CNS-Specific Ribozyme Expression

Targeted genetic deletion is a powerful tool for analysis of gene function, but the standard approaches carry certain inescapable disadvantages. First, deletion is ubiquitous; tissue-specific knockout cannot be obtained. Second, temporal regulation of depletion is unattainable; the deleted functions are absent throughout the animal's development. As a consequence, during ontogeny, other gene products may be able to compensate, filling the functional gap. Furthermore bifunctional proteins exist that fulfill one role during development and another in the mature organism; deletion will remove the early function and, if this is lethal, the later function will remain undetected. Third, if genes utilize alternative splicing to control protein expression, it is difficult to target one spliced mRNA while leaving intact its related, but different, siblings. We review how these problems may be circumvented using ribozymes to diminish gene expression in a tissue-specific and temporally regulated manner and provide guidelines for the design and delivery of active ribozymesin vivo.Such methods may be particularly useful for analysis of genes involved in ontogeny and function of the central nervous system, in which individual genes may be expressed with alternative splicing patterns, or at differentially regulated levels, at different stages of CNS development.     

Ribozyme speed limits

The speed at which RNA molecules decompose is a critical determinant of many biological processes, including those directly involved in the storage and expression of genetic information. One mechanism for RNA cleavage involves internal phosphoester transfer, wherein the 2'-oxygen atom carries out an SN2-like nucleophilic attack on the adjacent phosphorus center (transesterification). In this article, we discuss fundamental principles of RNA transesterification and define a conceptual framework that can be used to assess the catalytic power of enzymes that cleave RNA. We deduce that certain ribozymes and deoxyribozymes, like their protein enzyme counterparts, can bring about enormous rate enhancements.     

Loss of pregnancy-associated plasma protein A extends lifespan in mice

Genetic deletion in mice of pregnancy-associated plasma protein A (PAPP-A), a recently identified metalloproteinase in the insulin-like growth factor system, extends by 30-40% both mean and maximum lifespan with no reduction in food intake or secondary endocrine abnormalities. Furthermore, these mice have markedly reduced incidence of spontaneous tumors. The findings implicate PAPP-A as a critical regulator of lifespan and age-related diseases, and suggest PAPP-A as a possible target to promote longevity.     

A Faster Triphosphorylation Ribozyme

In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5''-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min^-1 under selection conditions (50 mM Tmp, 100 mM MgCl₂, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme''s structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl₂, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min^-1.     

The origin of the genetic code and protein synthesis

A model for a parallel evolution of the genetic code and protein synthesis is presented. The main tenet of this model is that the genetic code, that is, a correspondence between nucleotide and aminoacid coding units, originated from sequence-specific interaction between abiotically synthesized polynucleotides and polypeptides. A sequence-specific binding between oligonucleotides and oligopeptides is supported by experimental findings. Moreover, it is parsimonious enough to be consistent with the relatively simple chemistry of a primordial environment. Proximity between peptides and RNA increased the rate of formation of ester bonds between them. This lead to the accumulation of sequence-specific polypeptide-polynucleotide pairs, that is, of primordial-loaded tRNA. Condensation of short polypeptides into longer products could be catalyzed by a sequence-specific juxtaposition of loaded tRNA over complementary RNA, originating the core of protein synthesis. The accumulation of useful encoded products, for example, catalysts for tRNA loading (primordial aminoacyl-tRNA synthetases) or stabilizers of tRNA-mRNA interactions (primordial ribosomes), permitted the subsequent evolution of protein synthesis and of the genetic code to their mature form. This occurred via a parallel reduction in length of the interacting polynucleotides and polypeptides. Thus, it maintained the correct reading frame of mRNA from the preceding stages of evolution. Received: 27 September 1996 / Accepted: 17 May 1997     

The role of the genetic code in protein evolution

The arrangement and degeneracy of triplets in the genetic code play an important but not inclusive part in protein evolution. In particular, data indicate that the genetic code is insufficient to account for the existence of homologous proteins. A novel theory is proposed suggesting that homologous proteins are the existing part of a discrete probability class whose members are genetically intermutable and whose size is delimited by natural selection.     

发夹核酶的研究与应用

核酶 (ｒｉｂｏｚｙｍｅ)是既能特异识别又能特异切割小分子ＲＮＡ的核酸内切酶 ,其本身也是ＲＮＡ ,主要包括发夹核酶、锤头核酶、丁肝病毒、链孢霉属ＶＳ和铅依赖性ＲＮＡ ,共同特点是可逆地切割底物ＲＮＡ的磷酸二酯键 ,生成 5′ ＯＨ和 2′ ,3′ 环磷酸末端。虽然催化产物相似 ,但它们的结构和催化机制却是很不相同的。发夹核酶 (ｈａｉｒｐｉｎｒｉｂｏｚｙｍｅ)发现于三种不同植物ＲＮＡ病毒 ,即烟草环点病毒 (ｔｏｂａｃ ｃｏｒｉｎｇｓｐｏｔｖｉｒｕｓ) ,菊苣黄色斑点病毒型 (ｃｈｉｃｏ ｒｙｙｅｌｌｏｗｍｏｔｔｌｅｖｉｒｕｓｔ…     

Amino acid code of protein secondary structure

The calculation of protein three-dimensional structure from the amino acid sequence is a fundamental problem to be solved. This paper presents principles of the code theory of protein secondary structure, and their consequence--the amino acid code of protein secondary structure. The doublet code model of protein secondary structure, developed earlier by the author (Shestopalov, 1990), is part of this theory. The theory basis are: 1) the name secondary structure is assigned to the conformation, stabilized only by the nearest (intraresidual) and middle-range (at a distance no more than that between residues i and i + 5) interactions; 2) the secondary structure consists of regular (alpha-helical and beta-structural) and irregular (coil) segments; 3) the alpha-helices, beta-strands and coil segments are encoded, respectively, by residue pairs (i, i + 4), (i, i + 2), (i, i = 1), according to the numbers of residues per period, 3.6, 2, 1; 4) all such pairs in the amino acid sequence are codons for elementary structural elements, or structurons; 5) the codons are divided into 21 types depending on their strength, i.e. their encoding capability; 6) overlappings of structurons of one and the same structure generate the longer segments of this structure; 7) overlapping of structurons of different structures is forbidden, and therefore selection of codons is required, the codon selection is hierarchic; 8) the code theory of protein secondary structure generates six variants of the amino acid code of protein secondary structure. There are two possible kinds of model construction based on the theory: the physical one using physical properties of amino acid residues, and the statistical one using results of statistical analysis of a great body of structural data. Some evident consequences of the theory are: a) the theory can be used for calculating the secondary structure from the amino acid sequence as a partial solution of the problem of calculation of protein three-dimensional structure from the amino acid sequence, and the calculated secondary structure and codon strength distribution can be used for simulating the next step of protein folding; b) one can propose that the same secondary structures can be folded into different tertiary structures and, vice versa, different secondary structures can be folded into the same tertiary structures, provided codon distributions are considered also; c) codons can be considered as first elements of protein three-dimensional structure language.     

Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles.

Electron microscopy revealed structures consisting of long fibers topped with knobs extending from the surfaces of virions of mammalian reoviruses. The morphology of these structures was reminiscent of the fiber protein of adenovirus. Fibers were also seen extending from the reovirus top component and intermediate subviral particles but not from cores, suggesting that the fibers consist of either the mu 1C or sigma 1 outer capsid protein. Amino acid sequence analysis predicts that the reovirus cell attachment protein sigma 1 contains an extended fiber domain (R. Bassel-Duby, A. Jayasuriya, D. Chatterjee, N. Sonenberg, J. V. Maizell, Jr., and B. N. Fields, Nature [London] 315:421-423, 1985). When sigma 1 protein was released from viral particles with mild heat and subsequently obtained in isolation, it was found to have a morphology identical to that of the fiber structures seen extending from the viral particles. The identification of an extended form of sigma 1 has important implications for its function in cell attachment. Other evidence suggests that sigma 1 protein may occur in virions in both an extended and an unextended state.     

Role of the code redundancy in determining cotranslational protein folding

It has been demonstrated earlier in our laboratory that rare codon clusters can determine the boundaries of the polypeptide chain fragments of the same secondary structure type during the co-translational protein folding. According to this data, co-translational protein folding can occur under condition of a correlation between the frequency of codon choice in mRNAs and the relative abundance of their isoaccepting tRNAs. The alterations in the spectrum and concentrations of the isoaccepting tRNAs in different cells were demonstrated by many authors. The existence of a mechanism of the coordinate regulation of the levels (activities) of the isoaccepting tRNAs, corresponding aminoacyl-tRNA synthetases and mRNAs predominantly translated at a given moment of time can be suggested. Such a mechanism can ensure the needed accuracy of the protein folding process. Analysis of gene sequences of various pro- and eukaryotic organisms carried out in the present work revealed that the codon usage frequency spectra of simultaneously synthesized proteins are similar. The relative appearance of the most rare and frequent codons in investigated gene sequences displays a high degree of conservatism. It has also been found that structural-homologous proteins from different organisms (cytochromes c, myoglobins) have very similar codon frequency distribution profiles. This property retains despite the significant variations in the codon usage spectra in the investigated gene sequences. The data obtained indicate that the codon distribution in mRNAs whose diversity is mainly conditioned by the genetic code redundance is a program that determines translational rates of different mRNA parts thus controlling the spatial folding of the synthesized peptide chain.     

Early assignments of the genetic code dependent upon protein structure

Orgel (1972) has suggested that polynucleotides with sequences of alternating purine/pyrimidine are likely to have predominated in prebiotic conditions. Therefore, in any early template-directed protein synthesis, the number of available codons would have been limited. However, for any self-organizing system to survive and propagate, some feedback must occur from the products of the synthesis to the control of the synthetic procedure itself; i.e. the protein synthesized should have catalysed some step in the initiation of template-directed synthesis. A given protein structure with a characteristic conformation and function would be optimal for the product of such a synthesis, and this in turn would limit the number of nucleotide sequences of those available able to give rise to a functioning synthetic assembly. A possible candidate for such an early polypeptide is the ferredoxin group of proteins and it is shown that with the present-day code the corresponding nucleotides do have a high percentage of alternating purine/pyrimidine sequences. Hence these combined restraints on the primitive synthetic machinery would direct the possible assignments of the genetic code helping to explain its regularity and universality.     

第二遗传密码与蛋白质结构优化算法

核苷酸通过“三联密码”决定氨基酸顺序 ,这就是第一遗传密码。多肽链中氨基酸的一定顺序就是蛋白质的一级结构。 2 0世纪 5 0年代Ａｎｆｉｎｓｅｎ提出假说 ,认为蛋白质特定的三维空间结构是由氨基酸排列顺序所决定的 ,现在已被广泛接受。从无结构的氨基酸序列到有特定功能的蛋白质的信息传递 ,即蛋白质中的氨基酸序列与其空间结构的对应关系 ,被称为第二遗传密码。收稿日期 :2 0 0 2 - 0 6 - 1 1 ;修回日期 :2 0 0 2 - 0 9- 1 8作者简介 :王华伟 (1 978- ) ,男 ,湖北孝感人 ,硕士生 ,从事生物信息学、ＤＮＡ分子生物计算研究。许进 (1 959…     

Ribozyme inhibition of alphavirus replication

A model system to examine the expression and antiviral activity of trans-acting ribozymes in mammalian cells has been developed and evaluated. Hairpin ribozymes were engineered to cleave a specific site, identified by a combinatorial activity-based selection method, within genomic and subgenomic RNA species of Sindbis virus. Transiently transfected cells expressed moderate levels of ribozyme (approximately 50,000 molecules/cell) with predominant nuclear localization and a short half-life (23 min). Stable cell lines expressed ribozymes at modest levels (approximately 2,000 molecules/cell). Ribozyme-mediated RNA cleavage activity was detected in cell extracts. Clonal cell lines were challenged with recombinant Sindbis virus, and viral replication was examined using plaque formation and green fluorescent protein assays. Significant inhibition of viral replication was observed in cells expressing the active antiviral ribozyme, and lower levels of inhibition in control cells expressing inactive or irrelevant ribozymes. These findings are consistent with a model in which inhibition of viral replication occurs via ribozyme cleavage of viral RNAs, suggesting that ribozymes may represent useful antiviral agents.     

核酶的22年

核酶发现至今已22年了。1989年的诺贝尔化学奖授于核酶的发现S.Altman和T.Cech。到2004年2月发表有5000多篇有关核酶的研究论和综述。自然界留存的核酶不多,但已能制造出许许多多人工核酶。从1997年人造出‘肽基转移核酶’到2000年根据一系列证据提出‘核糖体是一种核酶’,在理论上和应用上都具有深远意义。核酶出现早于酶（蛋白质）,后来让位于酶的观点,已为数人所接受。在应用上,人们已经设计和制造出各种样的核酶对付各样的疾病,但目前临床应用的极少或几乎没有。     

Ribozyme catalysis via orbital steering

Orbital steering is invoked to explain how the three-dimensional structure of a small self-cleaving RNA, the hammerhead ribozyme, both prevents and enhances RNA autocatalysis. Within the conserved catalytic core of the ribozyme, the position of the 2' oxygen atom of the G8 ribose is observed to be aligned almost perfectly with the phosphorus atom and the 5' oxygen atom of the adjacent A9 phosphate group for self-cleavage via an in-line attack mechanism. Despite this apparent near-perfect atomic positioning, no cleavage takes place. The explanation proposed is that a network of hydrogen bonds in the ribozyme core orients or steers the orbitals containing the electron lone pairs of the attacking nucleophile (the 2' oxygen atom) away from the A9 phosphorus atom, eliminating overlap with the vacant phosphorus d-orbitals despite the near-perfect in-line positioning of the oxygen atom, thus preventing catalysis. Because of the near-perfect atomic positioning of the 2' oxygen atom relative to the phosphate group, orbital steering effects in this case are fortuitously uncoupled from conformational, distance and orientation effects, allowing an assessment of the catalytic power due purely to orbital steering. In contrast, a conformational change at the cleavage site is required to bring the 2' oxygen atom and the scissile phosphate group into atomic positions amenable to an in-line attack mechanism. In addition, the conformationally changed structure must then steer the lone-pair orbitals of the correctly positioned 2' oxygen atom toward the scissile phosphorus atom in order for cleavage to take place. We estimate that fulfillment of each of these two required changes may contribute separately an approximately 1000-fold rate enhancement, potentially accounting for a significant fraction of the catalytic power of this ribozyme. Orbital steering therefore appears to be a general phenomenon that may help to explain catalysis in both ribozymes and protein enzymes in a unified manner.