A minor arginine tRNA mutant limits translation preferentially of a protein dependent on the cognate codon.

The Escherichia coli argU gene encodes a rare arginine tRNA (anticodon UCU) that translates the similarly rare AGA codon. The argU10(Ts) mutation is a transition that changes the first nucleotide of the mature tRNA from G to A, presumably destabilizing the acceptor stem. This mutation, when present in haploid condition in the chromosome, reduces the growth rate at 30 degrees C and results in cessation of growth after 60 to 90 min at 43 degrees C. The mutation also preferentially limits (compared with total protein synthesis) translation of an induced gene that depends on five AGA codons, i.e., the lambda cI repressor gene. Translation of another inducible protein, beta-galactosidase, which does not involve AGA codons, was inhibited to a much lesser extent. The chromosomal argU(Ts) mutation also confers the Pin phenotype, that is, loss of ability of the host, as a P2 lysogen, to inhibit growth of bacteriophage lambda, probably the result of reduced translation of the P2 old gene, which contains five AGA codons (E. Hagg?rd-Ljungquist, V. Barreiro, R. Calendar, D. M. Kurnit, and H. Cheng, Gene 85:25-33, 1989).     

Effects of a minor isoleucyl tRNA on heterologous protein translation in Escherichia coli.

In Escherichia coli, the isoleucine codon AUA occurs at a frequency of about 0.4% and is the fifth rarest codon in E. coli mRNA. Since there is a correlation between the frequency of codon usage and the level of its cognate tRNA, translational problems might be expected when the mRNA contains high levels of AUA codons. When a hemagglutinin from the influenza virus, a 304-amino-acid protein with 12 (3.9%) AUA codons and 1 tandem codon, and a mupirocin-resistant isoleucyl tRNA synthetase, a 1,024-amino-acid protein, with 33 (3.2%) AUA codons and 2 tandem codons, were expressed in E. coli, product accumulation was highly variable and dependent to some degree on the growth medium. In rich medium, the flu antigen represented about 16% of total cell protein, whereas in minimal medium, it was only 2 to 3% of total cell protein. In the presence of the cloned ileX, which encodes the cognate tRNA for AUA, however, the antigen was 25 to 30% of total cell protein in cells grown in minimal medium. Alternatively, the isoleucyl tRNA synthetase did not accumulate to detectable levels in cells grown in Luria broth unless the ileX tRNA was coexpressed when it accounted for 7 to 9% of total cell protein. These results indicate that the rare isoleucine AUA codon, like the rare arginine codons AGG and AGA, can interfere with the efficient expression of cloned proteins.     

A yeast tRNA mutant that causes pseudohyphal growth exhibits reduced rates of CAG codon translation

In Saccharomyces cerevisiae, the SUP70 gene encodes the CAG‐decoding tRNA^Gln_CUG. A mutant allele, sup70‐65, induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, 10 novel sup70 UAG suppressor alleles were identified, defining positions in the tRNA^Gln_CUG anticodon stem that restrict first base wobble. However, none conferred pseudohyphal growth, showing altered CUG anticodon presentation cannot itself induce pseudohyphal growth. Northern blot analysis revealed the sup70‐65 tRNA^Gln_CUG is unstable, inefficiently charged, and 80% reduced in its effective concentration. A stochastic model simulation of translation predicted compromised expression of CAG‐rich ORFs in the tRNA^Gln_CUG‐depleted sup70‐65 mutant. This prediction was validated by demonstrating that luciferase expression in the mutant was 60% reduced by introducing multiple tandem CAG (but not CAA) codons into this ORF. In addition, the sup70‐65 pseudohyphal phenotype was partly complemented by overexpressing CAA‐decoding tRNA^Gln_UUG, an inefficient wobble‐decoder of CAG. We thus show that introducing codons decoded by a rare tRNA near the 5′ end of an ORF can reduce eukaryote translational expression, and that the mutant tRNA_CUG^Gln constitutive pseudohyphal differentiation phenotype correlates strongly with reduced CAG decoding efficiency.     

Coupling between codon usage, translation and protein export in Escherichia coli

Proteins destined for export via the Sec-dependent pathway are synthesized with a short N-terminal signal peptide. A requirement for export is that the proteins are in a translocationally competent state. This is a loosely folded state that allows the protein to pass through the SecYEG apparatus and pass into the periplasm. In order to maintain pre-secretory proteins in an export-competent state, there are many factors that slow the folding of the pre-secretory protein in the cytoplasm. These include cytoplasmic chaperones, such as SecB, and the signal recognition particle, which bind the pre-secretory protein and direct it to the cytoplasmic membrane for export. Recently, evidence has been published that non-optimal codons in the signal sequence are important for a time-critical early event to allow the correct folding of pre-secretory proteins. This review details the recent developments in folding of the signal peptide and the pre-secretory protein.     

Nucleotide modifications and tRNA anticodon-mRNA codon interactions on the ribosome

We have carried out molecular dynamics simulations of the tRNA anticodon and mRNA codon, inside the ribosome, to study the effect of the common tRNA modifications cmo(5)U34 and m(6)A37. In tRNA(Val), these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. Previous data suggest that entropic effects are mainly responsible for the extended reading capabilities, but detailed mechanisms have remained unknown. We have performed a wide range of simulations to elucidate the details of these mechanisms at the atomic level and quantify their effects: extensive free energy perturbation coupled with umbrella sampling, entropy calculations of tRNA (free and bound to the ribosome), and thorough structural analysis of the ribosomal decoding center. No prestructuring effect on the tRNA anticodon stem-loop from the two modifications could be observed, but we identified two mechanisms that may contribute to the expanded decoding capability by the modifications: The further reach of the cmo(5)U34 allows an alternative outer conformation to be formed for the noncognate base pairs, and the modification results in increased contacts between tRNA, mRNA, and the ribosome.     

Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation

To investigate the possible influence of the local rates of translation on protein folding, 16 consecutive rare (in Escherichia coli) codons in the chloramphenicol acetyltransferase (CAT) gene have been replaced by frequent ones. Site-directed silent mutagenesis reduced the pauses in translation of CAT in E. coli S30 extract cell-free system and led to the acceleration of the overall rate of CAT protein synthesis. At the same time, the silently mutated protein (with unaltered protein sequence) synthesized in the E. coli S30 extract system was shown to possess 20% lower specific activity. The data suggest that kinetics of protein translation can affect the in vivo protein-folding pathway, leading to increased levels of protein misfolding.     

Recognition and selection of tRNA in translation

Aminoacyl-tRNA (aa-tRNA) is delivered to the ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. The stepwise movement of aa-tRNA from EF-Tu into the ribosomal A site entails a number of intermediates. The ribosome recognizes aa-tRNA through shape discrimination of the codon-anticodon duplex and regulates the rates of GTP hydrolysis by EF-Tu and aa-tRNA accommodation in the A site by an induced fit mechanism. Recent results of kinetic measurements, ribosome crystallography, single molecule FRET measurements, and cryo-electron microscopy suggest the mechanism of tRNA recognition and selection.     

Combining models of protein translation and population genetics to predict protein production rates from codon usage patterns

Genes are often biased in their codon usage. The degree of bias displayed often changes with expression level and intragenic position. Numerous indices, such as the codon adaptation index, have been developed to measure this bias. Although the expression level of a gene and index values are correlated, the heuristic nature of these metrics limits their ability to explain this relationship. As an alternative approach, this study integrates mechanistic models of cellular and population processes in a nested manner to develop a stochastic evolutionary model of a protein's production rate (SEMPPR). SEMPPR assumes that the evolution of codon bias is driven by selection to reduce the cost of nonsense errors and that this selection is counteracted by mutation and drift. Through the application of Bayes' theorem, SEMPPR generates a posterior probability distribution for the protein production rate of a given gene. Conceptually, SEMPPR's predictions are based on the degree of adaptation to reduce the cost of nonsense errors observed in the codon usage pattern of the gene. As an illustration, SEMPPR was parameterized using the Saccharomyces cerevisiae genome and its predictions tested using available empirical data. The results indicate that SEMPPR's predictions are as reliable index based ones. In addition, SEMPPR's output is more easily interpreted and its predictions could be improved through refinements of the models upon which it is built.     

Distortion of tRNA upon near-cognate codon recognition on the ribosome

The accurate decoding of the genetic information by the ribosome relies on the communication between the decoding center of the ribosome, where the tRNA anticodon interacts with the codon, and the GTPase center of EF-Tu, where GTP hydrolysis takes place. In the A/T state of decoding, the tRNA undergoes a large conformational change that results in a more open, distorted tRNA structure. Here we use a real-time transient fluorescence quenching approach to monitor the timing and the extent of the tRNA distortion upon reading cognate or near-cognate codons. The tRNA is distorted upon codon recognition and remains in that conformation until the tRNA is released from EF-Tu, although the extent of distortion gradually changes upon transition from the pre- to the post-hydrolysis steps of decoding. The timing and extent of the rearrangement is similar on cognate and near-cognate codons, suggesting that the tRNA distortion alone does not provide a specific switch for the preferential activation of GTP hydrolysis on the cognate codon. Thus, although the tRNA plays an active role in signal transmission between the decoding and GTPase centers, other regulators of signaling must be involved.     

tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon.

Tryptophanase (tna) operon expression in Escherichia coli is induced by tryptophan. This response is mediated by features of a 319-base-pair leader region preceding the major structural genes of the operon. Translation of the coding region (tnaC) for a 24-amino-acid leader peptide is essential for induction. We have used site-directed mutagenesis to investigate the role of the single Trp codon, at position 12 in tnaC, in regulation of the operon. Codon 12 was changed to either a UAG or UGA stop codon or to a CGG arginine codon. Induction by tryptophan was eliminated by any of these changes. Studies with suppressor tRNAs indicated that tRNA(Trp) translation of codon 12 in tnaC is essential for induction of the operon. Reduction of tna expression by a miaA mutation supports a role for translation by tRNA(Trp) in regulation of the operon. Frameshift mutations and suppression that allows translation of tnaC to proceed beyond the normal stop codon result in constitutive tna operon expression. Deletion of a potential site for Rho factor utilization just beyond tnaC also results in partial constitutive expression. These studies suggest possible models for tryptophan induction of tna operon expression involving tRNA(Trp)-mediated frame shifting or readthrough at the tnaC stop codon.     

tRNA selection and kinetic proofreading in translation

Using single-molecule methods we observed the stepwise movement of aminoacyl-tRNA (aa-tRNA) into the ribosome during selection and kinetic proofreading using single-molecule fluorescence resonance energy transfer (smFRET). Intermediate states in the pathway of tRNA delivery were observed using antibiotics and nonhydrolyzable GTP analogs. We identified three unambiguous FRET states corresponding to initial codon recognition, GTPase-activated and fully accommodated states. The antibiotic tetracycline blocks progression of aa-tRNA from the initial codon recognition state, whereas cleavage of the sarcin-ricin loop impedes progression from the GTPase-activated state. Our data support a model in which ribosomal recognition of correct codon-anticodon pairs drives rotational movement of the incoming complex of EF-Tu-GTP-aa-tRNA toward peptidyl-tRNA during selection on the ribosome. We propose a mechanistic model of initial selection and proofreading.     

Mechanistic insights into tRNA cleavage by a contact-dependent growth inhibitor protein and translation factors

Contact-dependent growth inhibition is a mechanism of interbacterial competition mediated by delivery of the C-terminal toxin domain of CdiA protein (CdiA–CT) into neighboring bacteria. The CdiA–CT of enterohemorrhagic Escherichia coli EC869 (CdiA–CT^EC869) cleaves the 3′-acceptor regions of specific tRNAs in a reaction that requires the translation factors Tu/Ts and GTP. Here, we show that CdiA–CT^EC869 has an intrinsic ability to recognize a specific sequence in substrate tRNAs, and Tu:Ts complex promotes tRNA cleavage by CdiA–CT^EC869. Uncharged and aminoacylated tRNAs (aa-tRNAs) were cleaved by CdiA–CT^EC869 to the same extent in the presence of Tu/Ts, and the CdiA–CT^EC869:Tu:Ts:tRNA(aa-tRNA) complex formed in the presence of GTP. CdiA–CT^EC869 interacts with domain II of Tu, thereby preventing the 3′-moiety of tRNA to bind to Tu as in canonical Tu:GTP:aa-tRNA complexes. Superimposition of the Tu:GTP:aa-tRNA structure onto the CdiA–CT^EC869:Tu structure suggests that the 3′-portion of tRNA relocates into the CdiA–CT^EC869 active site, located on the opposite side to the CdiA–CT^EC869 :Tu interface, for tRNA cleavage. Thus, CdiA–CT^EC869 is recruited to Tu:GTP:Ts, and CdiA–CT:Tu:GTP:Ts recognizes substrate tRNAs and cleaves them. Tu:GTP:Ts serves as a reaction scaffold that increases the affinity of CdiA–CT^EC869 for substrate tRNAs and induces a structural change of tRNAs for efficient cleavage by CdiA–CT^EC869.