide chain release factor eRF1

The high-resolution NMR structure of the N-domain of human eRF1, responsible for stop codon recognition, has been determined in solution. The overall fold of the protein is the same as that found in the crystal structure. However, the structures of several loops, including those participating in stop codon decoding, are different. Analysis of the NMR relaxation data reveals that most of the regions with the highest structural discrepancy between the solution and solid states undergo internal motions on the ps-ns and ms time scales. The NMR data show that the N-domain of human eRF1 exists in two conformational states. The distribution of the residues having the largest chemical shift differences between the two forms indicates that helices α2 and α3, with the NIKS loop between them, can switch their orientation relative to the β-core of the protein. Such structural plasticity may be essential for stop codon recognition by human eRF1.     

Decoding the translational termination signal: the polypeptide chain release factor in Escherichia coli crosslinks to the base following the stop codon.

Protein release factors act like tRNA analogues in decoding translational stop signals. Statistical analysis of the sequences at translational stop sites and functional studies with particular signals indicate this mimicry involves an increase in the length of the signal in the mRNA. The base following the stop codon (+4 base) is of particular interest because it has a strong influence on the competitiveness of the stop signal at recoding sites, suggesting it might form part of the release factor recognition element. Site-directed crosslinking from the +4 base showed that it is in close proximity to the Escherichia coli release factor-2 in a termination complex, a prerequisite for the +4 base being part of the recognition element. Fingerprinting analysis indicates that crosslinking to the release factor occurred from both +1 and +4 positions of the stop signal in the same RNA molecule. This provides more evidence that the +4 base may be an integral part of the decoding signature in the mRNA during the termination phase of protein biosynthesis.     

A mechanism for stop codon recognition by the ribosome: a bioinformatic approach.

Protein synthesis in ribosomes requires two kinds of tRNAs: initiation and elongation. The former initiates the process (formylmethionine tRNA in prokaryotes and special methionine tRNA in eukaryotes). The latter participates in the synthesis proper, recognizing the sense codons. Synthesis is also assisted by special proteins: initiation, elongation, and termination factors. The termination factors are necessary to recognize stop codons (UAG, UGA, and UAA) and to release the complete protein chain from the elongation tRNA preceding a stop codon. No termination tRNA capable of recognizing stop codons by their anticodons is known. The termination factors are thought to do this. In the large ribosomal RNA, we found two sites that, like tRNAs, contain the anticodon hairpin but with triplets complementary to stop codons. One site is hairpin 69 from domain IV; the other site is hairpin 89, domain V. By analogy, we call them termination tRNAs: Ter-tRNA1 and Ter-tRNA2, respectively, even though they transport no amino acids, and suggest that they directly pair to stop codons. The termination factors only aid in this recognition, making it specific and reliable. A strong argument in favor of our hypothesis comes from vertebrate mitochondria. They are known to acquire two new stop codons, AGA and AGG. In the standard code, these are two out of six arginine codons. We revealed that the corresponding anticodons, UCU and CCU, have evolved in Ter-tRNA1 of these mitochondria.     

Inhibition of translation termination mediated by an interaction of eukaryotic release factor 1 with a nascent peptidyl-tRNA

Expression of the human cytomegalovirus UL4 gene is inhibited by translation of a 22-codon-upstream open reading frame (uORF2). The peptide product of uORF2 acts in a sequence-dependent manner to inhibit its own translation termination, resulting in persistence of the uORF2 peptidyl-tRNA linkage. Consequently, ribosomes stall at the uORF2 termination codon and obstruct downstream translation. Since termination appears to be the critical step affected by translation of uORF2, we examined the role of eukaryotic release factors 1 and 3 (eRF1 and eRF3) in the inhibitory mechanism. In support of the hypothesis that an interaction between eRF1 and uORF2 contributes to uORF2 inhibitory activity, specific residues in each protein, glycines 183 and 184 of the eRF1 GGQ motif and prolines 21 and 22 of the uORF2 peptide, were found to be necessary for full inhibition of downstream translation. Immunoblot analyses revealed that eRF1, but not eRF3, accumulated in the uORF2-stalled ribosome complex. Finally, increased puromycin sensitivity was observed after depletion of eRF1 from the stalled ribosome complex, consistent with inhibition of peptidyl-tRNA hydrolysis resulting from an eRF1-uORF2 peptidyl-tRNA interaction. These results reveal the paradoxical potential for interactions between a nascent peptide and eRF1 to obstruct the translation termination cascade.