大肠杆菌tRNASec关键核苷酸位点

在原核生物中,硒蛋白合成需要tRNA~(Sec) (SelC)与硒代半胱氨酸合成(Sec synthase, SelA)、硒代半胱氨酸特异性延伸因子(Sec-specificelongationfactor,SelB)之间相互作用。【目的】基于大肠杆菌掺硒机器,寻找tRNA~(Sec)骨架上关键核苷酸位点,为解决硒蛋白目前面临的掺硒效率较低、产量低的问题提供新思路。【方法】以大鼠细胞质型硫氧还蛋白还原酶(thioredoxinreductase1,TrxR1)为掺硒模式蛋白为定点突变tRNA~(Sec),转化至BL21 (DE3) gor-获得阳性重组菌株(携带pET-TRSter/pSUABC’),用于表达大鼠硒蛋白TrxR1,然后使用2￠,5￠ADP-Sepharose亲和层析和凝胶过滤两步法分离纯化TrxR1,最后利用经典硒依赖型DTNB还原反应测定TrxR1的酶活,分析关键核苷酸位点,评价掺硒效率。【结果】在存在SECIS元件的前提下,当SelA、SelB、tRNA~(Sec)共表达时,与野生型相比,携带突变型tRNA~(Sec)所共表达的TrxR1酶活力呈现不同程度的降低,其中E.colitRNA~(Sec)的G18、G19这两个位点的所有的TrxR1酶活远低于野生型(10%);然而,a26和b7的酶活相对较高。【结论】E. coli tRNA~(Sec)骨架上G18和G19位点对于维持tRNA稳定性和灵活性发挥了关键作用,位点突变引起tRNA结构变化会影响tRNA~(Sec)与掺硒元件的互作,因此有望通过改造tRNA核苷酸位点来提高硒蛋白的掺硒效率。     

Conformational Change of the L-Shaped tRNAPhe Molecule

Abstract

Fluorophore of proflavine was introduced onto the 3′-terminal ribose moiety of yeast tRNA^Phe. The distance between the fluorophore and the fluorescent Y base in the anticodon of yeast tRNA^Phe was measured by a singlet-singlet energy transfer. Conformational changes of tRNA^Phe with binding of tRNA^Glu ₂, which has the anticodon UUC complementary to the anticodon GAA of tRNA^Phe, were investigated. The distance obtained at the ionic strength of 100 mM K⁺ and 10 mM Mg²⁺ is very close to the distance from x-ray diffraction, while the distance obtained in the presence of tRNA^Glu ₂ is significantly smaller. Further, using a fluorescent probe of 4-bromomethl-7-methoxycoumarin introduced onto pseudouridine residue Ψ₅₅ in the TΨC loop of tRNA^Phe, Stern-Volmer quenching experiments for the probe with or without added tRNA^Glu ₂were carried out. The results showed greater access of the probe to the quencher with added tRNA^Glu ₂. These results suggest that both arms of the L-shaped tRNA structure tend to bend inside with binding of tRNA^Glu ₂ and some structural collapse occurs at the corner of the L-shaped structure.     

Primary structure of Bacillus subtilis tRNAsTyr

tRNA_I^Tyr and tRNA_II^Tyr have been purified from B.subtilis and their nucleotide sequence determined. tRNA_I^Tyr differs from tRNA_II^Tyr only by the extent of modification of the adenosine in 3′ position adjacent to the anticodon, i⁶A and ms²i⁶A respectively.     

The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC)

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo⁵UAC) contains a unique modification, N⁶-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA₁^Val. Inactivation of yfiC gene abolishes m⁶A formation in tRNA₁^Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA₁^Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m⁶A in tRNA₁^Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.     

On the Possible Role of tRNA Base Modifications in the Evolution of Codon Usage: Queuosine and Drosophila

The best documented selection-based hypothesis to explain unequal usage of codons is based on the relative abundance of isoaccepting tRNAs. In unicellular organisms the most used codons are optimally translated by the most abundant tRNAs. The chemical bonding energies are affected by modification of the four traditional bases, in particular in the first anti-codon corresponding to the third codon position. One nearly universal modification is queuosine (Q) for guanine (G) in tRNA^His, tRNA^Asp, tRNA^Asn, and tRNA^Tyr; this changes the optimal binding from codons ending in C to no preference or a slight preference for U-ending codons. Among species of Drosophila, codon usage is constant with the exception of the Drosophila willistoni lineage which has shifted primary usage from C-ending codons to U/T ending codons only for these four amino acids. In Drosophila melanogaster Q containing tRNAs only predominate in old adults. We asked the question whether in D. willistoni these Q containing tRNAs might predominate earlier in development. As a surrogate for levels of modification we studied the expression of the gene (tgt) coding for the enzyme that catalyzes the substitution of Q for G in different life stages of D. melanogaster, D. pseudoobscura, and D. willistoni. Unlike the other two species, the highest tgt expression in D. willistoni is in young females producing eggs. Because tRNAs laid down in eggs persist through the early stages of development, this implies that Q modification occurs earlier in development in D. willistoni than in other Drosophila.     

Sequences of three transfer RNAs from mosquito mitochondria

The sequences of three transfer RNAs from mosquito cell mitochondria, tRNA_UCG^Arg, tRNA_GUC^Asp, and tRNA_GAU^Ile, determined using a combination of rapid ladder and fingerprinting procedures are reported. These were compared with hamster mitochondrial tRNA_UCG^Arg and tRNA_GUC^Asp determined similarly, and a bovine mitochondrial tRNA_GAU^Ile determined using a somewhat different approach. The primary sequences of the mosquito tRNAs were 35 to 65% homologous to the corresponding mammalian mitochondrial species, and bore little homology to “conventional” (bacterial or eucaryotic cytoplasmic) tRNA. The modification status of the mosquito mitochondrial tRNAs resembled that of mammalian mitochondrial tRNA. The results contribute to the generalization that metazoan mitochondrial tRNA constitutes a distinctive, albeit loosely structured, phylogenetic group.     

Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment

Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNA^Opt. We suspected a modification of the tRNA^Opt_AUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNA^Opt_AUG is converted to inosine. We identified tRNA^Opt_AUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms.     

Evolutionary repair reveals an unexpected role of the tRNA modification m1G37 in aminoacylation

The tRNA modification m¹G37, introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria because it suppresses translational frameshift errors at proline codons. However, because bacteria can tolerate high levels of mistranslation, it is unclear why loss of m¹G37 is not tolerated. Here, we addressed this question through experimental evolution of trmD mutant strains of Escherichia coli. Surprisingly, trmD mutant strains were viable even if the m¹G37 modification was completely abolished, and showed rapid recovery of growth rate, mainly via duplication or mutation of the proline-tRNA ligase gene proS. Growth assays and in vitro aminoacylation assays showed that G37-unmodified tRNA^Pro is aminoacylated less efficiently than m¹G37-modified tRNA^Pro, and that growth of trmD mutant strains can be largely restored by single mutations in proS that restore aminoacylation of G37-unmodified tRNA^Pro. These results show that inefficient aminoacylation of tRNA^Pro is the main reason for growth defects observed in trmD mutant strains and that proS may act as a gatekeeper of translational accuracy, preventing the use of error-prone unmodified tRNA^Pro in translation. Our work shows the utility of experimental evolution for uncovering the hidden functions of essential genes and has implications for the development of antibiotics targeting TrmD.     

Cell growth and tRNA-lys4 synthesis in mouse 3T3 cells

The RPC-5 chromatographic profiles of lys-tRNA were analyzed during the growth of 3T3 cells in culture. An inverse relationship was seen between tRNA₂^lys and tRNA₄^lys which was markedly influenced by medium changes. This interchange of tRNA₂^lys and tRNA₄^lys could be controlled by altering the levels of serum in the medium, or more precisely by altering the serum to cell ratio. A different change in lys-tRNA distribution was seen when the cells reached confluency. The amounts of tRNA₂^lys, tRNA₃^lys and tRNA₄^lys all decreased with a corresponding increase in either tRNA₅^lys or tRNA₆^lys. An identical change in lys-tRNA could be produced by shifting sparse cells into a medium containing 10% calf plasma instead of 10% serum. Both tRNA^lys profiles and cell growth were returned to normal when the cells were returned to medium with 10% fetal calf serum (FCS) or 10% calf plasma and fibroblast growth factor (FGF). A third alteration in tRNA^lys profiles was seen by the addition of cAMP to the cultures. A decrease in tRNA₅^lys and a corresponding increase in tRNA₆^lys was seen upon the addition of 10^?3 M db-cAMP and was accentuated by the simultaneous addition of 10^?3 M methyl isobutylxanthine.These data are consistent with an ordered sequence of tRNA^lys modification involving tRNA₂^lys, tRNA₃^lys, tRNA₄^lys, tRNA_5B^lys and tRNA₆^lys. Several of the factors which control proliferation appear to control the activity of different tRNA-modifying enzymes in this tRNA^lys pathway thereby controlling the levels of tRNA₄^lys, a tRNA previously shown to correlate directly with the proliferative rate of cells.     

Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.     

Cell growth and tRNA4 synthesis in mouse 3T3 cells

The RPC-5 chromatographic profiles of lys-tRNA were analyzed during the growth of 3T3 cells in culture. An inverse relationship was seen between tRNA₂^lys and tRNA₄^lys which was markedly influenced by medium changes. This interchange of tRNA₂^lys and tRNA₄^lys could be controlled by altering the levels of serum in the medium, or more precisely by altering the serum to cell ratio. A different change in lys-tRNA distribution was seen when the cells reached confluency. The amounts of tRNA₂^lys, tRNA₃^lys and tRNA₄^lys all decreased with a corresponding increase in either tRNA₅^lys or tRNA₆^lys. An identical change in lys-tRNA could be produced by shifting sparse cells into a medium containing 10% calf plasma instead of 10% serum. Both tRNA^lys profiles and cell growth were returned to normal when the cells were returned to medium with 10% fetal calf serum (FCS) or 10% calf plasma and fibroblast growth factor (FGF). A third alteration in tRNA^lys profiles was seen by the addition of cAMP to the cultures. A decrease in tRNA₅^lys and a corresponding increase in tRNA₆^lys was seen upon the addition of 10⁻³ M db-cAMP and was accentuated by the simultaneous addition of 10⁻³ M methyl isobutylxanthine.These data are consistent with an ordered sequence of tRNA^lys modification involving tRNA₂^lys, tRNA₃^lys, tRNA₄^lys, tRNA_5B^lys and tRNA₆^lys. Several of the factors which control proliferation appear to control the activity of different tRNA-modifying enzymes in this tRNA^lys pathway thereby controlling the levels of tRNA₄^lys, a tRNA previously shown to correlate directly with the proliferative rate of cells.     

N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network

N⁷-methylguanine at position 46 (m⁷G46) in tRNA is produced by tRNA (m⁷G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (ΔtrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the ΔtrmB strain and the lack of the m⁷G46 modification in tRNA^Phe were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the ΔtrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m⁷G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m¹G37, suggesting that the m⁷G46 positively affects their formations. Although the lack of the m⁷G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA^Phe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNA^Phe and tRNA^Ile. ³⁵S-Met incorporation into proteins revealed that protein synthesis in ΔtrmB cells is depressed above 70°C. At 80°C, the ΔtrmB strain exhibits a severe growth defect. Thus, the m⁷G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m⁷G46 modification supports introduction of other modifications.     

Commonality and diversity in tRNA substrate recognition in t6A biogenesis by eukaryotic KEOPSs

N ⁶-Threonylcarbamoyladenosine (t⁶A) is a universal and pivotal tRNA modification. KEOPS in eukaryotes participates in its biogenesis, whose mutations are connected with Galloway-Mowat syndrome. However, the tRNA substrate selection mechanism by KEOPS and t⁶A modification function in mammalian cells remain unclear. Here, we confirmed that all ANN-decoding human cytoplasmic tRNAs harbor a t⁶A moiety. Using t⁶A modification systems from various eukaryotes, we proposed the possible coevolution of position 33 of initiator tRNA^Met and modification enzymes. The role of the universal CCA end in t⁶A biogenesis varied among species. However, all KEOPSs critically depended on C32 and two base pairs in the D-stem. Knockdown of the catalytic subunit OSGEP in HEK293T cells had no effect on the steady-state abundance of cytoplasmic tRNAs but selectively inhibited tRNA^Ile aminoacylation. Combined with in vitro aminoacylation assays, we revealed that t⁶A functions as a tRNA^Ile isoacceptor-specific positive determinant for human cytoplasmic isoleucyl-tRNA synthetase (IARS1). t⁶A deficiency had divergent effects on decoding efficiency at ANN codons and promoted +1 frameshifting. Altogether, our results shed light on the tRNA recognition mechanism, revealing both commonality and diversity in substrate recognition by eukaryotic KEOPSs, and elucidated the critical role of t⁶A in tRNA^Ile aminoacylation and codon decoding in human cells.     

NSUN3 and ABH1 modify the wobble position of mt‐tRNAMet to expand codon recognition in mitochondrial translation

Mitochondrial gene expression uses a non‐universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt‐)tRNA^Met mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt‐tRNA^Met to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m⁵C34 of mt‐tRNA^Met to generate an f⁵C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m⁵C34 mt‐tRNA^Met in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt‐tRNA^Met function. Together, our data reveal how modifications in mt‐tRNA^Met are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNA^Met to recognise the different codons encoding methionine.     

Structural Changes of Yeast tRNATyr Caused by the Binding of Divalent Ions in the Presence of Spermine

Abstract

The existence of specific sites in tRNA for the binding of divalent cations has been seriously questioned by electrostatic considerations [Leroy & Guéron (1979) Biopolymers, 16, 2429–2446], However, our earlier studies of the binding of Mg²⁺ and Mn²⁺ to yeast tRNA^Tyr have indicated that spermine creates new binding sites for divalent cations [Weygand-Durasevi? et al. (1977) Biochim. Biophys. Acta, 479, 332–344; Nöthig-Laslo et al. (1981) Eur. J. Biochem. 117, 263–267]. We have now used yeast tRNA^Tyr, spin labeled at the hypermodified purine (i⁶A-37) in the anticodon loop, to study the effect of spermine on the binding of manganese ions. The presence of eight spermine molecules per tRNA^Tyr at high ionic strength (0.2 M NaCl, 0.05 M triethanolamine-HCl) and at low temperature (7°C) enhances the binding of manganese to tRNA^Tyr. This effect could not be explained by electrostatic binding. The initial binding of manganese to tRNA^Tyr affects the motional properties of the spin label indicating a change of the conformation of the anticodon loop. From the absence of the paramagnetic effect of manganese on the ESR spectra of the spin label one can conclude that the first binding site for manganese is at a distance from i⁶A-37, influencing the spin label motion through a long-range effect. The enhancement of the binding of manganese to tRNA^Tyr by spermine is lost upon destruction of its specific macromolecular structure and it does not occur in single stranded or in double-stranded polynucleotides. The observed effect can be explained by the binding of Mn²⁺ to new sites, created by the binding of spermine, which are specific for the macromolecular structure of tRNA.