翻译速率与蛋白质二级结构的关系

统计分析了人的 119种蛋白质和大肠杆菌的 92种蛋白质密码子翻译速率和蛋白质二级结构的关系。据m 密码子片段在不同二级结构中的频数分布 ,我们发现人和大肠杆菌中翻译速率与蛋白质二级结构之间有一定关系 :高翻译速率时倾向编码α螺旋、不倾向编码线团 (coil) ;低翻译速率时倾向编码线团、不倾向编码α螺旋 ;β折叠结构则随翻译速率表现出明显的振荡。同时 ,密码子的使用在不同片段内一般也是不均匀的 :在α螺旋片段内 ,结构尾部偏向使用高翻译速率密码子 ;中部倾向使用中翻译速率密码子 ;而头部使用的密码子翻译速率偏低。这样的倾向性不大可能归结为随机起伏的影响。     

Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52.

Purified human Rad51 protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair. Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 protein. Joint molecule formation required the presence of a 3' or 5'-overhang on the duplex DNA substrate and initiated preferentially at the 5'-end of the complementaryx strand. These results are consistent with a preference for strand transfer in the 3'-5' direction relative to the single-stranded DNA. The human single-strand DNA-binding protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E. coli single-strand DNA-binding protein in RecA-mediated strand exchange reactions. Indeed, E. coli single-strand DNA-binding protein could substitute for hRP-A in hRad51-mediated reactions. Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions. The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA.     

Histone-like proteins in the purified Escherichia coli deoxyribonucleoprotein

Analysis of E.coli chromosomes isolated under conditions similar to those used for isolation of eukaryotic chromatin has shown that: 1) The proteins of highly purified E.coli deoxyribonucleoprotein are mainly in addition to RNA polymerase two specific histone-like proteins of apparent molecular weight of 17,000 and 9,000 (proteins 1 and 2, respectively). 2) Proteins 1 and 2 occur in approximately equal molar amounts in the isolated E.coli chromosome, and their relative content corresponds to one molecule of protein 1 plus one molecule of protein 2 per 150-200 base pairs of DNA. 3) There are no long stretches of naked DNA in the purified E.coli deoxyribonucleoprotein suggesting a fairly uniform distribution of the proteins 1 and 2 along DNA. 4) The protein 2 is apparently identical to the DNA-binding protein HU which was isolated previously /1/ from extracts of E.coli cells. 5) Digestion of the isolated E.coli chromosomes with staphylococcal nuclease proceeds through discrete deoxyribonucleoprotein intermediates (in particular, at approximately 120 base pairs) which contain both proteins 1 and 2. However, since no repeating multimer structure was observed so far in nuclease digests of the E.coli chromosome, it seems premature to draw definite conclusions about possible similarities between the nucleosomal organization of the eukaryotic chromatin and the E.coli chromatin structure.Images     

Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein.

Single-stranded DNA binding proteins (SSBs) have been isolated from many organisms, including Escherichia coli, Saccharomyces cerevisiae and humans. Characterization of these proteins suggests they are required for DNA replication and are active in homologous recombination. As an initial step towards understanding the role of the eukaryotic SSBs in DNA replication and recombination, we examined the DNA binding and strand exchange stimulation properties of the S. cerevisiae single-strand binding protein y-RPA (yeast replication protein A). y-RPA was found to bind to single-stranded DNA (ssDNA) as a 115,000 M(r) heterotrimer containing 70,000, 36,000 and 14,000 M(r) subunits. It saturated ssDNA at a stoichiometry of one heterotrimer per 90 to 100 nucleotides and binding occurred with high affinity (K omega greater than 10(9) M-1) and co-operativity (omega = 10,000 to 100,000). Electron microscopic analysis revealed that y-RPA binding was highly co-operative and that the ssDNA present in y-RPA-ssDNA complexes was compacted fourfold, arranged into nucleosome-like structures, and was free of secondary structure. y-RPA was also tested for its ability to stimulate the yeast Sepl and E. coli RecA strand-exchange proteins. In an assay that measures the pairing of circular ssDNA with homologous linear duplex DNA, y-RPA stimulated the strand-exchange activity of Sepl approximately threefold and the activity of RecA protein to the same extent as did E. coli SSB. Maximal stimulation of Sepl occurred at a stoichiometry of one y-RPA heterotrimer per 95 nucleotides of ssDNA. y-RPA stimulated RecA and Sepl mediated strand exchange reactions in a manner similar to that observed for the stimulation of RecA by E. coli SSB; in both of these reactions, y-RPA inhibited the aggregation of ssDNA and promoted the co-aggregation of single-stranded and double-stranded linear DNA. These results demonstrate that the E. coli and yeast SSBs display similar DNA-binding properties and support a model in which y-RPA functions as an E. coli SSB-like protein in yeast.     

同义密码子的反常蛋白质二级结构偏好性

统计分析了 119种人蛋白质和 92种大肠杆菌蛋白质的mRNA序列和蛋白质二级结构的关系 .从二肽频数出发 ,研究了同义密码子使用对蛋白质二级结构的影响 ,证明其影响在 10 %到 2 0 %的量级 .对于人和大肠杆菌 ,在 90 %置信水平上 ,4 0 0对二肽中分别有 79对和 6 0对 ,在 95 %置信水平上 ,分别有 4 5对和 36对二肽的相应密码子二联体具有不同于氨基酸的反常二级结构偏好性 ,并且这种反常不能归因于随机涨落     

The signal for the termination of protein synthesis in procaryotes.

The sequences around the stop codons of 862 Escherichia coli genes have been analysed to identify any additional features which contribute to the signal for the termination of protein synthesis. Highly significant deviations from the expected nucleotide distribution were observed, both before and after the stop codon. Immediately prior to UAA stop codons in E. coli there is a preference for codons of the form NAR (any base, adenine, purine), and in particular those that code for glutamine or the basic amino acids. In contrast, codons for threonine or branched nonpolar amino acids were under-represented. Uridine was over-represented in the nucleotide position immediately following all three stop codons, whereas adenine and cytosine were under-represented. This pattern is accentuated in highly expressed genes, but is not as marked in either lowly expressed genes or those that terminate in UAG, the codon specifically recognised by polypeptide chain release factor-1. These observations suggest that for the efficient termination of protein synthesis in E. coli, the 'stop signal' may be a tetranucleotide, rather than simply a tri-nucleotide codon, and that polypeptide chain release factor-2 recognises this extended signal. The sequence following stop codons was analysed in genes from several other procaryotes and bacteriophages. Salmonella typhimurium, Bacillus subtilis, bacteriophages and the methanogenic archaebacteria showed a similar bias to E. coli.     

Aspartate transcarbamylase from the deep-sea hyperthermophilic archaeon Pyrococcus abyssi: genetic organization, structure, and expression in Escherichia coli.

The genes coding for aspartate transcarbamylase (ATCase) in the deep-sea hyperthermophilic archaeon Pyrococcus abyssi were cloned by complementation of a pyrB Escherichia coli mutant. The sequence revealed the existence of a pyrBI operon, coding for a catalytic chain and a regulatory chain, as in Enterobacteriaceae. Comparison of primary sequences of the polypeptides encoded by the pyrB and pyrI genes with those of homologous eubacterial and eukaryotic chains showed a high degree of conservation of the residues which in E. coli ATCase are involved in catalysis and allosteric regulation. The regulatory chain shows more-extensive divergence with respect to that of E. coli and other Enterobacteriaceae than the catalytic chain. Several substitutions suggest the existence in P. abyssi ATCase of additional hydrophobic interactions and ionic bonds which are probably involved in protein stabilization at high temperatures. The catalytic chain presents a secondary structure similar to that of the E. coli enzyme. Modeling of the tridimensional structure of this chain provides a folding close to that of the E. coli protein in spite of several significant differences. Conservation of numerous pairs of residues involved in the interfaces between different chains or subunits in E. coli ATCase suggests that the P. abyssi enzyme has a quaternary structure similar to that of the E. coli enzyme. P. abyssi ATCase expressed in transgenic E. coli cells exhibited reduced cooperativity for aspartate binding and sensitivity to allosteric effectors, as well as a decreased thermostability and barostability, suggesting that in P. abyssi cells this enzyme is further stabilized through its association with other cellular components.     

Heat labile ribonuclease HI from a psychrotrophic bacterium: gene cloning, characterization and site-directed mutagenesis.

The rnhA gene encoding RNase HI from a psychrotrophic bacterium, Shewanella sp. SIB1, was cloned, sequenced and overexpressed in an rnh mutant strain of Escherichia coli. SIB1 RNase HI is composed of 157 amino acid residues and shows 63% amino acid sequence identity to E.coli RNase HI. Upon induction, the recombinant protein accumulated in the cells in an insoluble form. This protein was solubilized and purified in the presence of 7 M urea and refolded by removing urea. Determination of the enzymatic activity using M13 DNA-RNA hybrid as a substrate revealed that the enzymatic properties of SIB1 RNase HI, such as divalent cation requirement, pH optimum and cleavage mode of a substrate, are similar to those of E.coli RNase HI. However, SIB1 RNase HI was much less stable than E.coli RNase HI and the temperature (T(1/2)) at which the enzyme loses half of its activity upon incubation for 10 min was approximately 25 degrees C for SIB1 RNase HI and approximately 60 degrees C for E.coli RNase HI. The optimum temperature for the SIB1 RNase HI activity was also shifted downward by 20 degrees C compared with that of E.coli RNase HI. Nevertheless, SIB1 RNase HI was less active than E.coli RNase HI even at low temperatures. The specific activity determined at 10 degrees C was 0.29 units/mg for SIB1 RNase HI and 1.3 units/mg for E.coli RNase HI. Site-directed mutagenesis studies suggest that the amino acid substitution in the middle of the alphaI-helix (Pro52 for SIB1 RNase HI and Ala52 for E.coli RNase HI) partly accounts for the difference in the stability and activity between SIB1 and E.coli RNases HI.     

Identification of RNase HII from psychrotrophic bacterium, Shewanella sp. SIB1 as a high-activity type RNase H

The gene encoding RNase HII from the psychrotrophic bacterium, Shewanella sp. SIB1 was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RNase HII is a monomeric protein with 212 amino acid residues and shows an amino acid sequence identity of 64% to E. coli RNase HII. The enzymatic properties of SIB1 RNase HII, such as metal ion preference, pH optimum, and cleavage mode of substrate, were similar to those of E. coli RNase HII. SIB1 RNase HII was less stable than E. coli RNase HII, but the difference was marginal. The half-lives of SIB1 and E. coli RNases HII at 30 degrees C were approximately 30 and 45 min, respectively. The midpoint of the urea denaturation curve and optimum temperature of SIB1 RNase HII were lower than those of E. coli RNase HII by approximately 0.2 M and approximately 5 degrees C, respectively. However, SIB1 RNase HII was much more active than E. coli RNase HII at all temperatures studied. The specific activity of SIB1 RNase HII at 30 degrees C was 20 times that of E. coli RNase HII. Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII represent low- and high-activity type RNases H, respectively, in SIB1. In contrast, RNases HI and HII represent high- and low-activity type RNases H, respectively, in E. coli. We propose that bacterial cells usually contain low- and high-activity type RNases H, but these types are not correlated with RNase H families.     

Projection structure of NhaA, a secondary transporter from Escherichia coli, at 4.0 A resolution.

Electron cryomicroscopy of frozen-hydrated two-dimensional crystals of NhaA, a Na+/H+ antiporter from Escherichia coli predicted to have 12 transmembrane alpha-helices, has facilitated the calculation of a projection map of NhaA at 4.0 A resolution. NhaA was homologously expressed in E.coli with a His6 tag, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two-dimensional crystals were obtained after reconstitution of purified protein with E.coli lipids. The projection map reveals that this secondary transporter has a highly asymmetric structure in projection. NhaA exhibits overall dimensions of approximately 38x48 A with a ring-shaped density feature probably corresponding to a bundle of tilted helices, adjacent to an elongated region of density containing several peaks indicative of transmembrane helices. Two crystal forms with p22121 symmetry show tightly packed dimers of NhaA which differ in the interactions between adjacent dimers. This work provides the first direct glimpse into the structure of a secondary transporter.     

The purification, characterization, and primary structure of a small redox protein from Methanobacterium thermoautotrophicum, an archaebacterium.

A small redox-active protein has been purified to homogeneity from cell-free extracts of the strictly anaerobic thermophilic methanogen, Methanobacterium thermoautotrophicum (strain Marburg). The purification consisted of streptomycin sulfate and acid treatments and three chromatographic steps using Sephadex G-75, Mono Q HR 10/10, and Superose 12 HR 10/30 columns. When these procedures were carried out under strictly anaerobic conditions, approximately 3 mg of this protein could be isolated from 45 g of wet cell paste. Like the thioredoxins and glutaredoxins, it is a small acidic protein (pI = 4.2) consisting of 83 amino acids (M(r) = 9136). In the presence of dithiothreitol or dihydrolipoate, the protein serves as a hydrogen donor for the ribonucleotide reductase from Escherichia coli, and it catalyzes the reduction of insulin. However, it does not interact with the thioredoxin reductases from E. coli or Corynebacterium nephridii and does not function as a hydrogen donor for the ribonucleotide reductase of C. nephridii. The amino acid sequences determined by automated Edman degradation of the 14C-carboxymethylated protein and of peptides derived from trypsin and chymotrypsin digestions show a redox-active site -Cys-Pro-Tyr-Cys-, typical of the glutaredoxins. Its amino acid sequence shows moderate identity with the known glutaredoxins (E. coli, yeast, rabbit bone marrow, calf thymus, and pig liver) when the proteins are aligned at the active site. The secondary structure of the glutaredoxin-like protein predicted by the Chou-Fasman procedure shows that it is similar to the known glutaredoxins. However, surprisingly, the protein does not function as a glutathione-disulfide oxidoreductase in the presence of glutathione and glutathione reductase. This glutaredoxin-like protein may be a component of a ribonucleotide-reducing system distinct from the previously described systems utilizing thioredoxin or glutaredoxin.     

Bacillus subtilis alkaline phosphatases III and IV. Cloning, sequencing, and comparisons of deduced amino acid sequence with Escherichia coli alkaline phosphatase three-dimensional structure

Bacillus subtilis has an alkaline phosphatase multigene family. Two members of this gene family, phoAIII and phoAIV, were cloned, taking advantage of in vitro constructed strains containing a plasmid insertion within one or the other of the structural genes. The DNA sequences of the two genes showed approximately 64% identity at the DNA level and 63% identity in the deduced primary amino acid sequences. The phoAIII and phoAIV genes code for predicted proteins of 47,149 and 45,935 Da, respectively. Comparison of the deduced primary amino acid sequence of the mature proteins with other sequenced alkaline phosphatases from Escherichia coli, yeast, and humans shows 25-30% identity. Based on the refined crystal structure of E. coli alkaline phosphatase, it appears that the active site and the core of the structure are retained in both Bacillus alkaline phosphatases. However, both proteins are truncated at the amino terminus compared with other mature alkaline phosphatases, three sizable surface loops of E. coli are deleted, and a minidomain is replaced with a larger domain in the model. Neither Bacillus alkaline phosphatase sequenced contains any cysteine residues, an amino acid implicated in intrachain disulfide bond formation in other alkaline phosphatases.     

Complete 1H, 13C, and 15N NMR resonance assignments and secondary structure of human glutaredoxin in the fully reduced form.

Human glutaredoxin is a member of the glutaredoxin family, which is characterized by a glutathione binding site and a redox-active dithiol/disulfide in the active site. Unlike Escherichia coli glutaredoxin-1, this protein has additional cysteine residues that have been suggested to play a regulatory role in its activity. Human glutaredoxin (106 amino acid residues, M(r) = 12,000) has been purified from a pET expression vector with both uniform 15N labeling and 13C/15N double labeling. The combination of three-dimensional 15N-edited TOCSY, 15N-edited NOESY, HNCA, HN(CO)CA, and gradient sensitivity-enhanced HNCACB and HNCO spectra were used to obtain sequential assignments for residues 2-106 of the protein. The gradient-enhanced version of the HCCH-TOCSY pulse sequence and HCCH-COSY were used to obtain side chain 1H and 13C assignments. The secondary structural elements in the reduced protein were identified based on NOE information, amide proton exchange data, and chemical shift index data. Human glutaredoxin contains five helices extending approximately from residues 4-10, 24-36, 53-64, 83-92, and 94-104. The secondary structure also shows four beta-strands comprised of residues 15-19, 43-48, 71-75, 78-80, which form a beta-sheet almost identical to that found in E. coli glutaredoxin-1. Complete 1H, 13C, and 15N assignments and the secondary structure of fully reduced human glutaredoxin are presented. Comparison to the structures of other glutaredoxins is presented and differences in the secondary structure elements are discussed.     

A study of the thermal stability of ribosomes and biologically active subribosomal particles

1. The ability of Escherichia coli ribosomes to function in poly(U)-directed protein synthesis was measured at elevated temperatures by using thermostable supernatant factors from Bacillus stearothermophilus. The amount of polyphenylalanine synthesized at 55 degrees C was about the same as at 37 degrees C, but the rate of synthesis was increased approximately fivefold. At 60 degrees C the activity of the ribosomes was halved. 2. E. coli ribosomes can sustain the loss of approx. 10% of the double-helical secondary structure of RNA without losing activity. 3. Within the active ribosome the double-helical secondary structure of the rRNA moiety is stabilized compared with isolated rRNA, as judged by enzymic hydrolysis and by measurements of E(260). 4. The main products, over the range 0-55 degrees C, of ribonuclease T(1) digestion of the smaller subribosomal particle of E. coli were two fragments (s(0) (20,w) 15S and 25.3S) of approximately one-quarter and three-quarters of the size of the intact molecule, revealing the presence of a ;weak spot' where intramolecular bonds appear insufficient to hold the fragments together. 5. Subribosomal particles of B. stearothermophilus were more stable to heating, by approx. 10 degrees C, than those of E. coli, and the stabilization of double-helical secondary structure of the RNA moiety was more striking. 6. Rabbit reticulocyte ribosomes were active in poly(U)-directed protein synthesis at 45 degrees C, and half the activity was lost after heating to 53 degrees C. Active subribosomal particles of rabbit reticulocytes and of oocytes of Xenopus laevis, like the bacterial subribosomal particles, underwent a conformational change to a slower-sedimenting form on heating. The temperature range of the transition depended on the species. 7. Slower-sedimenting particles, whether produced by EDTA treatment or by heating, had different ;melting' profiles compared with active subribosomal particles, providing another indication of conformational differences. 8. Comparison of the properties of the various subribosomal particles revealed greater variation in the secondary structure of the protein moieties (judged by measurement of circular dichroism) than in the secondary structure of the RNA moieties, which appeared to have features in common.     

Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host

Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm ("codon harmonization") for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the "recoded" genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli.     

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.