Reversibility and efficiency in coding protein information

Why the genetic code has a fixed length? Protein information is transferred by coding each amino acid using codons whose length equals 3 for all amino acids. Hence the most probable and the least probable amino acid get a codeword with an equal length. Moreover, the distributions of amino acids found in nature are not uniform and therefore the efficiency of such codes is sub-optimal. The origins of these apparently non-efficient codes are yet unclear. In this paper we propose an a priori argument for the energy efficiency of such codes resulting from their reversibility, in contrast to their time inefficiency. Such codes are reversible in the sense that a primitive processor, reading three letters in each step, can always reverse its operation, undoing its process.We examine the codes for the distributions of amino acids that exist in nature and show that they could not be both time efficient and reversible. We investigate a family of Zipf-type distributions and present their efficient (non-fixed length) prefix code, their graphs, and the condition for their reversibility. We prove that for a large family of such distributions, if the code is time efficient, it could not be reversible. In other words, if pre-biotic processes demand reversibility, the protein code could not be time efficient. The benefits of reversibility are clear: reversible processes are adiabatic, namely, they dissipate a very small amount of energy. Such processes must be done slowly enough; therefore time efficiency is non-important. It is reasonable to assume that early biochemical complexes were more prone towards energy efficiency, where forward and backward processes were almost symmetrical.     

tRNA structure from a graph and quantum theoretical perspective

One of the objectives of theoretical biochemistry is to find a suitable representation of molecules allowing us to encode what we know about their structures, interactions and reactivity. Particularly, tRNA structure is involved in some processes like aminoacylation and genetic code translation, and for this reason these molecules represent a biochemical object of the utmost importance requiring characterization. We propose here two fundamental aspects for characterizing and modeling them. The first takes into consideration the connectivity patterns, i.e. the set of linkages between atoms or molecular fragments (a key tool for this purpose is the use of graph theory), and the second one requires the knowledge of some properties related to the interactions taking place within the molecule, at least in an approximate way, and perhaps of its reactivity in certain means. We used quantum mechanics to achieve this goal; specifically, we have used partial charges as a manifestation of the reply to structural changes. These charges were appropriately modified to be used as weighted factors for elements constituting the molecular graph. This new graph-tRNA context allow us to detect some structure-function relationships.     

Genetic-code evolution for protein synthesis with non-natural amino acids

The genetic encoding of synthetic or “non-natural” amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG “stop” triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites.     

Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.     

ATM protein physically and functionally interacts with proliferating cell nuclear antigen to regulate DNA synthesis

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner.     

DNA序列分析中的信息熵应用现状

信息熵理论是生物信息学研究的一个重要工具,它在DNA序列分析中有着广泛的应用。本文详细介绍了近年来诸多DNA序列分析问题中信息熵应用的研究进展,并分析了未来该问题的研究方向。     

The universal dinucleotide asymmetry rules in DNA and the amino acid codon choice

Summary Natural DNA sequences were recently found to contain distinct nearest neighbor patterns. Hetero-dinucleotides were demonstrated to appear consistently more (less) than their mirror-image counterparts. This paper shows that this assymmetric behavior does not stem from the coding requirements of the DNA. It also shows some codon patterns in prokaryotic and eukaryotic genomes which came up in the course of this work.     

The relationship of DNA base composition and individual protein composition in micro-organisms

Summary To investigate the dependence of protein composition on DNA base composition, a set of data on individual proteins with known amino acid compositions from a spectrum of bacterial species has been compiled. It is found that similar relationships of amino acid frequency to G + C content exist for these proteins as for the bulk proteins studied by Sueoka (1961). The data are analysed by linear and cubic regression, and a measure of the proportions of A + T-rich and G + C-rich codons in the underlying messenger RNAs is put forward. The theoretical limits on the G + C content of coding DNA are discussed, and inference are made about the various selective forces acting on DNAs of different G + C contents.     

Identification of amino acids stabilizing the tetramerization of the single stranded DNA binding protein from Escherichia coli

Mutating the histidine at position 55 present at the subunit interface of the tetrameric E. coli single stranded DNA binding (SSB) protein to tyrosine or lysine leads to cells which are UV- and temperature-sensitive. The defects of both ssbH55Y (ssb-1) and ssbH55K can be overcome by increasing protein concentration, with the ssbH55K mutation producing a less stable, readily dissociating protein whose more severe replication and repair phenotypes were less easily ameliorated by protein amplification. In this study we selected and analyzed E. coli strains where the temperature sensitivity caused by the ssbH55K mutation was suppressed by spontaneous mutations that changed the glutamine at position 76 or 110 to leucine. Using guanidinium chloride denaturation monitored by sedimentation diffusion equilibrium experiments in the analytical ultracentrifuge, we demonstrate that the double mutant SSBH55KQ76L and SSBH55KQ110L proteins form more stable homotetramers as compared to the SSBH55K single mutant protein although they are less stable than wild-type SSB. Additionally, the single mutant proteins SSBQ76L and SSBQ110L form tetramers which are more resistant to guanidinium denaturation than wild-type SSB protein.     

Isolation and identification of novel protein kinase genes from the round-spotted pufferfish(Tetraodon fluviatilis) genomic DNA

The round-spotted pufferfishTetraodon fluviatilis has a genome size of 380 Mb which is slightly smaller than that of another pufferfish,Fugu rubripes rubripes (Fugu). Due to their compact genome and small introns, both pufferfishes have been proposed as model organisms for genome studies. In this study, we have used genomic DNA as template to perform PCR to screen for protein kinase (pk) genes. Forty-oneT. fluviatilis pk genes encoding 7 receptor tyrosine kinases, 14 nonreceptor tyrosine kinases, 16 serine/threonine kinases, 1 dual kinase and 3 novel kinases have been identified. The success of this approach depends on the size and location of the introns. Most of the identifiedpk gene fragments contain introns, ranging from 71 to 300 bp, with an average of 120 bp. It is noteworthy that the intron/exon boundaries of certain genes which belong to the same family are identical. We also analyzed by specific RT-PCR primers the expression profile of those 3 novel genes as well as some selectedpk genes in a variety of tissues. We found thaterbB3,pku , mrk, CaMK I,CaMKII, and two novel kinase genes (133 and 3–26) are expressed in all tissues examined. However, the novel clone 146 is strongly expressed in the brain and weakly in the intestine, kidney and heart.     

Indexes to the reassociation and stability of solution DNA hybrids

Summary The computation, assumptions, and properties of DNA-hybrid stability and reassociation indexes were reviewed. Different methods of computing the same index typically yielded similar values. However, because dissociation curves change from asymmetric to symmetric as increasingly divergent DNAs are compared, adequate determination of mode required fitting a complex function. Delta Tm, delta mode, and delta T50H correlated well up to ca. 12, and all were found to be useful indexes of genomic similarity in that range. They also exhibited similar levels of error, even though T50H comprises a percent reassociation component with relatively large variance. At greater distances, the delta Tm scale became markedly compressed because of the boundary imposed by the temperature of hybrid formation (incubation temperature). Though not compressed or technically limited by it, delta mode and delta T50H could not be extrapolated with certainty below the incubation temperature. Among theoretical problems discussed: Tm and mode index an increasingly small percentage of the genome as the extent of reassociation decreases, and they may compare different genomic segments as DNAs become highly diverged. T50H relies upon the assumptions that all sequences evolve at a constant rate and that reassociation behavior is the same among all sequences regardless of their extent of divergence. Tm and T50H may be biased by selfhybridization of repetitive elements or cross-hybridization of paralogous sequences. Delta mode is free of such biases as long as the genomes under comparison are not too diverged. No index was found to be best in all circumstances.     

Histone-like protein HU from Deinococcus radiodurans binds preferentially to four-way DNA junctions

The histone-like protein HU from Escherichia coli is involved in DNA compaction and in processes such as DNA repair and recombination. Its participation in these events is reflected in its ability to bend DNA and in its preferred binding to DNA junctions and DNA with single-strand breaks. Deinococcus radiodurans is unique in its ability to reconstitute its genome from double strand breaks incurred after exposure to ionizing radiation. Using electrophoretic mobility shift assays (EMSA), we show that D.radiodurans HU (DrHU) binds preferentially only to DNA junctions, with half-maximal saturation of 18 nM. In distinct contrast to E.coli HU, DrHU does not exhibit a marked preference for DNA with nicks or gaps compared to perfect duplex DNA, nor is it able to mediate circularization of linear duplex DNA. These unexpected properties identify DrHU as the first member of the HU protein family not to serve an architectural role and point to its potential participation in DNA recombination events. Our data also point to a mechanism whereby differential target site selection by HU proteins is achieved and suggest that the substrate specificity of HU proteins should be expected to vary as a consequence of their individual capacity for inducing the required DNA bend.     

Electrochemical biosensor based on base-stacking-dependent DNA hybridization assay for protein detection

An antibody-based electrochemical biosensing platform has been developed and used for the detection of protein. In the presence of the target, an antibody pair binds to the protein simultaneously, which causes two oligo-DNAs conjugated with the antibody pair to hybridize to each other and become a big “stem–loop” structure. Subsequently, the longer oligo-DNA of the stem, with a methylene blue (MB) label at the terminal, hybridizes stably with capture DNA owing to the enhancement of base stacking. The strong redox current signal of MB is used for protein quantification. Using α-fetoprotein (AFP) as a model, the proposed method could detect AFP at a concentration as low as 2 pg ml⁻¹ with a dynamic range of 4 orders of magnitude, which approaches traditional assays such as enzyme-linked immunosorbent assay.     

Use of Chou-Fasman amino acid conformational parameters to analyze the organization of the genetic code and to construct protein genealogies

Summary Chou-Fasman parameters, measuring preferences of each amino acid for different conformational regions in proteins, were used to obtain an amino acid difference index of conformational parameter distance (CPD) values. CPD values were found to be significantly lower for amino acid exchanges representing in the genetic code transitions of purines, GA than for exchanges representing either transitions of pyrimidines, CU, or transversions of purines and pyrimidines. Inasmuch as the distribution of CPD values in these non GA exchanges resembles that obtained for amino acid pairs with double or triple base differences in their underlying codons, we conclude that the genetic code was not particularly designed to minimize effects of mutation on protein conformation. That natural selection minimizes these changes, however, was shown by tabulating results obtained by the maximum parsimony method for eight protein genealogies with a total occurrence of 4574 base substitutions. At the beginning position of the codons GA transitions were in very great excess over other base substitutions, and, conversely, CU transitions were deficient. At the middle position of the codons only fast evolving proteins showed an excess of GA transitions, as though selection mainly preserved conformation in these proteins while weeding out mutations affecting chemical properties of functional sites in slow evolving proteins. In both fast and slow evolving proteins the net direction of transitions and transversions was found to be from G beginning codons to non-G beginning codons resulting in more commonly occurring amino acids, especially alanine with its generalized conformational properties, being replaced at suitable sites by amino acids with more specialized conformational and chemical properties. Historical circumstances pertaining to the origin of the genetic code and the nature of primordial proteins could account for such directional changes leading to increases in the functional density of proteins.In order to further explore the course of protein evolution, a modified parsimony algorithm was developed for constructing protein genealogies on the basis of minimum CPD length. The algorithm's ability to judge with finer discrimination that in protein evolution certain pathways of amino acid substitution should occur more readily than others was considered a potential advantage over strict maximum parsimony. In developing this CPD algorithm, the path of minimum CPD length through intermediate amino acids allowed by the genetic code for each pair of amino acids was determined. It was found that amino acid exchanges representing two base changes have a considerably lower average CPD value per base substitution than the amino acid exchanges representing single base changes. Amino acid exchanges representing three base changes have yet a further marked reduction in CPD per base change. This shows how extreme constraining effects of stabilizing selection can be circumvented, for by way of intermediate amino acids almost any amino acid can ultimately be substituted for another without damage to an evolving protein's conformation during the process.     

Self-assembling protein arrays on DNA chips by auto-labeling fusion proteins with a single DNA address

The high-throughput deposition of recombinant proteins on chips, beads or biosensor devices would be greatly facilitated by the implementation of self-assembly concepts. DNA-directed immobilization via conjugation of proteins to an oligonucleotide would be preeminently suited for this purpose. Here, we present a unique method to attach a single DNA address to proteins in one step during the purification from the E. coli lysate by fusion to human O6-alkylguanine-DNA-alkyltransferase (SNAP-tag) and the Avitag. Use of the conjugates in converting a DNA chip into a protein chip by self assembly is demonstrated.