Radiation Inhibition of Amino Acid Uptake by Escherichia coli

The inhibition of macromolecular synthesis in Escherichia coli by ionizing radiation has been investigated. The survival of the ability to incorporate arginine, leucine, isoleucine, histidine, uracil, and glucose after various doses of gamma radiation, deuteron and alpha particle bombardment has been measured. All amino acids are incorporated by processes which show the same radiation sensitivity. The sensitivity of uracil corresponds to a volume which is roughly spherical, of radius about 160A, whereas the amino acids possess sensitive regions which are long and thin in character. The uptake of glucose is concerned with a smaller, roughly spherical unit. The possible identification of the radiation-sensitive targets with cellular constituents is discussed. The long thin character observed for amino acids suggests that the sensitive region affected by radiation is an unfolded form of a ribosome, or alternatively a long nucleic acid molecule. For uracil the sensitive region fits with a 70S ribosome, while for glucose a smaller particle would fit the data.     

Chimeras between single-stranded DNA-binding proteins from Escherichia coli and Mycobacterium tuberculosis reveal that their C-terminal domains interact with uracil DNA glycosylases

Uracil, a promutagenic base in DNA can arise by spontaneous deamination of cytosine or incorporation of dUMP by DNA polymerase. Uracil is removed from DNA by uracil DNA glycosylase (UDG), the first enzyme in the uracil excision repair pathway. We recently reported that the Escherichia coli single-stranded DNA binding protein (SSB) facilitated uracil excision from certain structured substrates by E. coli UDG (EcoUDG) and suggested the existence of interaction between SSB and UDG. In this study, we have made use of the chimeric proteins obtained by fusion of N- and C-terminal domains of SSBs from E. coli and Mycobacterium tuberculosis to investigate interactions between SSBs and UDGs. The EcoSSB or a chimera containing its C-terminal domain interacts with EcoUDG in a binary (SSB-UDG) or a ternary (DNA-SSB-UDG) complex. However, the chimera containing the N-terminal domain from EcoSSB showed no interactions with EcoUDG. Thus, the C-terminal domain (48 amino acids) of EcoSSB is necessary and sufficient for interaction with EcoUDG. The data also suggest that the C-terminal domain (34 amino acids) of MtuSSB is a predominant determinant for mediating its interaction with MtuUDG. The mechanism of how the interactions between SSB and UDG could be important in uracil excision repair pathway has been discussed.     

A multivariate study of the relationship between the genetic code and the physical-chemical properties of amino acids

Summary The 20 naturally occurring amino acids are characterized by 20 variables: pK_{NH 2}, pK_COOH, pI, molecular weight, substituent van der Waals volume, seven¹H and¹³C nuclear magnetic resonance shift variables, and eight hydrophobicity-hydrophilicity scales. The 20-dimensional data set is reduced to a few new dimensions by principal components analysis. The three first principal components reveal relationships between the properties of the amino acids and the genetic code. Thus the amino acids coded for by adenosine (A), uracil (U), or cytosine (C) in their second codon position (corresponding to U, A, or G in the second anticodon position) are grouped in these components. No grouping was detected for the amino acids coded for by guanine (G) in the second codon position (corresponding to C in the second anticodon position). The results show that a relationship exists between the physical-chemical properties of the amino acids and which of the A (U), U (A), or C (G) nucleotide is used in the second codon (anticodon) position. The amino acids coded for by G (C) in the second codon (anticodon) position do not participate in this relationship.     

Nutritional induction and suppression of fruiting in Myxococcus xanthus FBa

A defined agar medium (A agar) containing 15 amino acids in concentrations between 0.5 and 2 mm was developed for studying the fruiting cycle of Myxococcus xanthus FBa. Cells grew only vegetatively in this medium unless the initial concentration of one of nine required or stimulatory amino acids was lowered about 50-fold. In the latter circumstance, fruiting bodies developed after several days of vegetative growth. The conclusion was that fruiting occurred when any amino acid required for normal growth became limiting in the environment. High concentrations (10 mm) of phenylalanine, tryptophan, or methionine prevented fruiting without affecting growth. Mutants requiring arginine, thymidine, or adenine could not be induced to fruit by limiting their unique requirement although they responded to the same deprivations which brought about fruiting of the wild type. A histidine auxotroph formed fruiting bodies when histidine was lowered to growth-limiting concentrations, provided that the medium was supplemented with purines. A uracil auxotroph was isolated that, perhaps secondarily, had lost some of the mechanisms which control the formation of fruiting bodies; if uracil was present, it formed fruits even when no amino acid was limiting. No concentration of uracil was sufficient to prevent fruiting. Fruiting bodies were formed when mixtures of the uracil auxotroph and wild-type cells were inoculated on A agar plus uracil, even when 75% of the cells were wild type. Microcysts of both strains were present in the fruiting bodies.     

Genetic code: codon bases--the symbols of amino acid synthesis and catabolism pathways

The correlations between genetic codes of amino acids and pathways of synthesis and catabolism of carbon backbone of amino acids are considered. Codes of amino acids which are synthesized from oxoacids of glycolysis, the Krebs cycle and glyoxalic cycle via transamination without any additional chemical reactions, are initiated with guanine (alanine, glutamic and aspartic acids, glycine). Codons of amino acids which are formed on the branches of glycolysis at the level of compounds with three carbon atoms, begin with uracil (phenylalanine, serine, leucine, tyrosine, cysteine, tryptophan). Codes of amino acids formed from aspartate begin with adenine (methionine, isoleucine, threonine, asparagine, lysine, serine), while those of the amino acids formed from the compounds with five carbon atoms (glutamic acid and phosphoribosyl pyrophosphate) begin with cytosine (arginine, proline, glutamine, histidine). The second letter of codons is linked to catabolic pathways of amino acids: most of amino acids entering glycolysis and the Krebs cycle through even-numbered carbon compounds, have adenine and uracil at the second position of codes (A-U type); most of amino acids entering the glycolysis and the Krebs cycle via odd-numbered carbon compounds, have codons with guanine and cytidine at the second position (G-C type). The usage of purine and pyrimidine as the third letter of weak codones in most of amino acids is linked to the enthropy of amino acid formation. A hypothesis claiming that the linear genetic code was assembled from the purine and pyrimidine derivatives which have acted as participants of primitive control of amino acid synthesis and catabolism, is suggested.     

The complementarity of the chemical parameters of amino acids and anticodons in the genetic code

Chemical language of the genetic code is suggested in which elementary information code units are presented by functional groups of amino acids and nucleotides. Using this language, the existence of correspondence and conformity of chemical parameters of amino acids and of central nucleotides of their anticodons was demonstrated. These findings confirm the idea that the genetic code is determined by chemical properties of amino acids and nucleotides and that this determination is the result of direct specific interactions between amino acids and nucleotide triplets at the stage of the origin of the code. The data obtained reveal primary role of anticodon triplets in the origin of the code. Key role of the central nucleotide in triplets for amino acid coding is confirmed.     

Synthesis of prebiotic molecules — Role of some carbonyl compounds in prebiotic chemistry

Methyleneaminoacetonitrile(MAAN) resulting from the interaction of formaldehyde, ammonia and hydrogen cyanide on hydrolysis under mildly alkaline conditions gives a number of amino acids and peptides. Various aldehydes react with glycine to give corresponding hydroxyalkyl amino acids, which on reduction with formic acid are converted to reduced amino acids. Formaldehyde reacts with uracil to give 5-hydroxymethyl uracil which on reduction with formic acid yields thymine. Pyrrole formed by heating serine reacts with aldehydes to form porphyrins. Clays do not seem to influence most of these reactions, except the uracil-formaldehyde — formic acid reaction which results in enhanced yield of thymine.     

Minicells of Bacillus subtilis a unique system for transport studies

Ultrasound-purified minicells produced by Bacillus subtilis mutant div IV-Bl have been studied for their ability to transport and incorporate into macromolecules a variety of amino acids, uracil and thymine. Minicells transport all 12 amino acids examined, but are unable to incorporate them into macromolecules. No significant differences were found in the initial uptake rates of glutamic acid, aspartic acid, and alanine by minicells and parental cells. The uptake of methionine and proline by minicells was shown to be inhibited by metabolic poisons, indicating an energy-metabolism requirement for transport in this system. The proline pool in minicells was found to be readily exchangeable with exogenous proline. In contrast metabolically poisoned minicells only slowly lose their pool proline, indicating an energy requirement for pool maintenance. Packed-cell experiments reveal that minicells accumulate proline against a concentration gradient.In addition to amino acids, minicells are able to transport uracil but cannot incorporate uracil into acid-precipitable material (RNA). Neither thymine transport nor its incorporation into macromolecules can be demonstrated in minicells.Minicells appear to be a new system, therefore, in which transport may be studied in the absence of macromolecular biosynthesis.     

The Uptake and Utilization of Bacteria, Amino Acids and Nucleic Acid Components by the Rumen Ciliate Eudiplodinium maggii

Washed suspensions of the rumen ciliate protozoon Eudiplodinium maggii grown in vitro and incubated anaerobically engulfed all the bacteria tested except for Bacteroides ruminicola and Klebsiella aerogenes. There was considerable variation (160–9100 bacteria/h/protozoon at an external concentration of 10¹⁰ bacteria/ml) in the rate at which the bacteria were engulfed, but Eu. maggii showed some preference for bacteria of rumen origin. Some of the bacteria were digested with the release of soluble materials into the medium. Free amino acids were incorporated from an 0.1 mM solution at rates of 0.13 to 0.84 pmol/h/protozoon. Evidence is presented that Eu. maggii could obtain half the amino acids required for growth by the engulfment and digestion of bacteria and half by the uptake of free amino acids. Eudiplodinium maggii incorporated uridine 5' monophosphate and also hydrolysed this to uridine and then to uracil which was reduced to dihydrouracil. These products all appeared in the medium. Ribose was incorporated by the protozoon and appeared as glucose in protozoal and bacterial polysaccharide; none was incorporated as such into protozoal nucleic acid.     

Complexes of polyadenylic acid and the methyl esters of amino acids

This report includes studies of the binding of the methyl esters of a series of amino acids to polyadenylic acid. The principal data were obtained using proton NMR; however, some additional data were obtained through the study of insoluble complexes and through ultraviolet spectroscopy. The binding constants are in the order Phe>Ile?Leu>Val>Gly, and show a direct correlation with the hydrophobicities of the amino acids. In most cases they are essentially double the binding constants found by Reuben and Polk (1980) for monomeric AMP. All of these amino acids, except Gly, have A as the middle letter of their anticodons, and Phe is the only one with XAA as its only anticodon. It has the anticodon richest in A and has the highest binding constant for A. These results, coupled with other data, continue to support a model of the origin of the code which is based on weak, but selective affinities between amino acids and their anticodons.     

Uracil recognition in archaeal DNA polymerases captured by X-ray crystallography

Archaeal family B DNA polymerases bind tightly to template-strand uracil and stall replication on encountering the pro-mutagenic base. This article describes an X-ray crystal structure, at 2.8 Å resolution, of Thermococcus gorgonarius polymerase in complex with a DNA primer-template containing uracil in the single-stranded region. The DNA backbone is distorted to position the uracil deeply within a pocket, located in the amino-terminal domain of the polymerase. Specificity arises from a combination of hydrogen bonds between the protein backbone and uracil, with the pocket shaped to prevent the stable binding of the four standard DNA bases. Strong interactions are seen with the two phosphates that flank the uracil and the structure gives clues concerning the coupling of uracil binding to the halting of replication. The importance of key amino acids, identified by the analysis of the structure and their conservation between archaeal polymerases, was confirmed by site-directed mutagenesis. The crystal structure of V93Q, a polymerase variant that no longer recognises uracil, is also reported, explaining the V93Q phenotype by the steric exclusion of uracil from the pocket.     

Determinants of affinity and mode of DNA binding at the carboxy terminus of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1.

The role of the carboxy-terminal amino acids of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1, in DNA binding was analyzed. Chain-terminating mutations truncating the normally 99-amino-acid TF1 at amino acids 96, 97, and 98 were constructed, as were missense mutations substituting cysteine, arginine, and serine for phenylalanine at amino acid 97 and tryptophan for lysine at amino acid 99. The binding of the resulting proteins to a synthetic 44-bp binding site in 5-(hydroxymethyl)uracil DNA, to binding sites in larger SPO1 [5-(hydroxymethyl)uracil-containing] DNA fragments, and to thymine-containing homologous DNA was analyzed by gel retardation and also by DNase I and hydroxy radical footprinting. We conclude that the C tail up to and including phenylalanine at amino acid 97 is essential for DNA binding and that the two C-terminal amino acids, 98 and 99, are involved in protein-protein interactions between TF1 dimers bound to DNA.     

Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil

Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3′–5′ proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase–DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase.     

Compressing Proteomes: The Relevance of Medium Range Correlations

We study the nonrandomness of proteome sequences by analysing the correlations that arise between amino acids at a short and medium range, more specifically, between amino acids located 10 or 100 residues apart; respectively. We show that statistical models that consider these two types of correlation are more likely to seize the information contained in protein sequences and thus achieve good compression rates. Finally, we propose that the cause for this redundancy is related to the evolutionary origin of proteomes and protein sequences.     

De novo synthesis of uracil nucleotides in mouse liver and intestine studied using [15N]alanine

The amount of newly synthesized uracil nucleotides in mouse liver and intestine was determined by analysis of 15N incorporation into the uracil nucleotide pool of these tissues after intraperitoneal infusion of 15N-labelled amino acids. The appearance of newly synthesized uracil nucleotides was linear with time, and essentially independent of the rate of infusion of L-[15N]alanine. Varying the amino acid used in the infusion could affect the enrichment in the uracil ring nitrogens, but had no significant effect on the calculated amount of de novo synthesis. These results demonstrate the utility of this method in measuring de novo uracil nucleotide synthesis in mouse liver and intestine in vivo. The method should be a valuable tool in the effort to understand the regulation and pharmacological manipulation of de novo uracil nucleotide synthesis.     

A new theoretical model for the origin of amino acid homochirality

Amino acid homochirality, as a unique behavior of life, could have originated synchronously with the genetic code. In this paper, phosphoryl amino-acid-5′-nucleosides with P-N bond are postulated to be a chiral origin model in prebiotic molecular evolution. The enthalpy change in the intramolecular interaction between the nucleotide base and the amino-acid side-chain determines the stability of the particular complex, resulting in a preferred conformation associated with the chirality of amino acids. Based on the theoretical model, our experiments and calculations show that the chiral selection of the earliest amino acids for L-enantiomers seems to be a strict stereochemical/physicochemical determinism. As other amino acids developed biosynthetically from the earliest amino acids, we infer that the chirality of the later amino acids was inherited from the precursor amino acids. This idea probably goes far back in history, but it is hoped that it will be a guide for further experiments in this area.     

A hypothesis and an experiment on the origin of the genetic coding process

From thermodynamic considerations it is argued that the earliest templates for protein synthesis were very unlike present nucleic acids. It is suggested that they were composed of linked uracil derivatives bearing hypothetical side chains that could specifically recognize and interact with individual amino acid side chains (Fig. 1). An experiment supporting the possibility of such a specific interaction is described. The hypothesis accounts for the grouping of amino acids in the present codon table according to their relative hydrophobic character.     

On the Physical Basis of the Amino Acid Polar Requirement

Understanding how codons became associated with their specific amino acids is fundamental to deriving a theory for the origin of the genetic code. Carl Woese and coworkers designed a series of experiments to test associations between amino acids and nucleobases that may have played a role in establishing the genetic code. Through these experiments it was found that a property of amino acids called the polar requirement (PR) is correlated with the organization of the codon table. No other property of amino acids has been found that correlates with the codon table as well as PR, indicating that PR is uniquely related to the modern genetic code. Using molecular dynamics simulations of amino acids in solutions of water and dimethylpyridine used to experimentally measure PR, we show that variations in the partitioning between the two phases as described by radial distribution functions correlate well with the measured PRs. Partition coefficients based on probability densities of the amino acids in each phase have the linear behavior with base concentration as suggested by PR experiments.     

Membrane topology of the yeast uracil permease

The uracil permease of Saccharomyces cerevisia e is a 633 residue polytopic plasma membrane protein. Hydropathy profile analysis indicates that this protein has long hydrophilic N-and C-termini and 10–12 potential transmembrane segments. Previous results based on analysis of hybrid proteins allowed identification of the first transmembrane segment of uracil permease, and provided a preliminary indication of the cytoplasmic orientation of its N-terminus. In this work, other experimental approaches were used to confirm this orientation, and to determine that of the C-terminus. Epitopes in the N-and the C-termini of the protein were protected against trypsin degradation on intact protoplasts, but readily digested on permeabilized protoplasts. Immunofluorescent analysis showed that antibodies to the last 10 amino acids of uracil permease bind to detergent-treated protoplasts, but not to intact ones. Carboxypeptidase digested the C-terminus of uracil permease inserted into sealed dog-pancreas microsomes. These results establish that both N- and C-termini are cytoplasmic, the permease polypeptide spanning the membrane an even number of times. The orientation of several hydrophilic loops with respect to the membrane was investigated by introducing potential glycosylation sites into these regions. We checked whether the resulting mutant proteins were glycosylated when expressed in the presence of dog-pancreas microsomes. Our data show that two loops of the protein are lumenal. Together with previous results, this work indicates that uracil permease is a 10 membrane-spanning protein, with rather small external loops and three main cytoplasmic regions (the N-and C-termini and a central 60-residue loop).     

Toxoplasma gondii: the enzymic defect of a mutant resistant to 5-fluorodeoxyuridine.

We have previously described a mutant of Toxoplasma gondii that was 100-fold more resistant to 5-fluorodeoxyuridine, as measured by growth in human fibroblast cultures. Various pyrimidine salvage enzymes were measured in the wild type and the mutant parasites to determine the biochemical basis for resistance to fluorodeoxyuridine. Both the resistant mutant and the wild type parasite had little or no uridine kinase, an enzyme readily detectable in the human fibroblast host cells. Uridine and deoxyuridine phosphorylases were found in both parasites while human fibroblasts had much less of these enzymes. The critical difference between the mutant and the wild type parasites proved to be a 100-fold lower concentration of uracil phosphoribosyltransferase in the fluorodeoxyuridine-resistant mutant. A back mutant of the resistant strain, selected for its ability to use uracil, simultaneously regained uracil phosphoribosyltransferase and sensitivity to fluorodeoxyuridine. This enzymic evidence together with previously published data show that in wild type T. gondii, deoxyuridine is incorporated into nucleic acids through a phosphorolysis to produce uracil which is then converted to uridylic acid by uracil phosphoribosyltransferase.