Regulation of the Escherichiacoli tryptophan operon by readthrough of UGA termination codons

The regulation of the synthesis of $\text{trp}$ operon enzymes was studied in streptomycin-resistant $\text{Escherichia}\text{coli}$ mutants temperature-sensitive for UGA suppression by normal tRNA^Trp. Our mutants carry a $\text{trp}\text{R}{}^{\mathrm{+}}$ allele that when transferred to a different genetic background causes repression of trp operon enzyme synthesis at both low (35°C) and high (42°C) temperatures; however, in our mutants with an excess of tryptophan and at increased temperatures $\text{trp}$ enzyme synthesis is derepressed. Based on our results and the sequence data of the $\text{trp}$R gene [Singleton et al. (1980) Nucleic Acids Res., 8, 1551–1560], we offer a model for the involvement of the limited misreading of UGA codons by normal charged tRNA^Trp in the autogenous regulation of the $\text{trp}$R gene expression. The UGA readthrough process may be a regulatory amplifier of the effect of tryptophan starvation.     

Modulation of Escherichia coli tryptophan (trp) attenuation by the UGA readthrough process

Summary We constructed plasmid pAtrp46 in which lacZ gene expression is regulated by the attenuator of the Escherichia coli tryptophan (trp) operon. The attenuation of trp, which occurs in the presence of an excess of tryptophan, is reflected by a decrease in the expression of the lacZ gene of pAtrp46 in a trpR^- strain. Experiments with pAtrp46 further support our previous results (Engelberg-Kulka et al. 1982b) that suppression of a UGA termination codon by normal charged tRNA^Trp, a process called UGA readthrough, is a necessary mechanism in trp attenuation. Our experiments also suggest that plasmid pAtrp46 is useful for studies of other aspects of trp attenuation.     

Helix 69 in 23S rRNA modulates decoding by wild type and suppressor tRNAs

Helix 69 of 23S rRNA forms one of the major inter-subunit bridges of the 70S ribosome and interacts with A- and P-site tRNAs and translation factors. Despite the proximity of h69 to the decoding center and tRNAs, the contribution of h69 to the tRNA selection process is unclear: previous genetic analyses have shown that h69 mutations increase frameshifting and readthrough of stop codons. However, a complete deletion of h69 does not affect the selection of cognate tRNAs in vitro. To address these discrepancies, the in vivo effects of a range of single- and multi-base h69 mutations in Escherichia coli 23S rRNA on various translation errors have been determined. While a majority of the h69 mutations examined here affected readthrough of stop codons and frameshifting, the ΔA1916 single base deletion mutation uniquely influenced missense decoding. Different h69 mutants had either increased or decreased levels of stop codon readthrough. The h69 mutations that decreased UGA readthrough also decreased UGA reading by a mutant, near-cognate tRNA^Trp carrying a G24A substitution in the D arm, but had far less effect on UGA reading by a suppressor tRNA with a complementary anticodon. These results suggest that h69 interactions with release factors contribute significantly to termination efficiency and that interaction with the D arm of A-site tRNA is important for discrimination between cognate and near-cognate tRNAs.     

Functional Interaction Between Release Factor One and P-site Peptidyl-tRNA on the Ribosome

Translation termination at UAG is influenced by the nature of the 5′ flanking codon inEscherichia coli. Readthrough of the stop codon is always higher in a strain with mutant (prfA1) as compared to wild-type (prfA⁺) release factor one (RF1). Isocodons, which differ in the last base and are decoded by the same tRNA species, affect termination at UAG differently in strains with mutant or wild-type RF1. No general preference of the last codon base to favour readthrough or termination can be found. The data suggest that RF1 is sensitive to the nature of the wobble base anticodon-codon interaction at the ribosomal peptidyl-tRNA binding site (P-site). For some isoaccepting P-site tRNAs (tRNA₃^ProversustRNA₂^Pro, tRNA₄^ThrversustRNA_1,3^Thr) the effect is different on mutant and wild-type RF1, suggesting an interaction between RF1 at the aminoacyl-tRNA acceptor site (A-site) and the P-site tRNA itself. The glycine codons GGA (tRNA₂^Gly) and GGG (tRNA_2,3^Gly) at the ribosomal P-site are associated with an almost threefold higher readthrough of UAG than any of the other 42 codons tested, including the glycine codons GGU/C, in a strain with wild-type RF1. This differential response to the glycine codons is lost in the strain with the mutant form of RF1 since readthrough is increased to a similar high level for all four glycine codons. High α-helix propensity of the last amino acid residue at the C-terminal end of the nascent peptide is correlated with an increased termination at UAG. The effect is stronger on mutant compared to wild-type RF1. The data suggest that RF1-mediated termination at UAG is sensitive to the nature of the codon-anticodon interaction of the wobble base, the last amino acid residue of the nascent peptide chain, and the tRNA at the ribosomal P-site.     

Role of stoichiometry between mRNA, translation factor SeIB and selenocysteyl-tRNA in selenoprotein synthesis

The specialized translation factor SeIB forms a quaternary complex in vitro with selenocysteyl-tRNA^Sec, the selenoprotein mRNA and guanine nucleotides. To gain information on whether this complex is required for selenocysteine insertion in vivo we have studied the effect of unbalanced ratios of the individual components of the complex on UGA readthrough. It was found that overproduction of SeIB in an otherwise wild-type genetic background reduced UGA read-through to less than 1 %. Concomitant overexpression of seIC (the gene for selenocysteine-specific tRNA^Sec) completely reversed the inhibition. Truncation of SeIB from the C-terminal end abolished function as a translation factor but the truncated molecules, when overproduced, were still able to suppress UGA read-through. The inhibition was also reversed by overproduction of tRNA^Sec. The most plausible explanation is that overproduction of SeIB impairs the statistics of formation of the quaternary complex and that the C-terminally truncated molecules are still able to bind selenocysteyl-tRNA^Sec and remove it from the pool. The mRNA-binding capacity, therefore, is physically separated from the selenocysteyl-tRNA-binding domain.     

Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB

Selenocysteine (Sec), the 21^st amino acid in translation, uses its specific tRNA (tRNA^Sec) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNA^Sec (Sec-tRNA^Sec) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1–3), followed by four winged-helix domains (WHD1–4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNA^Sec is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNA^Sec, SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNA^Sec allows SelB to specifically recognize tRNA^Sec and characteristically place it at the ribosomal A-site.     

Factors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in Escherichia coli

Selenoprotein synthesis in Escherichia coli strictly depends on the presence of a specific selenocysteine insertion sequence (SECIS) following the selenocysteine-encoding UGA codon of the respective mRNA. It is recognized by the selenocysteine-specific elongation factor SelB, leading to cotranslational insertion of selenocysteine into the nascent polypeptide chain. The synthesis of three different selenoproteins from the gram-positive anaerobe Eubacterium acidaminophilum in E. coli was studied. Incorporation of ⁷⁵Se into glycine reductase protein B (GrdB1), the peroxiredoxin PrxU, and selenophosphate synthetase (SelD1) was negligible in an E. coli wild-type strain and was fully absent in an E. coli SelB mutant. Selenoprotein synthesis, however, was strongly increased if selB and selC (tRNA^Sec) from E. acidaminophilum were coexpressed. Putative secondary structures downstream of the UGA codons did not show any sequence similarity to each other or to the E. coli SECIS element. However, mutations in these structures strongly reduced the amount of ⁷⁵Se-labeled protein, indicating that they indeed act as SECIS elements. UGA readthrough mediated by the three different SECIS elements was further analyzed using gst-lacZ translational fusions. In the presence of selB and selC from E. acidaminophilum, UGA readthrough was 36 to 64% compared to the respective cysteine-encoding UGC variant. UGA readthrough of SECIS elements present in Desulfomicrobium baculatum (hydV), Treponema denticola (selD), and Campylobacter jejuni (selW-like gene) was also considerably enhanced in the presence of E. acidaminophilum selB and selC. This indicates recognition of these SECIS elements and might open new perspectives for heterologous selenoprotein synthesis in E. coli.     

Tryptophan residue is essential for immunoreactivity of a diagnostically relevant peptide epitope of A. fumigatus

The role of tryptophan (Trp¹⁷) in immunoreactivity of P1, the diagnostically relevant peptide from a major allergen/antigen of Aspergillus fumigatus, was evaluated by chemically modifying tryptophanyl residue of P1. In BIAcore kinetic studies, unmodified P1 showed a 100-fold higher binding with ABPA (Allergic Bronchopulmonary Aspergillosis) patients’ IgG [K_D (equilibrium dissociation constant) = 2.74 e⁻⁸ ± 0.13 M] than the controls’ IgG (K_D = 2.97 e⁻⁶± 0.14 M), whereas chemically-modified P1 showed similar binding [K_D patients’ IgG = 3.25 e⁻⁷± 0.16 M, K_D controls’ IgG = 3.86 e⁻⁷± 0.19 M] indicating loss of specific immunoreactivity of P1 on tryptophan modification. Modified P1 showed loss of specific binding to IgE and IgG antibodies of ABPA patients in ELISA (Enzyme-Linked Immunosorbent Assay). The study infers that tryptophan residue (Trp¹⁷) is essential for immunoreactivity of P1.     

Human Cells Have a Limited Set of tRNA Anticodon Loop Substrates of the tRNA Isopentenyltransferase TRIT1 Tumor Suppressor

Human TRIT1 is a tRNA isopentenyltransferase (IPTase) homologue of Escherichia coli MiaA, Saccharomyces cerevisiae Mod5, Schizosaccharomyces pombe Tit1, and Caenorhabditis elegans GRO-1 that adds isopentenyl groups to adenosine 37 (i6A37) of substrate tRNAs. Prior studies indicate that i6A37 increases translation fidelity and efficiency in codon-specific ways. TRIT1 is a tumor suppressor whose mutant alleles are associated with cancer progression. We report the systematic identification of i6A37-containing tRNAs in a higher eukaryote, performed using small interfering RNA knockdown and other methods to examine TRIT1 activity in HeLa cells. Although several potential substrates contained the IPTase recognition sequence A36A37A38 in the anticodon loop, only tRNA^SerAGA, tRNA^SerCGA, tRNA^SerUGA, and selenocysteine tRNA with UCA (tRNA^[Ser]SecUCA) contained i6A37. This subset is a significantly more restricted than that for two distant yeasts (S. cerevisiae and S. pombe), the only other organisms comprehensively examined. Unlike the fully i6A37-modified tRNAs for Ser, tRNA^[Ser]SecUCA is partially (∼40%) modified. Exogenous selenium and other treatments that decreased the i6A37 content of tRNA^[Ser]SecUCA led to increased levels of the tRNA^[Ser]SecUCA. Of the human mitochondrion (mt)-encoded tRNAs with A36A37A38, only mt tRNAs tRNA^SerUGA and tRNA^TrpUCA contained detectable i6A37. Moreover, while tRNA^Ser levels were unaffected by TRIT1 knockdown, the tRNA^[Ser]SecUCA level was increased and the mt tRNA^SerUGA level was decreased, suggesting that TRIT1 may control the levels of some tRNAs as well as their specific activity.     

Ion and acid–base regulation in the freshwater mummichog (Fundulus heteroclitus): a departure from the standard model for freshwater teleosts

Ion and acid–base balance were examined in the freshwater-adapted mummichog (Fundulus heteroclitus) using a series of treatments designed to perturb the coupling mechanisms. Unidirectional Cl⁻ uptake (J^Cl_in) was extremely low whereas J^Na_in was substantial (three- to sixfold higher); comparable differences occurred in unidirectional efflux rates (J^Cl_out, J^Na_out). J^Cl_in was refractory to all treatments, suggesting that Cl⁻/base exchange was unimportant or absent. Indeed, no base excretion or modulation of ion fluxes occurred for acid–base balance for up to 8 h after NaHCO₃⁻ loading (injections of 1000 or 3000 nequiv.·g⁻¹). Acute environmental low pH (4.5) and amiloride (10⁻⁴ M) treatments caused concurrent inhibition of J^Na_in and net H⁺ excretion (J^H+_net), indicating the presence of Na⁺/H⁺ exchange. J^Na_in was elevated and J^H+_net restored during recovery from both treatments, but this exchange did not appear to be dynamically adjusted for acid–base homeostasis. High external ammonia exposure (1 mmol·l⁻¹) initially blocked ammonia excretion (J^Amm_net) but had no effect on J^Na_in, whereas high pH (9.4) reduced both J^Amm_net and J^Na_in. Inhibition of J^Na_in by the low pH and amiloride treatments had no effect on J^Amm_net. These results indicate that ammonia excretion is entirely diffusive and independent of both Na⁺uptake and the protons that are transported via the Na⁺/H⁺ coupling. In addition, ureagenesis served as a compensatory mechanism during high external ammonia exposure, as a marked elevation in urea excretion partially replaced the inhibited J^Amm_net. In all treatments, changes in the Na⁺–Cl⁻ net flux differential were consistent with changes in J^H+_net measured by traditional water titration techniques, indicating that the former can be used as an estimate of the acid–base status of the fish. Overall, the results demonstrate that the freshwater-adapted F. heteroclitus does not conform to the ion/acid–base relationships described in the standard model based on commonly studied species such as trout, goldfish, and catfish.     

New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae

Stop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5′ nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNA^Gln were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.     

A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids

There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA^Ala-based body (tRNA^AlaB) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNA^PheB body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNA^AlaB body than from the tRNA^PheB body. At ∼1 µM EF-Tu, tRNA^AlaB conferred considerably faster incorporation kinetics than tRNA^PheB, especially in the case of the bulky bK. In contrast, the swap to the tRNA^AlaB body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNA^AlaB and tRNA^PheB bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.     

Box C/D RNA-guided 2'-O methylations and the intron of tRNATrp are not essential for the viability of Haloferax volcanii

Deleting the box C/D RNA-containing intron in the Haloferax volcanii tRNA^Trp gene abolishes RNA-guided 2′-O methylations of C34 and U39 residues of tRNA^Trp. However, this deletion does not affect growth under standard conditions.     

Translation of the UGA triplet in vitro by tryptophan transfer RNA's

Tryptophan transfer RNA from the UGA-suppressing strain of Escherichia coli CAJ64 was purified and assayed for suppressor activity in vitro in two ways: by translation of the bacteriophage T4 lysozyme messenger RNA bearing a UGA mutation, and by translation of poly(U-G-A). Purified tRNA^Trp, and no other fraction, stimulates lysozyme synthesis 30-fold above the level seen when comparable amounts of tryptophan tRNA from the non-suppressing strain, CA244, were added; it also translates poly(U-G-A) as polytryptophan more efficiently than the su⁻ tRNA. Tryptophan tRNA from the non-suppressing strain is active in the assays but far less so than CAJ64 tRNA^Trp, and this is consistent with the leakiness of su⁻ strains. Since the nucleotide sequences of these tryptophan tRNA's are known (Hirsh, 1971), it is concluded that tRNA with a CCA anticodon recognizes the UGA triplet and this recognition is improved by a nucleotide change elsewhere in the molecule.     

In situ measurement of fine root water absorption in three temperate tree species—Temporal variability and control by soil and atmospheric factors

Miniature heat balance-sap flow gauges were used to measure water flows in small-diameter roots (3–4 mm) in the undisturbed soil of a mature beech–oak–spruce mixed stand. By relating sap flow to the surface area of all branch fine roots distal to the gauge, we were able to calculate real time water uptake rates per root surface area (J_s) for individual fine root systems of 0.5–1.0 m in length. Study aims were (i) to quantify root water uptake of mature trees under field conditions with respect to average rates, and diurnal and seasonal changes of J_s, and (ii) to investigate the relationship between uptake and soil moisture θ, atmospheric saturation deficit D, and radiation I. On most days, water uptake followed the diurnal course of D with a mid-day peak and low night flow. Neighbouring roots of the same species differed up to 10-fold in their daily totals of J_s (<100–2000 g m⁻² d⁻¹) indicating a large spatial heterogeneity in uptake. Beech, oak and spruce roots revealed different seasonal patterns of water uptake although they were extracting water from the same soil volume. Multiple regression analyses on the influence of D, I and θ on root water uptake showed that D was the single most influential environmental factor in beech and oak (variable selection in 77% and 79% of the investigated roots), whereas D was less important in spruce roots (50% variable selection). A comparison of root water uptake with synchronous leaf transpiration (porometer data) indicated that average water fluxes per surface area in the beech and oak trees were about 2.5 and 5.5 times smaller on the uptake side (roots) than on the loss side (leaves) given that all branch roots <2 mm were equally participating in uptake. Beech fine roots showed maximal uptake rates on mid-summer days in the range of 48–205 g m⁻² h⁻¹ (i.e. 0.7–3.2 mmol m⁻² s⁻¹), oak of 12–160 g m⁻² h⁻¹ (0.2–2.5 mmol m⁻² s⁻¹). Maximal transpiration rates ranged from 3 to 5 and from 5 to 6 mmol m⁻² s⁻¹ for sun canopy leaves of beech and oak, respectively. We conclude that instantaneous rates of root water uptake in beech, oak and spruce trees are above all controlled by atmospheric factors. The effects of different root conductivities, soil moisture, and soil hydraulic properties become increasingly important if time spans longer than a week are considered.     

Uniform affinity-tuning of N-methyl-aminoacyl-tRNAs to EF-Tu enhances their multiple incorporation

In ribosomal translation, the accommodation of aminoacyl-tRNAs into the ribosome is mediated by elongation factor thermo unstable (EF-Tu). The structures of proteinogenic aminoacyl-tRNAs (pAA-tRNAs) are fine-tuned to have uniform binding affinities to EF-Tu in order that all proteinogenic amino acids can be incorporated into the nascent peptide chain with similar efficiencies. Although genetic code reprogramming has enabled the incorporation of non-proteinogenic amino acids (npAAs) into the nascent peptide chain, the incorporation of some npAAs, such as N-methyl-amino acids (^MeAAs), is less efficient, especially when ^MeAAs frequently and/or consecutively appear in a peptide sequence. Such poor incorporation efficiencies can be attributed to inadequate affinities of ^MeAA-tRNAs to EF-Tu. Taking advantage of flexizymes, here we have experimentally verified that the affinities of ^MeAA-tRNAs to EF-Tu are indeed weaker than those of pAA-tRNAs. Since the T-stem of tRNA plays a major role in interacting with EF-Tu, we have engineered the T-stem sequence to tune the affinity of ^MeAA-tRNAs to EF-Tu. The uniform affinity-tuning of the individual pairs has successfully enhanced the incorporation of ^MeAAs, achieving the incorporation of nine distinct ^MeAAs into both linear and thioether-macrocyclic peptide scaffolds.     

Full Implementation of the Genetic Code by Tryptophanyl-tRNA Synthetase Requires Intermodular Coupling

Tryptophanyl-tRNA Synthetase (TrpRS) Urzyme (fragments A and C), a 130-residue construct containing only secondary structures positioning the HIGH and KMSKS active site signatures and the specificity helix, accelerates tRNA^Trp aminoacylation with ∼10-fold specificity toward tryptophan, relative to structurally related tyrosine. We proposed that including the 76-residue connecting peptide 1 insertion (Fragment B) might enhance tryptophan affinity and hence amino acid specificity, because that subdomain constrains the orientation of the specificity helix. We test that hypothesis by characterizing two new constructs: the catalytic domain (fragments A–C) and the Urzyme supplemented with the anticodon-binding domain (fragments A, C, and D). The three constructs, together with the full-length enzyme (fragments A–D), comprise a factorial experiment from which we deduce individual and combined contributions of the two modules to the steady-state kinetics parameters for tryptophan-dependent ³²PP_i exchange, specificity for tryptophan versus tyrosine, and aminoacylation of tRNA^Trp. Factorial design directly measures the energetic coupling between the two more recent modules in the contemporary enzyme and demonstrates its functionality. Combining the TrpRS Urzyme individually in cis with each module affords an analysis of long term evolution of amino acid specificity and tRNA aminoacylation, both essential for expanding the genetic code. Either module significantly enhances tryptophan activation but unexpectedly eliminates amino acid specificity for tryptophan, relative to tyrosine, and significantly reduces tRNA aminoacylation. Exclusive dependence of both enhanced functionalities of full-length TrpRS on interdomain coupling energies between the two new modules argues that independent recruitment of connecting peptide 1 and the anticodon-binding domain during evolutionary development of Urzymes would have entailed significant losses of fitness.