Changing the acceptor identity of a transfer RNA by altering nucleotides in a "variable pocket"

The specificity of tRNA(Arg) (arginine transfer RNA) for aminoacylation (its acceptor identity) were first identified by computer analysis and then examined with amber suppressor tRNAs in Escherichia coli. On replacing two nucleotides in tRNA(Phe) (phenylalanine transfer RNA) with the corresponding nucleotides from tRNA(Arg), the acceptor identity of the resulting tRNA was changed to that of tRNA(Arg). The nucleotides used in the identity transformation occupy a "variable pocket" structure on the surface of the tRNA molecule where two single-stranded loop segments interact. The middle nucleotide in the anticodon also probably contributes to the interaction, since an amber suppressor of tRNA(Arg) had an acceptor identity for lysine as well as arginine.     

Association of transfer RNA acceptor identity with a helical irregularity

The aminoacylation specificity ("acceptor identity") of transfer RNAs (tRNAs) has previously been associated with the position of particular nucleotides, as opposed to distinctive elements of three-dimensional structure. The contribution of a G.U wobble pair in the acceptor helix of tRNA(Ala) to acceptor identity was examined with synthetic amber suppressor tRNAs in Escherichia coli. The acceptor identity was not affected by replacing the G.U wobble pair in tRNA(Ala) with a G.A, C.A, or U.U wobble pair. Furthermore, a tRNA(Ala) acceptor identity was conferred on tRNA(Lys) when the same site in the acceptor helix was replaced with any of several wobble pairs. Additional data with tRNA(Ala) show that a substantial acceptor identity was retained when the G.U wobble pair was translocated to another site in the acceptor helix. These results suggest that the G.U wobble pair induces an irregularity in the acceptor helix of tRNA(Ala) to match a complementary structure in the aminoacylating enzyme.     

Autoreactive epitope defined as the anticodon region of alanine transfer RNA

Autoantibodies to aminoacyl-transfer RNA (tRNA) synthetases are common in the human autoimmune diseases polymyositis and dermatomyositis. Sera of the PL-12 specificity contain separate antibodies reacting with alanyl-tRNA synthetase and alanine tRNA (tRNAAla). The antibodies to tRNA recognize at least six distinguishable human tRNAAla species grouped into two sequence families. The antibody-reactive determinants on the tRNA were identified through ribonuclease protection and oligonucleotide binding experiments. The antibody binding site is a seven- to nine-nucleotide sequence containing the anticodon loop and requires an intact anticodon. No requirement for anticodon stem structure or sequence is observed, although the 5' portion of the stem is protected from nuclease attack. Antibodies from several patients appear to share the same specificitym, indicating that the antibodies are induced by a unique sequence feature in the immunogen.     

The anticodon contains a major element of the identity of arginine transfer RNAs

The contribution of the anticodon to the discrimination between cognate and noncognate tRNAs by Escherichia coli Arg-tRNA synthetase has been investigated by in vitro synthesis and aminoacylation of elongator methionine tRNA (tRNA(mMet) mutants. Substitution of the Arg anticodon CCG for the Met anticodon CAU leads to a dramatic increase in Arg acceptance by tRNA(mMet). A nucleotide (A20) previously identified by others in the dihydrouridine loop of tRNA(Arg)s makes a smaller contribution to the conversion of tRNA(mMet) identity from Met to Arg. The combined anticodon and dihydrouridine loop mutations yield a tRNA(mMet) derivative that is aminoacylated with near-normal kinetics by the Arg-tRNA synthetase.     

Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution

The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) complexed with its cognate glutaminyl transfer RNA (tRNA(Gln] and adenosine triphosphate (ATP) has been derived from a 2.8 angstrom resolution electron density map and the known protein and tRNA sequences. The 63.4-kilodalton monomeric enzyme consists of four domains arranged to give an elongated molecule with an axial ratio greater than 3 to 1. Its interactions with the tRNA extend from the anticodon to the acceptor stem along the entire inside of the L of the tRNA. The complexed tRNA retains the overall conformation of the yeast phenylalanine tRNA (tRNA(Phe] with two major differences: the 3' acceptor strand of tRNA(Gln) makes a hairpin turn toward the inside of the L, with the disruption of the final base pair of the acceptor stem, and the anticodon loop adopts a conformation not seen in any of the previously determined tRNA structures. Specific recognition elements identified so far include (i) enzyme contacts with the 2-amino groups of guanine via the tRNA minor groove in the acceptor stem at G2 and G3; (ii) interactions between the enzyme and the anticodon nucleotides; and (iii) the ability of the nucleotides G73 and U1.A72 of the cognate tRNA to assume a conformation stabilized by the protein at a lower free energy cost than noncognate sequences. The central domain of this synthetase binds ATP, glutamine, and the acceptor end of the tRNA as well as making specific interactions with the acceptor stem.2+t is     

Nucleotide sequence of a lysine transfer ribonucleic Acid from bakers' yeast

The nucleotide sequence of one of the two major lysine transfer RNA's from bakers' yeast has been determined. Its structure is compared to that of a lysine tRNA from a haploid yeast. A total of 21 nucleotides differ in the two molecules. Only the T-psi-C-G (thymidine-pseudouridine-cytidine-guanosine) loop and its supporting stem are identical.     

Nucleotide sequence of the "denaturable" leucine transfer RNA from yeast

The nucleotide sequence of " denaturable"leucine acceptor transfer RNA (tRNA(Leu)(3)) from baker's yeast was determined on (32)P-labeled material. The molecule is 85 nucleotides long and can be folded into the "cloverleaf" model for secondary structure. The basis on which the sequence was deduced from the products of complete enzymatic digestion, prior to its unambiguous determination, is presented.     

Nucleotides in yeast tRNAPhe required for the specific recognition by its cognate synthetase

An analysis of the aminoacylation kinetics of unmodified yeast tRNAPhe mutants revealed that five single-stranded nucleotides are important for its recognition by yeast phenylalanyl-tRNA synthetase, provided they were positioned correctly in a properly folded tRNA structure. When four other tRNAs were changed to have these five nucleotides, they became near-normal substrates for the enzyme.     

Suppression of mutations in mitochondrial DNA by tRNAs imported from the cytoplasm

Mitochondrial import of a cytoplasmic transfer RNA (tRNA) in yeast requires the preprotein import machinery and cytosolic factors. We investigated whether the tRNA import pathway can be used to correct respiratory deficiencies due to mutations in the mitochondrial DNA and whether this system can be transferred into human cells. We show that cytoplasmic tRNAs with altered aminoacylation identity can be specifically targeted to the mitochondria and participate in mitochondrial translation. We also show that human mitochondria, which do not normally import tRNAs, are able to internalize yeast tRNA derivatives in vitro and that this import requires an essential yeast import factor.     

豆豉纤溶酶基因的克隆及其在大肠杆菌中的表达

根据已发表的豆豉纤溶酶基因DNA序列(GeneBank AY720895.2),设计并合成了一对引物,应用PCR技术以筛选的来自豆豉的产纤溶酶的芽孢杆菌的总DNA为模板,扩增出了豆豉纤溶酶基因,并将其克隆到pMD18-T载体上,进行了序列测定,测序结果经Genetool序列分析表明该基因长1 089 bp,编码363个氨基酸,与已发表Brevibacillus laterosporus豆豉纤溶酶基因序列在核苷酸水平上与所发表的序列有98%的同源性;在氨基酸水平上与已发表的芽孢杆菌豆豉纤溶酶同源性为100%。并将该基因克隆到大肠杆菌表达载体pET22b上,在大肠杆菌中实现了高效表达。     

Structural basis for misaminoacylation by mutant E. coli glutaminyl-tRNA synthetase enzymes

A single-site mutant of Escherichia coli glutaminyl-synthetase (D235N, GlnRS7) that incorrectly acylates in vivo the amber suppressor supF tyrosine transfer RNA (tRNA(Tyr] with glutamine has been described. Two additional mutant forms of the enzyme showing this misacylation property have now been isolated in vivo (D235G, GlnRS10; I129T, GlnRS15). All three mischarging mutant enzymes still retain a certain degree of tRNA specificity; in vivo they acylate supE glutaminyl tRNA (tRNA(Gln] and supF tRNA(Tyr) but not a number of other suppressor tRNA's. These genetic experiments define two positions in GlnRS where amino acid substitution results in a relaxed specificity of tRNA discrimination. The crystal structure of the GlnRS:tRNA(Gln) complex provides a structural basis for interpreting these data. In the wild-type enzyme Asp235 makes sequence-specific hydrogen bonds through its side chain carboxylate group with base pair G3.C70 in the minor groove of the acceptor stem of the tRNA. This observation implicates base pair 3.70 as one of the identity determinants of tRNA(Gln). Isoleucine 129 is positioned adjacent to the phosphate of nucleotide C74, which forms part of a hairpin structure adopted by the acceptor end of the complexed tRNA molecule. These results identify specific areas in the structure of the complex that are critical to accurate tRNA discrimination by GlnRS.     

莴苣花叶病毒北京分离物基因组3＇末端序列分析

笔者通过RT-PCR方法对LMV北京分离物(LMV-BJ)基因组3''端1620nt的核苷酸片段进行了克隆和序列分析(GenBank登录号为EF423619)。所获片段含有NIb基因3''端的574nt,编码NIb C-端190个氨基酸;完整的CP基因,全长为834nt,编码一个由277个氨基酸组成的分子量约为30kDa的结构蛋白;3''非编码区含有209nt。通过序列分析软件将LMV-BJ与已经报道的法国分离物O(X97704)、法国分离物E(X97705),美国分离物(X65652),巴西分离物(AJ278854)和余杭分离物(AJ306288)基因组3''末端和CP基因的核苷酸序列与氨基酸序列分别进行了比较。     

温州光唇鱼线粒体基因组结构及系统发育分析

采用已公布的鱼类线粒体基因组全序列,设计8对引物扩增、测定并注释温州光唇鱼(Acrossocheilus wenchowensis)线粒体基因组(GenBank登录号:KC_495074)。序列全长16 591 bp,包括13个蛋白质基因、22个tRNA基因、2个rRNA基因和1个非编码区,各基因的位置及组成与已公布的鲤科鱼类一致;37个基因中,1个蛋白质编码基因(ND6)和8个tRNA基因(tRNAGln、tRNAAla、tRNAAsn、tRNACys、tRNATyr、tRNASer(UCN)、tRNAGlu和tRNAPro)由L链编码,其余的均由H链编码。A、T、G、C碱基组成分别为30.92%、24.86%、16.41%、27.81%;除tRNASer(AGN)外,其它21个tRNA的二级结构均具有典型的三叶草结构;13个蛋白编码基因中,除COⅠ起始密码子为GTG外,其余均以ATG为起始密码子,而COⅡ、ND4和Cytb基因的终止密码子为不完整的T,其它10个基因均具有完整的终止密码子。利用鲤科共17属18种线粒体基因组13个蛋白质基因的氨基酸序列,从线粒体基因组水平探讨了温州光唇鱼在鲤科鱼类中的系统进化地位,为光唇鱼属乃至鲤科鱼类的系统分类学研究提供基础资料。     

柑橘黄龙病病原菌非洲种和美洲种23S-5S rDNA序列的克隆及分析

 【目的】克隆柑橘黄龙病病原菌非洲种和美洲种23S-5S rDNA序列并和亚洲种相应区域进行比对,阐明黄龙病3个种核糖体RNA操纵子之间的关系。【方法】根据亚洲种23S-5S rRNA基因区域序列的保守性设计引物,在非洲种和美洲种DNA样品上扩增,对PCR产物进行克隆和测序,并对新获得的序列进行序列验证和分析。【结果】 非洲种获得了3 057 bp 序列包括23S rRNA 基因、细胞壁脱氢酶假基因和5S rRNA 基因;美洲种获得了3 033 bp序列包括23S rRNA 基因、glpK基因和5S rRNA 基因。3个种核糖体基因顺序分别是：亚洲种16S rRNA、tRNAIle、tRNAAla、23S rRNA、细胞壁脱氢酶假基因、5S rRNA 和 tRNAMet;非洲种16S rRNA、tRNAIle、tRNAAla、23S rRNA、细胞壁脱氢酶假基因和 5S rRNA;美洲种16S rRNA、tRNAIle、tRNAAla、23S rRNA、glpK 基因和5S rRNA。【结论】黄龙病病原菌核糖体RNA基因有特殊的排列方式,即在23S rRNA和5S rRNA基因区间均有反向编码的细胞壁脱氢酶基因,其中亚洲种和非洲种为假基因,美洲种为glpK基因。     

Enzymatic modification of transfer RNA

The molecular events leading to the synthesis of mature tRNA are only now becoming amenable to experimental study. In bacterial and mammalian cells tRNA genes are transcribed into precursor tRNA. These molecules, when isolated, contain additional nucleotides at both ends (20) of the mature tRNA and lack most modified nucleosides. Presumably, specific nucleases ("trimming" enzymes) cut the precursor to proper tRNA size. The C-C-A nucleotide sequence of the amino acid acceptor end common to all tRNA's does not seem to be coded by tRNA genes (30), and may be added to the trimmed molecules by the tRNA-CMP-AMP-pyrophosphorylase (71). Modifications at the polynucleotide level of the heterocyclic bases or the sugar residues give rise to the modified nucleosides in tRNA. Although newly available substrates have allowed the detection of more of the enzymes involved in these reactions, there is still no knowledge about the sequence of modification or trimming events leading to the synthesis of active tRNA. Progress in these studies may not be easy because enzyme preparations free of nucleases or other tRNA modifying enzymes are required. The role of the modified nucleosides in the biological functions of tRNA is still unknown. Possibly pseudouridine is required for ribosome mediated protein synthesis; some other modified nucleosides in tRNA are not required for this reaction, but may enhance its rate. What might be the role of the large variety of modified nucleosides in tRNA? One is tempted to speculate that such nucleosides are important in other cellular processes in which tRNA is thought to participate such as virus infection, cell differentiation, and hormone action (2, 3). Mutants in a number of tRNA-modifying enzymes are needed in order to extend our knowledge of their purpose and of tRNA involvement in other biological processes. But unless tRNA-modifying enzymes specific for a particular tRNA species exist, no simple selection procedure can be devised. Possibly some of the regulatory mutants of amino acid biosynthesis may prove to affect tRNA-modifying enzymes (72). Transfer RNA's are macromolecules well suited for the study of nucleic acid-protein interactions. The tRNA molecules are structurally very similar, and they interact with a large number of enzymes or protein factors (2, 3). Each aminoacyl-tRNA synthetase, for instance, very precisely recognizes a set of cognate isoacceptor tRNA's (2, 73). The availability of the tRNA- modifying enzymes adds another dimension to the problem of the nature of specific recognition of tRNA by proteins. There are some tRNA-modifying enzymes, such as the uracil-tRNA methylase, which may recognize all tRNA species, while others, such as the isopentenyl-tRNA transferase, probably recognize only a selected set of tRNA molecules, even with different amino acid accepting capacities. With well-characterized RNA precursor and tRNA molecules we can hope to delineate those features of primary, secondary, and tertiary structure involved in the specific interactions of tRNA with these enzymes.     

绍兴鸭线粒体基因组全序列测定与分析

参照近源物种线粒体基因组序列设计15对引物,通过PCR扩增、测序、拼接,获得绍兴鸭(Anas platyrhychos)线粒体基因组全序列并进行序列分析。绍兴鸭线粒体基因组全长16 604 bp,碱基组成为29.20%A,22.19%T,15.78%G,32.83%C,包含13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码控制区(D-loop),基因组成及排列顺序与其他鸟类相似,表明鸟类线粒体DNA进化上有较高的保守性。     

建昌鸭线粒体基因组全序列分析

参照近源物种线粒体基因组序列设计引物15对,用PCR产物直接测序法测得建昌鸭(Anas platyrhychos)线粒体基因组全序列,初步分析其特点和各基因的定位.结果显示:建昌鸭线粒体基因组全长16 606 bp,碱基A、T、G、C的含量分别为29.21%、22.19%、15.77%、32.83%,包含13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码调控区(D-loop),基因组结构和已知雁形目鸟类的完全相同.基于线粒体13个蛋白质编码基因序列,采用邻接法构建雁形目8个物种的系统进化树,所得结果与传统的系统分类基本一致.