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【求助/交流】关于自我剪接肽
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最近看到有关蛋白质内含肽的文章，有很多疑惑，但是现在看到的文章有限，请大家帮忙给解释一下，谢谢了 蛋白质内含肽(intein) 是蛋白质中的一段多肽链. 其DNA 顺序与外显子一起转录和翻译,产生一条多肽链,然后从肽链中切除与内含肽对应的氨基酸顺序,再把与外显子对应的顺序连接起来,成为有功能的蛋白质。从这些话可以理解在翻译水平上内含肽将自己剪切掉，把前后的蛋白质再连在一起， ............................那样不就成融合蛋白了吗 实在是不明白 请高手赐教  |
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qingfengwu
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4楼2009-12-23 15:59:49
qingfengwu
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蛋白质翻译产物在成熟过程中剪切释放出来的一段氨基酸序列称为"intein"--即内含肽.它与前体蛋白以框内融合的形式共同翻译,并内嵌于前体蛋白序列中.内含肽的解离以及内含肽两侧氨基酸序列的连接是在内含肽自身催化作用下完成的. 蛋白质内含子(intein)是具有自我催化活性的蛋白质.翻译后,通过蛋白质剪接从蛋白质前体中去掉,并以肽键连接两侧蛋白质外显子(extein)形成成熟蛋白质.断裂蛋白质内含子(split intein)在蛋白质内含子中部区域特定位点发生断裂,形成N端片段和C端片段,分别由基因组上相距较远的两个基因编码.现在已知,它仅分布于蓝细菌和古细菌中.断裂蛋白质内含子的N端片段和C端片段通过非共价键(如静电作用)相互识别,重建催化活性中心,介导蛋白质反式剪接.断裂蛋白质内含子的发现进一步深化了人们对基因表达和蛋白质翻译后成熟过程复杂性的认识,而且它在蛋白质工程、蛋白质药物开发和蛋白质结构与功能研究等方面有非常广泛的应用. |
2楼2009-12-23 15:58:41
qingfengwu
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Inteins are selfish DNA elements inserted in-frame and translated together with their host proteins. This precursor protein undergoes an autocatalytic protein splicing reaction resulting in two products: the host protein and the intein. Protein-splicing scheme. A precursor protein is shown on the right, with intein protein-splicing domain shown in red and the host protein flanks (exteins) shown in blue. The intein protein-splicing domain autocatalyzes its excision and the ligation of its two flanks. Protein splicing was shown to occur in heterologous organisms, in different in-vitro systems and with various natural and engineered host flanks. Hence, protein splicing is probably independent of specific host cell factors. Most inteins have an endonuclease domain inserted in the protein splicing domain. The endonuclease activity of these inteins can mediate the specific transposition of their gene into unoccupied integration sites of intein-less homologs (homing). Inteins have a diverse and sporadic distribution across species and proteins. They occur in all three domains of life but so far have been found in just (relatively) few species. Inteins are currently known in more then 50 types of proteins with diverse in functions. These proteins include metabolic enzymes, DNA and RNA polymerases, proteases, ribonucleotide reductases, and vacuolar-type ATPases. Inteins integration points also vary in structure in function. Their only apparent common feature is being in highly conserved protein motifs. Intein protein domain family is part of the Hint superfamily, termed after the characteristic structure fold first identified in Hedgehogs and Intein protein domains (Hall et al. '97). Four characterized Hint domain families are currently known: Hog Hints, inteins, and two types of Bacterial intein-like (BIL) domains. Together with sharing the same structure fold and common sequence features, Hint domains have similar biochemical activities. The domains post-translationally process the proteins in which they are present by protein-splicing, self-cleavage or ligation activities. This site mainly introduces inteins and some their sister families. It explores the relation between the activities of these domains, their sequence motifs, and protein structure. We also show how are these related to the different biological roles and evolution modes of inteins and intein-like domains. A database of inteins is maintained at New England Biolabs. Intein registry, publications, sequence search and information on their mechanism can be found there. Intein proteins contain a number of conserved sequence motifs (blocks). The motifs can be grouped in three domains according to their location and inferred function. Intein structures show that the inteins protein-splicing and endonuclease active sites are formed from conserved motifs. The intein's domain organization, deduced by sequence analysis, exactly corresponds to the structural domains. Domain structure of a typical intein with a LAGLIDADG type endonuclease domainN domain EN domain (optional) C domain==-=----==----==----==--------==-==---==--------===scale: - 8 amino acids, = motif region Positions of the protein splicing motifs (in red) in an intein structure (Mxe GyrA). In green are N2 and N4 structural motifs. A single amino acid N-terminal to the intein is shown in blue. This intein does not have an endonuclease domain. The protein-splicing N-terminal (N) domain spans about 100-150 aa. Mutations in this domain affect the first step in protein splicing, an N-S/O acyl shift in the peptide bond connecting the N terminus of the intein and the N-terminal flank (intein flanks are termed exteins). The N domain motifs are similar in sequence and function to motifs found at the Hint domain of Hog regions found in the Hedgehog animal developmental proteins and a few related nematode protein families. Hog Hint domains self-process their precursor proteins, cleaving themselves off the N-terminal parts of the proteins. Cleavage was shown to utilize a cholesterol molecule that is consequently covalently attached to the cleavage site in the N-terminal part modulating the activity of that part ( Porter et al. '96). The relation between inteins and Hog Hint domains was first determined by their similarity in sequence and function. Determination of protein structures from both families provided final proof for the common origin of inteins and Hog Hint domains. More on the structure and relation of inteins and Hog Hint domains. The intein protein-splicing C-terminal (C) domain is composed of the two adjacent motifs in the C-terminal 25-40 aa (including the conserved aa immediately C' to the intein). Residues in these motifs are necessary for the catalyzing the next steps of protein splicing: the branch formation and its resolution. Most (but not all) inteins also include a central endonuclease (EN) domain. The EN domain is usually of the LAGLIDADG (dodecapeptide) homing endonucleases type. Intein LAGLIDADG EN domains are characterized by 4 motifs that probably form the endonuclease active site (Duan et al. '97). An intein from the cyanobacteria Synechocystis species PCC6803 (Ssp gyrB) has a different type of endonuclease domain. In this intein the endonuclease domain contains an HNH motif. This motif is found in various homing and other endonucleases (Shub et al. '94, Gorbalenya '94). The endonuclease domain is optional in inteins. Mutations in it affect the intein endonuclease activity but not the protein splicing activity, some inteins are missing this domain, and inteins were shown to protein splice with this domain removed (Chong and Xu '97, Derbyshire et al. '97). Functional inteins with no EN domain (minimal inteins), the relation of the protein splicing domain to other Hint domains and the presence of different EN domains in inteins all indicate that the primeval inteins had no EN domains. Different EN domains, perhaps from homing endonucleases, and DNA binding domains invaded intein genes to form the typical present day intein. Some present day minimal inteins clearly lost their EN domain (such as Mxe_gyrA, see Telenti et al. '97 and Klabunde et al. '98) and some maybe never acquired one. Inteins are found in all three domains of life: Archaea, Bacteria, and . EukaryotesHowever their distribution is sporadic in species and in hosts. Some species have no inteins, some just one and Methanococcus jannaschii has nineteen. For species with completely sequenced genomes like E.coli, M.jannaschii and S. cerevisiae we know the total number of inteins in the strain sequenced. For other species we can only estimate their number. Intein distribution seems most varied in archaea. This table compares the inteins found in archaea with fully sequenced genomes. One major group of organisms where inteins are not known in is multicellular , eukaryotesboth metazoa and plants. The multicellular red alga Porphyra does contain an intein but in its chloroplast genome. The reasons for this absence are not clear. Inteins may yet be found in these organisms and only turn out to be scarcer or perhaps difficult to detect. It is interesting to note that some intein-containing organisms, such as Mycobacteria tuberculosis and the CIV virus, are intra-cellular pathogens of metazoa. Thus, the opportunity for intein invasion into animal genomes does exist ( more details). Intein distribution May 2001. Species with inteins, fully sequenced genome species with no identified inteins, partially sequenced genome species with no identified inteins. The tree is based on phylogenetic data of Baldauf et al. '00 and Nelson et al. '00 . Species abbreviations: Bacteria- Aae: Aquifex aeolicus, Tma: Thermotoga maritima, Dvu: Desulfovibrio vulgaris, Hpy: Helicobacter pylori, Cje: Campylobacter jejuni, Ccr: Caulobacter crescentus, Rpr: Rickettsia prowazekii, Nme: Neisseria meningitidis, Ngo: Neisseria gonorrhoeae, Xfa: Xylella fastidiosa, Vch: Vibrio cholerae, Hin: Haemophilus influenzae, Eco: Escherichia coli, Pae: Pseudomonas aeruginosa, Bsu: Bacillus subtilis, Bha: Bacillus halodurans, Mpn: Mycoplasma pneumoniae, Mge: Mycoplasma genitalium, Uur: Ureaplasma urealyticum, Sau: Staphylococcus aureus, Cac: Clostridium acetobutylicum, Mtu: Mycobacterium tuberculosis, Mle: Mycobacterium leprae, Mav: Mycobacterium avium, Sco: Streptomyces coelicolor, Cpn: Chlamydia pneumoniae, Ctr: Chlamydia trachomatis, Ssp: Synechocystis sp. PCC6803, Pma: Prochlorococcus marinus, Pgi: Porphyromonas gingivalis, Cte: Chlorobium tepidum, Tde: Treponema denticola, Tpa: Treponema pallidum, Bbu: Borrelia burgdorferi, Det: Dehalococcoides ethenogenes, Dra: Deinococcus radiodurans, Archaea- Pae: Pyrobaculum aerophilum , Ape: Aeropyrum pernix, Sso: Sulfolobus solfataricus, Tac: Thermoplasma acidophilum, Hsp: Halobacterium sp. NRC-1, Pfu: Pyrococcus furiosus, Pho: Pyrococcus horikoshii, Pab: Pyrococcus abyssi, Afu: Archaeoglobus fulgidus, Mth: Methanobacterium thermoautotrophicum, Mja: Methanococcus jannaschii, Mba: Methanosarcina barkeri, Eukaryotes- Cel: Caenorhabditis elegans, Dme: Drosophila melanogaster, Hsa: Homo sapiens, Cal: Candida albicans, Sce: Saccharomyces cerevisiae, Spo: Schizosaccharomyces pombe, cpPpu: Porphyra purpurea chloroplast, Ath: Arabidopsis thaliana, Pfa: Plasmodium falciparum, Tbr: Trypanosoma brucei. Some protein families, such as ribonucleotide reductases and archaeal DNA polymerase type B, are more prone to contain inteins. These proteins contain inteins in different organisms and in different integration sites. Some of the ribonucleotide reductases and most of the DNA polymerases with inteins contain more than one intein. Currently (June 2004) about 200 inteins are identified in more than100 different species and strains, at more than 50 various families of protein hosts (details here). Inteins found at homologous integration sites are most probably homologous too. However, it is not clear in which cases this relation between the inteins is due to vertical transfer (the usual inheritance, from an organism to its progeny) or horizontal transfer (movement of DNA across species). Inteins at homologous integration sites are termed intein alleles. |
3楼2009-12-23 15:59:11
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An intein is a segment of a protein that is able to excise itself and rejoin the remaining portions (the exteins) with a peptide bond. Inteins have also been called "protein introns".[1] Most reported inteins also contain an endonuclease domain that plays a role in intein propagation. In fact, many genes have unrelated intein-coding segments inserted at different positions. For these and other reasons, inteins (or more properly, the gene segments coding for inteins) are sometimes called selfish genetic elements but it may be more accurate to call them parasitic. The difference is that "selfish genes" are "selfish" only insofar as to compete with other genes or alleles, but usually fulfill a function, whereas "parasitic genes" are always functionless. Intein-mediated protein splicing occurs after mRNA has been translated into a protein. This precursor protein contains three segments - an N-extein followed by the intein followed by a C-extein. After splicing has taken place, the result is also called an extein. The first intein was discovered in 1987. Since then, inteins have been found in all three domains of life (eukaryotes, bacteria, and archaea) and in viruses. Knowledge regarding the evolutionary situation of inteins and related elements is reviewed in Gogarten & Hilario (2006). The mechanism for the splicing effect is a naturally occurring analogy to the technique for chemically generating medium-sized proteins called native chemical ligation, which was developed at the same time as inteins were discovered. [edit] Inteins in biotechnology Inteins are very efficient at protein splicing and they have accordingly found an important role in biotechnology. There are more than 200 inteins identified to date, sizes range from 100-800 aa. Inteins have been engineered for particular applications such as protein synthesis,[2] and the selective labeling of protein segments, which is useful for NMR studies of large proteins.[3] Pharmaceutical inhibition of intein excision may be a useful tool for drug development, the protein that contains the intein will not carry out its normal function if the intein does not excise since its structure will be disrupted. It has been suggested that inteins could prove useful for achieving allotopic expression of certain highly hydrophobic proteins normally encoded by the mitochondrial genome, for example in gene therapy (de Grey 2000). The hydrophobicity of these proteins is an obstacle to their import into mitochondria. Therefore, the insertion of a non-hydrophobic intein may allow this import to proceed. Excision of the intein after import would then restore the protein to wild-type. [edit] Intein naming conventions The first part of an intein name is based on the scientific name of the organism in which it is found, and the second part is based on the name of the corresponding gene or extein. For example, the intein found in Thermoplasma acidophilum and associated with 'Vacuolar ATPase subunit A' (VMA) is called 'Tac VMA'. Normally, as in this example, just three letters suffice to specify the organism, but there are variations. For example, additional letters may be added to indicate a strain. If more than one intein is encoded in the corresponding gene, the inteins are given a numerical suffix starting from 5' to 3' or in order of their identification. For example, "Msm dnaB-1". The segment of the gene that encodes the intein is usually given the same name as the intein, but to avoid confusion, the name of the intein proper is usually capitalized (e.g. Pfu RIR1-1), whereas the name of the corresponding gene segment is italicized. [edit] Full and mini inteins Inteins can contain a homing endonuclease gene (HEG) domain in addition to the splicing domains. This domain is responsible for the spread of the intein by cleaving DNA at an intein free allele on the homologous chromosome, triggering the DNA double-stranded break repair (DSBR) system, which then repairs the break, thus copying the intein into a previously intein free site. The HEG domain is not necessary for intein splicing, and so it can be lost, forming a minimal, or mini intein. Several studies have demonstrated the modular nature of inteins by adding or removing HEG domains and determining the activity of the new construct. [edit] Split inteins Sometimes, the intein of the precursor protein comes from two genes. In this case, the intein is said to be a split intein. For example, in Cyanobacteria, DnaE, the catalytic subunit alpha of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. The dnaE-n product consists of an N-extein sequence followed by a 123-aa (amino acid) intein sequence, whereas the dnaE-c product consists of a 36-aa intein sequence followed by a C-extein sequence. |
5楼2009-12-23 16:00:21
