Misacylation of yeast amber suppressor tRNA(Tyr) by E. coli lysyl-tRNA synthetase and its effective repression by genetic engineering of the tRNA sequence

Through an exhaustive search for Escherichia coli aminoacyl-tRNA synthetase(s) responsible for the misacylation of yeast suppressor tRNA(Tyr), E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA(Tyr) (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA(Tyr)/tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid in E. coli translation system, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering. This "optimized" tRNA together with our mutant TyrRS should serve as an efficient and faithful tool for site-specific incorporation of unnatural amino acids into proteins in a protein-synthesizing system in vitro or in vivo.     

Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine

The Methanococcus jannaschii tRNA(Tyr)/TyrRS pair has been engineered to incorporate unnatural amino acids into proteins in E. coli. To reveal the structural basis for the altered specificity of mutant TyrRS for O-methyl-L-tyrosine (OMeTyr), the crystal structures for the apo wild-type and mutant M. jannaschii TyrRS were determined at 2.66 and 3.0 A, respectively, for comparison with the published structure of TyrRS complexed with tRNA(Tyr) and substrate tyrosine. A large conformational change was found for the anticodon recognition loop 257-263 of wild-type TyrRS upon tRNA binding in order to facilitate recognition of G34 of the anticodon loop through pi-stacking and hydrogen bonding interactions. Loop 133-143, which is close to the tRNA acceptor stem-binding site, also appears to be stabilized by interaction with the tRNA(Tyr). Binding of the substrate tyrosine results in subtle and cooperative movements of the side chains within the tyrosine-binding pocket. In the OMeTyr-specific mutant synthetase structure, the signature motif KMSKS loop and acceptor stem-binding loop 133-143 were surprisingly ordered in the absence of bound ATP and tRNA. The active-site mutations result in altered hydrogen bonding and steric interactions which favor binding of OMeTyr over L-tyrosine. The structure of the mutant and wild-type TyrRS now provide a basis for generating new active-site libraries to evolve synthetases specific for other unnatural amino acids.     

Changing the amino acid specificity of yeast tyrosyl-tRNA synthetase by genetic engineering

In an attempt to generate mutant aminoacyl-tRNA synthetases capable of charging non-canonical amino acids, a series of yeast tyrosyl-tRNA synthetase (TyrRS) mutants was constructed by site-specific mutagenesis of putative active site residues, which were deduced by analogy with those of Bacillus stearothermophilus TyrRS. Among these mutants, one with the replacement of tyrosine at position 43 by glycine, "Y43G," was found to be able to utilize several 3-substituted tyrosine analogues as substrates for aminoacylation. The catalytic efficiency (k(cat)/K(m)) of mutant Y43G for aminoacylation with L-tyrosine was about 400-fold decreased as compared to that of the wild-type TyrRS. On the other hand, the ability to utilize 3-iodo-L-tyrosine was newly generated in this mutant TyrRS, since the wild-type TyrRS could not accept 3-iodo-L-tyrosine at all under physiological conditions. This mutant TyrRS should serve as a new tool for site-specific incorporation of non-canonical amino acids, such as those in 3-substituted tyrosine analogues, into proteins in an appropriate translation system in vivo or in vitro.     

Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.     

Cooperative unfolding of Escherichia coli ribosome recycling factor originating from its domain-domain interaction and its implication for function

Cooperative unfolding of Escherichia coli ribosome recycling factor (RRF) and its implication for function were investigated by comparing the in vitro unfolding and the in vivo activity of wild-type E. coli RRF and its temperature-sensitive mutant RRF(V117D). The experiments show that mutation V117D at domain I could perturb the domain II structure as evidenced in the near-UV CD and tyrosine fluorescence spectra though no significant globular conformation change occurred. Both equilibrium unfolding induced by heat or denaturant and kinetic unfolding induced by denaturant obey the two-state transition model, indicating V117D mutation does not perturb the efficient interdomain interaction, which results in cooperative unfolding of the RRF protein. However, the mutation significantly destabilizes the E. coli RRF protein, moving the thermal unfolding transition temperature range from 50-65 to 35-50 degrees C, which spans the non-permissive temperature for the growth of E. coli LJ14 strain (frr(ts)). The in vivo activity assays showed that although V117D mutation results in a temperature sensitive phenotype of E. coli LJ14 strain (frr(ts)), over-expression of mutant RRF(V117D) can eliminate the temperature sensitive phenotype at the non-permissive temperature (42 degrees C). Taking all the results into consideration, it can be suggested that the mechanism of the temperature sensitive phenotype of the E. coli LJ14 cells is due to inactivation of mutant RRF(V117D) caused by unfolding at the non-permissive temperatures.     

可视化突变建模、定点突变和表达超耐热菌D-乳酸脱氢酶

目的 构建产天然防腐剂苯乳酸的工程菌。方法 分析超耐热菌（Aquifex aeolicus,A.aeolicus ）D-乳酸脱氢酶（D-LDH）的三维构象,并与构建的可视化突变体三维模型进行对比,通过比较酶活性中心氨基酸残基与底物的空间构象,优选最佳模型进行定点突变,克隆、表达和苯乳酸发酵实验。结果 优选到F49A和Y297S两个单突变模型和一个F49A/Y297S双突变模型;分别进行定点突变和工程菌构建,三个突变工程菌,均能发酵产生苯乳酸。结论 可视化定点突变乳酸脱氢酶可作为构建高产苯乳酸工程菌的有效方法。     

Mutants of Escherichia coli with an altered tyrosyl-transfer ribonucleic acid synthetase

We have isolated several mutants defective in the gene for tyrosyl-transfer ribonucleic acid (tRNA) synthetase (tyrS). One of these mutants is described in detail. It was isolated as a tyrosine auxotroph with defects both in the tyrosyl-tRNA synthetase and in the tyrosine biosynthetic enzyme, prephenate dehydrogenase. It also had derepressed levels of the tyrosine-specific 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase. The latter finding suggested that a wild-type tyrS gene was required for repression of the tyrosine biosynthetic enzymes. The following results demonstrated that this hypothesis was not correct. (i) When the defective tyrS gene was transferred to another strain, the tyrosine-specific DAHP synthetase in that strain was not derepressed, and (ii) two other mutants with defective tyrosyl-tRNA synthetases had repressed levels of the tyrosine biosynthetic enzymes. The tyrS gene was located near minute 32 on the Escherichia coli chromosome by interrupted mating experiments.     

Palmitoylation of either Cys-3 or Cys-5 is required for the biological activity of the Lck tyrosine protein kinase.

Palmitoylation can regulate both the affinity for membranes and the biological activity of proteins. To study the importance of the palmitoylation of the Src-like tyrosine protein kinase p56lck in the function of the protein, Cys-3, Cys-5, or both were mutated to serine, and the mutant proteins were expressed stably in fibroblasts and T cells. Both Cys-3 and Cys-5 were apparent sites of palmitoylation in Lck expressed in fibroblasts, as only the simultaneous mutation of both Cys-3 and Cys-5 caused a large reduction in the incorporation of [3H]palmitic acid. The double mutant S3/5Lck was no longer membrane bound when examined by either immunofluorescence or cell fractionation. This indicated that palmitoylation was required for association of Lck with the plasma membrane. Since the S3/5Lck protein was myristoylated, myristoylation of Lck is not sufficient for membrane binding. When Cys-3, Cys-5, or both Cys-3 and Cys-5 were changed to serine in activated F505Lck, palmitoylation of either Cys-3 or Cys-5 was found to be necessary and sufficient for the transformation of fibroblasts and for the induction of spontaneous, antigen-independent interleukin-2 production in the T-helper cell line DO-11.10. Nonpalmitoylated F505Lck exhibited little activity in vivo, where it did not induce elevated levels of tyrosine phosphorylation, and in vitro, where it was unable to phosphorylate angiotensin in an in vitro kinase assay. These findings suggest that F505Lck must be anchored stably to membranes to become activated. Because palmitoylation is dynamic, it may be involved in regulating the cellular localization of p56(lck), and consequently its activity, by altering the proximity of p56(lck) to its activators and/or targets.     

Genetic mapping of nth, a gene affecting endonuclease III (thymine glycol-DNA glycosylase) in Escherichia coli K-12.

The nth gene of Escherichia coli affects the production of endonuclease III, a glycosylase-endonuclease that attacks DNA damaged by oxidizing agents or by ionizing radiation. An nth insertion mutant and a deletion mutant were studied. nth is located between add and tyrS on the linkage map of E. coli K-12 and was 97% linked to tyrS in a transduction with phage P1.     

Selection and characterization of Escherichia coli variants capable of growth on an otherwise toxic tryptophan analogue

Escherichia coli isolates that were tolerant of incorporation of high proportions of 4-fluorotryptophan were evolved by serial growth. The resultant strain still preferred tryptophan for growth but showed improved growth relative to the parental strain on other tryptophan analogues. Evolved clones fully substituted fluorotryptophan for tryptophan in their proteomes within the limits of mass spectral and amino acid analyses. Of the genes sequenced, many genes were found to be unaltered in the evolved strain; however, three genes encoding enzymes involved in tryptophan uptake and utilization were altered: the aromatic amino acid permease (aroP) and tryptophanyl-tRNA synthetase (trpS) contained several amino acid substitutions, and the tyrosine repressor (tyrR) had a nonsense mutation. While kinetic analysis of the tryptophanyl-tRNA synthetase suggests discrimination against 4-fluorotryptophan, an analysis of the incorporation and growth patterns of the evolved bacteria suggest that other mutations also aid in the adaptation to the tryptophan analogue. These results suggest that the incorporation of unnatural amino acids into organismal proteomes may be possible but that extensive evolution may be required to reoptimize proteins and metabolism to accommodate such analogues.     

Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo

Proteins containing unnatural amino acids have immense potential in biotechnology and medicine. We prepared several histidine analogues including a novel histidine analogue, beta-(1,2,3-triazol-4-yl)-DL-alanine. These histidine analogues were assayed for translational activity in histidine-auxotrophic Escherichia coli strain UTH780. We observed that several histidine analogues, including our novel histidine analogue, were efficiently incorporated into the protein in vivo; however, other analogues were rejected. These results suggest that the hydrogen atom at a specific position seriously affects incorporation.     

Biosynthesis of the Branched-Chain Amino Acids in Yeast: a Trifluoroleucine-Resistant Mutant with Altered Regulation of Leucine Uptake

A trifluoroleucine-resistant mutant of yeast has been isolated that exhibits reduced incorporation of the analogue into protein (15%) of that in the wild type. In the mutant, uptake of the analogue and leucine into the expandable (water-extractable) pool is enhanced, passage from the expandable to the conversion (nonwater-, ethanol-extractable) pool is unaffected, and endogenous synthesis of leucine is normally regulated. Although the leucyl transfer ribonucluic acid (tRNA) synthetase appears normal, and the tRNA^leu has wild-type acceptor activities in vitro and in vivo, the level of the mutant trifluoroleucyl tRNA pool is only 2 to 3% of that in the wild type. The data support the idea of a mutation affecting passage between the conversion pool and the site of charging of the analogue. The mutation is dominant and exhibits pleiotropic effects: the first leucine biosynthetic enzyme appears nonrepressible, and the leucine, valine, and tyrosine uptake systems are constitutively elevated (three- to fourfold) in the absence of exogenous amino acids.     

Point mutation of a tyrosine in the linker region of Syk results in a gain of function

The protein tyrosine kinase Syk plays an essential role in Fc epsilon RI-mediated histamine release in mast cells by regulating the phosphorylation of other proteins. We investigated the functional role of a putative Syk phosphorylation site, Tyr317. This tyrosine in the linker region of Syk is a possible site for binding by the negative regulator Cbl. Syk with Tyr317 mutated to Phe (Y317F) was expressed in a Syk-negative variant of the RBL-2H3 mast cells. Compared with cells expressing wild-type Syk, expression of the Y317F mutant resulted in an increase in the Fc epsilon RI-mediated tyrosine phosphorylation of phospholipase C-gamma and a dramatic enhancement of histamine release. The in vivo Fc epsilon RI-induced tyrosine phosphorylation of wild-type Syk and that of the Y317F mutant were similar. Although the Fc epsilon RI-induced tyrosine phosphorylation of total cellular proteins was enhanced in the cells expressing the Y317F Syk, the phosphorylation of some other molecules, including the receptor subunits, Vav and mitogen-activated protein kinase, was not increased. The Fc epsilon RI-induced phosphorylation of Cbl was downstream of Syk kinase activity and was unchanged by expression of the Y317F mutation. These data indicate that Tyr317 in the linker region of Syk functions to negatively regulate the signals leading to degranulation.     

Ribosomes with large synthetic N-terminal extensions of protein S15 are active in vivo

The genes for ribosomal proteins S4, S13 or S15 were fused with the gene for staphylococcal protein A, or derivatives thereof (2A'-7A'). The gene fusions were introduced into Escherichia coli strains, mutated in the corresponding ribosomal protein gene, by transformation. These mutated ribosomal proteins cause a phenotype that can be complemented. Thus, the phenotype of the transformants was tested and the ribosomal proteins were analyzed. The S4 N-terminal fusion protein severely disturbed growth of both the mutant and the wild-type strains. The S13 C-terminal fusion protein was proteolyzed close to the fusion point, giving a ribosomal protein moiety that could assemble into the ribosome normally. S15 N-terminal fusion proteins complemented a cold-sensitive strain lacking protein S15 in its ribosomes. These fused proteins were assembled into active ribosomes. The position of S15 in the 30S ribosomal subunit is well known. Therefore, in structural studies of the ribosome in vivo, the S15 fusion proteins can be used as a physical reporter for S15.     

Dab1 tyrosine phosphorylation sites relay positional signals during mouse brain development

BACKGROUND: The extracellular protein Reln controls neuronal migrations in parts of the cortex, hippocampus and cerebellum. In vivo, absence of Reln correlates with up-regulation of the docking protein Dab1 and decreased Dab1 tyrosine phosphorylation. Loss of the Reln receptor proteins, apolipoprotein receptor 2 and very low density lipoprotein receptor, results in a Reln-like phenotype accompanied by increased Dab1 protein expression. Complete loss of Dab1, however, recapitulates the Reln phenotype. RESULTS: To determine whether Dab1 tyrosine phosphorylation affects Dab1 protein expression and positioning of embryonic neurons, we have identified Dab1 tyrosine phosphorylation sites. We then generated mice in which the Dab1 protein had all the potential tyrosine phosphorylation sites mutated. This mutant protein is not tyrosine phosphorylated during brain development and is not upregulated to the extent observed in the Reln or the apoER2 and VLDLR receptor mutants. Animals expressing the non-phosphorylated Dab1 protein have a phenotype similar to the dab1-null mutant. CONCLUSIONS: Dab1 is downregulated by the Reln signal in neurons in the absence of tyrosine phosphorylation. Dab1 tyrosine phosphorylation sites and not downregulation of Dab1 protein are required for Reln signaling.     

利用机器学习提高M. jannaschii酪氨酰tRNA合成酶底物特异性分子建模预测的准确度

设计结合不同化学结构底物的酶结合袋是一个巨大的挑战. 传统的湿实验要筛选成千上万甚至上百万个突变体来寻找对特定配体结合的突变体,此过程需要耗费大量的时间和资源. 为了加快筛选过程,我们提出了一种新的工作流程,将分子建模和数据驱动的机器学习方法相结合,生成具有高富集率的突变文库,用于高效筛选能识别特定底物的蛋白质突变体. M. jannaschii酪氨酰tRNA合成酶（Mj. TyrRS）能识别特定的非天然氨基酸并催化形成氨酰tRNA,其不同的突变体能够识别不同结构的非天然氨基酸,并且已经有了许多报道和数据的积累,因此我们使用TyrRS作为一个例子来进行此筛选流程的概念验证. 基于已知的多个Mj. TyrRS的晶体结构及分子建模的结果,我们发现D158G/P是影响残基158~163位α螺旋蛋白骨架变化的关键突变. 我们的模拟结果表明,在含有687个突变体的测试数据中,与随机突变相比,分子建模和打分函数计算排序可以将目标突变体的富集率提高2倍,而使用已知突变体和对应的非天然氨基酸数据训练的机器学习模型进行校准后,筛选富集率可提高11倍. 这种分子建模和机器学习相结合的计算和筛选流程非常有助于Mj.TyrRS的底物特异性设计,可以大大减少湿实验的时间和成本. 此外,这种新方法在蛋白质计算设计领域具有广泛的应用前景.     

Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: enhancement of PHA production and alteration of polymer molecular weight

In vitro evolution of the polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp. 61-3 (phaC1(Ps)) has been performed to generate highly active enzymes. In this study, a positive mutant of PHA synthase, Glu130Asp (E130D), was characterized in detail in vivo and in vitro. Recombinant Escherichia coli strain JM109 harboring the E130D mutant gene accumulated 10-fold higher (1.0 wt %) poly(3-hydroxybutyrate) [P(3HB)] from glucose, compared to recombinant E. coli harboring the wild-type PHA synthase gene (0.1 wt %). Recombinant E. coli strain LS5218 harboring the E130D PHA synthase gene grown on dodecanoate produced more poly(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] (20 wt %) copolymer than an LS5218 strain harboring the wild-type PHA synthase gene (13 wt %). The E130D mutation also resulted in the production of copolymer with a slight increase in 3HB composition, compared to copolymer produced by the wild-type PHA synthase. In vitro enzyme activities of the E130D PHA synthase toward various 3-hydroxyacyl-CoAs (4-10 carbons in length) were all higher than those of the wild-type enzyme. The combination of the E130D mutation with other beneficial mutations, such as Ser325Thr and Gln481Lys, exhibited a synergistic effect on in vivo PHA production and in vitro enzyme activity. Interestingly, gel-permeation chromatography analysis revealed that the E130D mutation also had a synergistic effect on the molecular weight of polymers produced in vivo.     

Synthetase competition and tRNA context determine the in vivo identify of tRNA discriminator mutants.

The discriminator nucleotide (position 73) in tRNA has long been thought to play a role in tRNA identity as it is the only variable single-stranded nucleotide that is found near the site of aminoacylation. For this reason, a complete mutagenic analysis of the discriminator in three Escherichia coli amber suppressor tRNA backgrounds was undertaken; supE and supE-G1C72 glutamine tRNAs, gluA glutamate tRNA and supF tyrosine tRNA. The effect of mutation of the discriminator base on the identity of these tRNAs in vivo was assayed by N-terminal protein sequencing of E. coli dihydrofolate reductase, which is the product of suppression by the mutated amber suppressors, and confirmed by amino acid specific suppression experiments. In addition, suppressor efficiency assays were used to estimate the efficiency of aminoacylation in vivo. Our results indicate that the supE glutamine tRNA context can tolerate multiple mutations (including mutation of the discriminator and first base-pair) and still remain predominantly glutamine-accepting. Discriminator mutants of gluA glutamate tRNA exhibit increased and altered specificity probably due to the reduced ability of other synthetases to compete with glutamyl-tRNA synthetase. In the course of these experiments, a glutamate-specific mutant amber suppressor, gluA-A73, was created. Finally, in the case of supF tyrosine tRNA, the discriminator is an important identity element with partial to complete loss of tyrosine specificity resulting from mutation at this position. It is clear from these experiments that it may not be possible to assign a specific role in tRNA identity to the discriminator. The identity of a tRNA in vivo is determined by competition among aminoacyl-tRNA synthetases, which is in turn modulated by the nucleotide substitution as well as the tRNA context.