Cleavage of 5' splice site and lariat formation are independent of 3' splice site in yeast mRNA splicing

Analysis of messenger RNA splicing in yeast and in metazoa has led to the identification of an RNA molecule in a lariat conformation. This structure has been found as an mRNA splicing intermediate in vitro and identical molecules have been identified in vivo. Lariat formation involves cleavage of the precursor at the 5' splice site (5' SS) and the formation of a 2'-5' phosphodiester bond between the guanosine residue at the 5' end of the intron and an adenosine within the intron. The yeast branchpoint is located within the absolutely conserved TACTAAC box (that is, the last A of the TACTAAC box is the site of formation of the 2'-5' phosphodiester bond with the 5' end of the intron)3,4. Moreover, efficient 5' SS cleavage and lariat formation require proper sequences at the 5' splice junction and within the TACTAAC box. Here we demonstrate that 5' SS cleavage and lariat formation take place in vitro in the absence of the 3' SS and much of the 3' junction. These results are discussed in light of possible differences between yeast and metazoan mRNA splicing.     

Characterization of the branch site in lariat RNAs produced by splicing of mRNA precursors

The branch site of lariat RNAs produced during the splicing of the first two late leader exons of adenovirus-2 has a structure of (formula; see text) There is a distinct complementarity between sequences preceding the adenosine at the branch site and the 5' terminus of the intervening sequence.     

Functional recognition of the 3' splice site AG by the splicing factor U2AF35

In metazoans, spliceosome assembly is initiated through recognition of the 5' splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF. U2AF is a heterodimer comprising a large subunit, U2AF65, and a small subunit, U2AF35. U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro. In comparison, the role of U2AF35 has been puzzling: U2AF35 is highly conserved and is required for viability, but can be dispensed with for splicing in vitro. Here we use site-specific crosslinking to show that very early during spliceosome assembly U2AF35 directly contacts the 3' splice site. Mutational analysis and in vitro genetic selection indicate that U2AF35 has a sequence-specific RNA-binding activity that recognizes the 3'-splice-site consensus, AG/G. We show that for introns with weak polypyrimidine tracts, the U2AF35-3'-splice-site interaction is critical for U2AF binding and splicing. Our results demonstrate a new biochemical activity of U2AF35, identify the factor that initially recognizes the 3' splice site, and explain why the AG dinucleotide is required for the first step of splicing for some but not all introns.     

Vesicle fusion following receptor-mediated endocytosis requires a protein active in Golgi transport

In reconstitution studies N-ethylmaleimide, a sulphydryl alkylating reagent, inhibits both fusion of endocytic vesicles and vesicular transport in the Golgi apparatus. We show here that the same N-ethylmaleimide-sensitive factor that catalyses the vesicle-mediated transport within Golgi stacks is also required for endocytic vesicle fusion. Thus, it is likely that a common mechanism for vesicle fusion exists for both the secretory and endocytic pathways of eukaryotic cells.     

Mutations in the active site of Escherichia coli phosphofructokinase

The enzyme-catalysed transfer of a phosphoryl group from ATP is an important reaction in a wide variety of biological processes. We demonstrate here the essential function of an aspartate group in the catalysis of phosphoryl transfer by Escherichia coli phosphofructokinase, and the minor role of an arginine residue. We have used oligonucleotide-directed mutagenesis to replace two amino-acid residues which X-ray analysis has shown to be close to the transferred phosphoryl group and we have analysed the forward and back reactions of the mutant enzymes by steady-state kinetics. Changing Asp 127 to Ser reduced the turnover number by a factor of 18,000 in the forward direction and 3,100 in the back reaction, and the Michaelis constant for fructose 1,6-bisphosphate in the reverse reaction by a factor of 45. This shows that this aspartate is a key residue in the rate enhancement by the enzyme, probably acting as a base in the reaction mechanism, and that it also destabilizes the product complex. Changing Arg 171 to Ser reduced the turnover numbers by about 3.4, showing that this arginine has only a minor effect on the catalysis.     

团簇Co_xFe_yB_2的催化活性位点

为了深入研究Co-Fe-B体系的催化性质,利用密度泛函理论(DFT)方法,在B3LYP/Lan12dz水平下,分别对团簇Co2Fe B2和Co Fe2B2的十余种可能存在的构型进行全参数优化计算和频率验证,通过比较Co和Fe原子比例的不同,得出如下结论:在团簇Co2Fe B2中,单重态的Co原子在HOMO的贡献大于Fe原子,三重态Fe原子在HOMO的贡献大于Co原子,Co和Fe原子都是其潜在的催化活性位点;在团簇Co Fe2B2中,Fe原子在HOMO和LUMO的贡献均大于Co原子,而且Co和Fe原子在HOMO和LUMO变化有此消彼长之势,Fe是主要的催化活性位点。     

Different substrate-dependent transition states in the active site of the ribosome

The active site of the ribosome, the peptidyl transferase centre, catalyses two reactions, namely, peptide bond formation between peptidyl-tRNA and aminoacyl-tRNA as well as the release-factor-dependent hydrolysis of peptidyl-tRNA. Unlike peptide bond formation, peptide release is strongly impaired by mutations of nucleotides within the active site, in particular by base exchanges at position A2602 (refs 1, 2). The 2'-OH group of A76 of the peptidyl-tRNA substrate seems to have a key role in peptide release. According to computational analysis, the 2'-OH may take part in a concerted 'proton shuttle' by which the leaving group is protonated, in analogy to similar current models of peptide bond formation. Here we report kinetic solvent isotope effects and proton inventories (reaction rates measured in buffers with increasing content of deuterated water, D(2)O) of the two reactions catalysed by the active site of the Escherichia coli ribosome. The transition state of the release factor 2 (RF2)-dependent hydrolysis reaction is characterized by the rate-limiting formation of a single strong hydrogen bond. This finding argues against a concerted proton shuttle in the transition state of the hydrolysis reaction. In comparison, the proton inventory for peptide bond formation indicates the rate-limiting formation of three hydrogen bonds with about equal contributions, consistent with a concerted eight-membered proton shuttle in the transition state. Thus, the ribosome supports different rate-limiting transition states for the two reactions that take place in the peptidyl transferase centre.     

人参皂甙Re的微生物降解研究

黑曲霉（ＡｓｐｅｒｇｉｌｌｕｓｎｉｇｅｒＡ１０３）可将人参皂甙Ｒｅ降解为其次生甙，经２８℃振荡培养６～１２０小时，此种降解力最强，其降解最佳ｐＨ为４．０～５．０，在发酵液中添加５％蛋白胨或１％Ｔｗｅｅｎ８０均可提高黑曲霉降解Ｒｅ的程度．经紫外线和氯化锂复合诱变后，我们获得一株具高降解Ｒｅ能力的变异株ＡＵ＿（１２），可降解Ｒｅ形成两种次生甙．     

Resistance to beta-lactam antibiotics by re-modelling the active site of an E. coli penicillin-binding protein

The beta-lactam antibiotics kill bacteria by inhibiting a set of penicillin-binding proteins (PBPs) that catalyse the final stages of peptidoglycan synthesis. In some bacteria the development of intrinsic resistance to beta-lactam antibiotics by the reduction in the affinity of PBPs causes serious clinical problems. The introduction of beta-lactam antibiotics that are resistant to hydrolysis by beta-lactamases may also result in the emergence of intrinsic resistance among the Enterobacteriaceae. The clinical problems that would arise from the emergence of resistant PBPs in enterobacteria have led us to examine the ease with which Escherichia coli can gain resistance to beta-lactams by the production of altered PBPs. The development of resistant PBPs also provides an interesting example of enzyme evolution, since it requires a subtle re-modeling of the enzyme active centre so that it retains affinity for its peptide substrate but excludes the structurally analogous beta-lactam antibiotics. We show here that only four amino-acid substitutions need to be introduced into PBP 3 of E. coli to produce a strain possessing substantial levels of resistance to a wide variety of cephalosporins. We also show that transfer of the gene encoding the resistant PBP 3 from the chromosome to a plasmid could result in the spread of intrinsic resistance not only to other strains of E. coli but also to other enterobacterial species.     

Calculation of electrostatic potentials in an enzyme active site

To be able to calculate the contributions of individual amino acids to the electrostatic field of a protein would be of considerable value in designing proteins of enhanced or altered function and stability. Recent studies on the serine protease subtilisin provide direct measurements of the electrostatic potential in the active site of the enzyme produced by two charged amino acids. We have used these results to test a recently developed method for the calculation of electrostatic interactions between two specific sites on a protein. The extent of agreement between the theoretical and experimental results suggests that the continuum solvent model used in the calculations reproduces the essential features of the interaction.     

NaHTe分解邻苯二甲酸二烷基酯的研究

本文报道了一种新方法(即在DMF中用NaHTe)分解邻苯二甲酸二烷基酯.     

复合材料层合板振动主动控制的方法研究

根据H am ilton原理,推导出复合材料层合板在四边简支的边界条件和各阶板理论下的振动控制方程,求得系统的频率、振幅。利用能量准则求出线性二次型最优调节器(LQR)方法中的权系数矩阵Q和R的值,求解R iccati方程后,得到位移和控制力;并利用M athem atic对系统进行仿真分析,得到各种因素对系统振动控制的影响,为以后控制和优化系统提供有力的根据。     

Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1

Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretases generates the amino and carboxy termini, respectively, of the A beta amyloidogenic peptides A beta40 and A beta42--the major constituents of the amyloid plaques in the brain parenchyma of Alzheimer's disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease increase the production of A beta42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a gamma-secretase cofactor, or helps to colocalize gamma-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent gamma-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of gamma-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.     

The essential light chains constitute part of the active site of smooth muscle myosin

Myosin, a major contractile protein, characteristically possesses a long coiled-coil alpha-helical tail and two heads. Each head contains both an actin binding site and an ATPase site and is formed from the NH2-terminal half of one of the two heavy chains (relative molecular mass, Mr, 200,000) and a pair of light chains; the so-called regulatory and essential light chains of approximately Mr 20,000 each. Recently we have identified Trp 130 of the myosin heavy chain from rabbit skeletal muscle as an active-site amino-acid residue after labelling with a new photoaffinity analogue of ADP, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (NANDP). Nonspecific labelling was eliminated by first trapping NANDP at the active site with thiol crosslinking agents. Exclusive labelling of the heavy chains with no labelling of the light chains agreed with previous findings that the heavy chains alone contain the actin-activated Mg-ATPase activity of rabbit skeletal myosin. Here we report similar photolabelling experiments with smooth muscle myosin (chicken gizzard) in which 3H-NANDP is trapped at the active site with vanadate and which show that both the heavy chains and the essential light chains are labelled. The results indicate that both chains contribute to the ATP binding site and represent the first direct evidence for participation of the essential light chains in the active site of any type of myosin.     

团簇V3BP成键及活性位点的研究

密度泛函理论结合拓扑学原理,在B3LYP/Lan12dz水平下对团簇V_3BP各个可能构型进行优化和频率计算,现对其成键、轨道贡献及活性位进行研究。结果表明金属-非金属键(V-P、V-B)是团簇V_3BP稳定性的主要贡献者。金属原子V是团簇V_3BP潜在的活性位点。构型1(4)~5(4)化学稳定性好,构型10(2)的反应活性好,多重度对相同构型团簇的化学反应活性有一定的影响。     

Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae.

An unusual protein splicing reaction joins the N-terminal segment (A) and the C-terminal segment (C) of the 119K primary translation product (ABC) of the yeast VMA1 gene to yield a 69K vacuolar H(+)-ATPase subunit (AC) and an internal 50K polypeptide (B). This 50K protein is a site-specific DNA endonuclease that shares 34% identity with the homothallic switching endonuclease. The site cleaved by the VMA1-derived endonuclease exists in a VMA1 allele that lacks the derived endonuclease segment of the open reading frame. Cleavage at this site only occurs during meiosis and initiates 'homing', a genetic event that converts a VMA1 allele lacking the endonuclease coding sequence into one that contains it.