Transfer RNA recognition by class I lysyl-tRNA synthetase from the Lyme disease pathogen Borrelia burgdorferi

Borrelia burgdorferi and other spirochetes contain a class I lysyl-tRNA synthetase (LysRS), in contrast to most eubacteria that have a canonical class II LysRS. We analyzed tRNA(Lys) recognition by B. burgdorferi LysRS, using two complementary approaches. First, the nucleotides of B. burgdorferi tRNA(Lys) in contact with B. burgdorferi LysRS were determined by enzymatic footprinting experiments. Second, the kinetic parameters for a series of variants of the B. burgdorferi tRNA(Lys) were then determined during aminoacylation by B. burgdorferi LysRS. The identity elements were found to be mostly located in the anticodon and in the acceptor stem. Transplantation of the identified identity elements into the Escherichia coli tRNA(Asp) scaffold endowed lysylation activity on the resulting chimera, indicating that a functional B. burgdorferi lysine tRNA identity set had been determined.     

Identification of novel inhibitors of methionyl-tRNA synthetase (MetRS) by virtual screening

Multiple inhibitors of the antibacterial target, Staphylococcus aureus MetRS, were identified by virtual screening. The process consisted of building a Catalyst^® pharmacophore from a ligand-S. aureus MetRS structure and using this pharmacophore to screen a commercial database. The top hits from this search were then docked into the S. aureus MetRS structure and this information was used to select compounds for testing. This resulted in a high hit rate of compounds that are in distinct structural classes from the known MetRS ligands.     

Cysteinyl-tRNA formation and prolyl-tRNA synthetase

Aminoacyl-tRNA (AA-tRNA) formation is a key step in protein biosynthesis. This reaction is catalyzed with remarkable accuracy by the AA-tRNA synthetases, a family of 20 evolutionarily conserved enzymes. The lack of cysteinyl-tRNA (Cys-tRNA) synthetase in some archaea gave rise to the discovery of the archaeal prolyl-tRNA (Pro-tRNA) synthetase, an enzyme capable of synthesizing Pro-tRNA and Cys-tRNA. Here we review our current knowledge of this fascinating process.     

氨酰tRNA 合成酶抑制剂作为新型抗感染药物的研究进展

细菌耐药性的不断上升对现有阶段的抗生素类药物提出了一个严峻的挑战,同时也掀起了针对于新靶标的抗菌药物的研究。氨酰tRNA合成酶(aaRS)催化特定氨基酸连接到相应的tRNA分子上,在蛋白质的合成过程中起着必不可少的作用。氨酰tRNA合成酶的抑制会导致蛋白质合成的停止,扰乱细菌和真菌的生长,因此氨酰tRNA合成酶是一类潜在的抗感染靶标。本文分别综述了天然产物及其衍生的aaRS抑制剂,底物和反应中间体模拟物,通过合成和通过虚拟筛选得到的aaRS抑制剂作为新型抗细菌和抗真菌药物的研究进展,并对aaRS的靶标特点、分类和催化机制作一简要介绍。     

Carotenoid Chemistry and Biochemistry : Edited by G. Britton and T.W. Goodwin Pergamon Press; Oxford, 1982 ix + 399 pages. £47.50

The dimeric tryptophanyl-tRNA synthetase from beef pancreas has been found to activate 2 tryptophans/mol enzyme [Eur. J. Biochem. (1982) 128, 389–398]. By using quenched-flow and stopped-flow methods under presteady-state conditions, we show that only one enzyme subunit operates at a time in the aminoacylation of tRNA^Trp and that the transfer reaction is not the rate-limiting step in the overall aminoacylation process.     

氨酰-tRNA合成酶的进化差异

大量研究显示,细菌与真核生物中的许多氨酰-tRNA合成酶(aaRS)在一些细菌与真核生物中的基因进化机制与模式、氨酰化途径和结构与功能的进化模式等方面往往有着明显的差异。通过对这些差异的深入研究,对于理解蛋白质的结构与功能的进化将是非常有帮助的。虽然造成这些差异的机制目前仍不清楚,但是,所有的这些差异似乎提示,在细菌与真核生物的一些基本生命活动过程中的某些方面,可能还存在着目前尚未被人们所认识到的较大差异。     

P, P-bis(5′-adenosyl)triphosphate (Ap3A) as a substrate and a product of mammalian tryptophanyl-tRNA synthetase

Bovine tryptophanyl-tRNA synthetase (TrpRS, E.C. 6.1.1.2) is unable to catalyze in vitro formation of Ap₄A in contrast to some other aminoacyl-tRNA synthetases. However, in the presence of -tryptophan, ATP-Mg²⁺ and ADP the enzyme catalyzes the Ap₃A synthesis via adenylate intermediate. Ap₃A (not Ap₄A) may serve as a substrate for TrpRS in the reaction of E·(Trp AMP) formation and in the tRNA^Trp charging. The K_m value for Ap₃A was higher than the K_m for ATP (approx. 1.00 vs. 0.22 mM) and V_max was 3 times lower than for ATP. The Zn²⁺-deficient enzyme catalyzes Ap₃A synthesis in the absence of exogenous ADP due to ATPase activity of Zn²⁺-deprived TrpRS. The inability of mammalian TrpRS to synthesize Ap₄A, might be considered as a molecular tool preventing the removal of Zn²⁺ due to chelation by Ap₄A and therefore preserving the enzyme activity.     

Searching tRNA sequences for relatedness to aminoacyl-tRNA synthetase families

tRNA sequences were analyzed for sequence features correlated with known classes of aminoacyl-tRNA synthetase enzymes. The tRNAs were searched for distinguishing nucleotides anywhere in their sequences. The analyses did not find nucleotides predictive of synthetase class membership. We conclude that such nucleotides never existed in tRNA sequences or that they existed and were lost from many of the tRNA sequences during evolution.Based on a presentation made at a workshop—Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: H.B. Nicholas, Jr.     

Processivity of translation in the eukaryote cell: Role of aminoacyl-tRNA synthetases

Several lines of evidence led to the conclusion that mammalian ribosomal protein synthesis is a highly organized biological process in vivo. A wealth of data support the concept according to which tRNA aminoacylation, formation of the ternary complex on EF1A and delivery of aminoacyl-tRNA to the ribosome is a processive mechanism where tRNA is vectorially transferred from one component to another. Polypeptide extensions, referred to as tRBDs (tRNA binding domains), are appended to mammalian and yeast aminoacyl-tRNA synthetases. The involvement of these domains in the capture of deacylated tRNA and in the sequestration of aminoacylated tRNA, suggests that cycling of tRNA in translation is mediated by the processivity of the consecutive steps. The possible origin of the tRBDs is discussed.     

通过基因组相减从悬铃木突变体中分离缺失DNA

悬铃木（Ｐｌａｔａｎｕｓ ｏｃｃｉｄｅｎｔａｌｉｓ Ｌ．）突变体是其种子经γ射线辐射培育出的一种营养生长正常而开花发育过程受阻的不育植株。经过１０多年的观察研究证明：突变体的花芽不能形成是因为突变体细胞染色体有部分缺失。应用基因组相减技术分离这些在突变体中缺失但在野生型中存在的ＤＮＡ,经过４次循环富集缺失片段后克隆了１个２．０ ｋｂ 的ＤＮＡ 片段,ＤＮＡ 杂交证明此片段只存在于野生型基因组中而不存在于突变体中。将基因组相减方法应用在复杂植物基因组中获得成功的研究还未见报道     

Identification of low-dye-binding (ldb) mutants of Saccharomyces cerevisiae

We have completed the identification of Saccharomyces cerevisiae genes that are defective in previously isolated ldb (low-dye-binding) mutants. This was done by complementation of the mutant's phenotype with DNA fragments from a genomic library and by running standard tests of allelism with single-gene deletion mutants of similar phenotype. The results were as follows: LDB2 is allelic to ERD1; LDB4 to SPC72; LDB5 to RLR1; LDB6 to GON7/YJL184W; LDB7 to YBL006C; LDB9 to ELM1; LDB10 to CWH36; LDB11 to COG1; LDB12 to OCH1; LDB13 to VAN1; LDB14 to BUD32; and LDB15 to PHO85. Since the precise function of some of the genes is not known, these data may contribute to the functional characterization of the S. cerevisiae genome.     

Speculations on the origin of the genetic code

The most primitive code is assumed to be a GC code: GG coding for glycine, CC coding for proline, GC coding for alanine, CG coding for arginine. The genetic code is assumed to have originated with the coupling of glycine to its anticodon CC mediated by a copper-montmorillonite. The polymerization of polyproline followed when it was coupled to its anticodon GG. In this case the aminoacyl-tRNA synthetase was a copper-montmorillonite. The first membrane is considered to be a sheet formed from polyglycine. As the code grew more complicated, the alternative hydrophobic-hydrophilic polypeptide (alanine-arginine) was coded for by the alternating CG copolymer. This alternating polypeptide (ala-arg) began to function as both a primitive membrane and as an aminoacyl-tRNA synthetase. The evolution of protein structure is tightly coupled to the evolution of the membrane. The a helix was evolved as lipids became part of the structure of biological membranes. The membrane finally became the fluid mosaic structure that is now universal.Based on a presentation made at a workshop-Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code-held at Berkeley, CA, July 17–20, 1994     

Comparative structural dynamics of Tyrosyl-tRNA synthetase complexed with different substrates explored by molecular dynamics

Several molecular dynamics simulations of S. aureus Tyrosyl-tRNA synthetase (TyrRS) in its free form and complexed with Tyr, ATP, tyrosyl adenylate and inhibitor respectively have been carried out to investigate the ligand-linked conformational stability changes associated with its catalytic cycle. The results show that unliganded S. aureus TyrRS samples a more relaxed conformational space than substrate-bound TyrRS. There are three high flexibility regions encompassing residues 114–118, 128–133, and 226–238 respectively. The region which includes the KMSKS motif (KFGKS in S. aureus TyrRS) shows the highest difference in fluctuations. Hydrogen bond network formed by Tyr, ATP, tyrosyl adenylate and inhibitor with S. aureus TyrRS is discussed. Our simulations suggest the induced-fit conformational changes of the KMSKS loop as follows: the KMSKS loop of substrate-free S. aureus TyrRS adopts an open conformation. The tyrosine binds in the pocket with the KMSKS loop balancing between semi-open and open forms. The ATP binding induces the KMSKS loop to the open form. After the Tyr-AMP is formed, the first two residues of KMSKS loop twists in an anticlockwise direction and drives the loop in a conformation similar to the closed one, while those of the last three GKS residues adopt a conformation between semi-open and open conformation. This conformational change may probably be involved in the initial tRNA binding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protein synthesis and loss of viability of rice seeds: Effect of polyamines on in vitro translation

The capacity of rice ( Oryza sativa L. cv. Rupsail) seeds to synthesize protein shows a gradual decline with loss of seed viability, and the lesion in post-ribosomal fraction is greater than that of ribosome. Spermine (0.4 m M ) is the most effective among the polyamines in enhancing the rate of protein synthesis. There are also indications that impairment of aminoacyl-tRNA synthetase may be one of the causes for this decreasing ability in protein synthesis by aged seeds.