A nascent polypeptide domain that can regulate translation elongation.
(9/39)
The evolutionarily conserved fungal arginine attenuator peptide (AAP), as a nascent peptide, stalls the translating ribosome in response to the presence of a high concentration of the amino acid arginine. Here we examine whether the AAP maintains regulatory function in fungal, plant, and animal cell-free translation systems when placed as a domain near the N terminus or internally within a large polypeptide. Pulse-chase analyses of the radiolabeled polypeptides synthesized in these systems indicated that wild-type AAP functions at either position to stall polypeptide synthesis in response to arginine. Toeprint analyses performed to map the positions of stalled ribosomes on transcripts introduced into the fungal system revealed that ribosome stalling required translation of the AAP coding sequence. The positions of the stalled ribosomes were consistent with the sizes of the radiolabeled polypeptide intermediates. These findings demonstrate that an internal polypeptide domain in a nascent chain can regulate eukaryotic translational elongation in response to a small molecule. Apparently the peptide-sensing features are conserved in fungal, plant, and animal ribosomes. These data provide precedents for translational strategies that would allow domains within nascent polypeptide chains to modulate gene expression. (+info)
A Bayesian method for identifying missing enzymes in predicted metabolic pathway databases.
(10/39)
BACKGROUND: The PathoLogic program constructs Pathway/Genome databases by using a genome's annotation to predict the set of metabolic pathways present in an organism. PathoLogic determines the set of reactions composing those pathways from the enzymes annotated in the organism's genome. Most annotation efforts fail to assign function to 40-60% of sequences. In addition, large numbers of sequences may have non-specific annotations (e.g., thiolase family protein). Pathway holes occur when a genome appears to lack the enzymes needed to catalyze reactions in a pathway. If a protein has not been assigned a specific function during the annotation process, any reaction catalyzed by that protein will appear as a missing enzyme or pathway hole in a Pathway/Genome database. RESULTS: We have developed a method that efficiently combines homology and pathway-based evidence to identify candidates for filling pathway holes in Pathway/Genome databases. Our program not only identifies potential candidate sequences for pathway holes, but combines data from multiple, heterogeneous sources to assess the likelihood that a candidate has the required function. Our algorithm emulates the manual sequence annotation process, considering not only evidence from homology searches, but also considering evidence from genomic context (i.e., is the gene part of an operon?) and functional context (e.g., are there functionally-related genes nearby in the genome?) to determine the posterior belief that a candidate has the required function. The method can be applied across an entire metabolic pathway network and is generally applicable to any pathway database. The program uses a set of sequences encoding the required activity in other genomes to identify candidate proteins in the genome of interest, and then evaluates each candidate by using a simple Bayes classifier to determine the probability that the candidate has the desired function. We achieved 71% precision at a probability threshold of 0.9 during cross-validation using known reactions in computationally-predicted pathway databases. After applying our method to 513 pathway holes in 333 pathways from three Pathway/Genome databases, we increased the number of complete pathways by 42%. We made putative assignments to 46% of the holes, including annotation of 17 sequences of previously unknown function. CONCLUSIONS: Our pathway hole filler can be used not only to increase the utility of Pathway/Genome databases to both experimental and computational researchers, but also to improve predictions of protein function. (+info)
Carbocyclic substrates for de novo purine biosynthesis. Enantiospecific synthesis and enantiospecificity of enzymatic utilization.
(11/39)
The carbocyclic analogues of phosphoribosylamine, glycinamide ribonucleotide, and formylglycinamide ribonucleotide have been prepared enantiospecifically from D-ribonic acid gamma-lactone. These carbocycles, which have the same absolute configuration as the natural D-ribose-derived intermediates of de novo purine biosynthesis, are utilized stoichiometrically by the initial enzymes of the pathway. A comparison of the enzymatic processing of the (-)-enantiomers with those of the racemates indicates that in some cases, the (+)-enantiomer acts to inhibit the enzymatic activity. (+info)
Genetic requirements for potassium ion-dependent colony spreading in Bacillus subtilis.
(12/39)
Undomesticated strains of Bacillus subtilis exhibit extensive colony spreading on certain soft agarose media: first the formation of dendritic clusters of cells, followed by spreading (pellicle-like) growth to cover the entire surface. These phases of colonization are dependent on the level of potassium ion (K(+)) but independent of flagella, as verified with a mutant with a hag gene replacement; this latter finding highlights the importance of sliding motility in colony spreading. Exploring the K(+) requirement, directed mutagenesis of the higher-affinity K(+) transporter KtrAB, but not the lower-affinity transporter KtrCD, was found to inhibit surface colonization unless sufficient KCl was added. To identify other genes involved in K(+)-dependent colony spreading, transposon insertion mutants in wild-type strain 3610 were screened. Disruption of genes for pyrimidine (pyrB) or purine (purD, purF, purH, purL, purM) biosynthetic pathways abolished the K(+)-dependent spreading phase. Consistent with a requirement for functional nucleic acid biosynthesis, disruption of purine synthesis with the folic acid antagonist sulfamethoxazole also inhibited spreading. Other transposon insertions disrupted acetoin biosynthesis (the alsS gene), acidifying the growth medium, glutamine synthetase (the glnA gene), and two surfactin biosynthetic genes (srfAA, srfAB). This work identified four classes of surface colonization mutants with defective (i) potassium transport, (ii) surfactin formation, (iii) growth rate or yield, or (iv) pH control. Overall, the ability of B. subtilis to colonize surfaces by spreading is highly dependent on balanced nucleotide biosynthesis and nutrient assimilation, which require sufficient K(+) ions, as well as growth conditions that promote sliding motility. (+info)
Extending top-down mass spectrometry to proteins with masses greater than 200 kilodaltons.
(13/39)
For characterization of sequence and posttranslational modifications, molecular and fragment ion mass data from ionizing and dissociating a protein in the mass spectrometer are far more specific than are masses of peptides from the protein's digestion. We extend the approximately 500-residue, approximately 50-kilodalton (kD) dissociation limitation of this top-down methodology by using electrospray additives, heated vaporization, and separate noncovalent and covalent bond dissociation. This process can cleave 287 interresidue bonds in the termini of a 1314-residue (144-kD) protein, specify previously unidentified disulfide bonds between 8 of 27 cysteines in a 1714-residue (200-kD) protein, and correct sequence predictions in two proteins, one with 2153 residues (229 kD). (+info)
Complexed structures of formylglycinamide ribonucleotide amidotransferase from Thermotoga maritima describe a novel ATP binding protein superfamily.
(14/39)
Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two types of PurL have been detected. The first type, found in eukaryotes and Gram-negative bacteria, consists of a single 140 kDa polypeptide chain and is designated large PurL (lgPurL). The second type, small PurL (smPurL), is found in archaea and Gram-positive bacteria and consists of an 80 kDa polypeptide chain. SmPurL requires two additional gene products, PurQ and PurS, for activity. PurL is a member of a protein superfamily that contains a novel ATP-binding domain. Structures of several members of this superfamily are available in the unliganded form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analogue, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. The availability of a ternary complex enabled mapping of the active site, thus identifying potential residues involved in catalysis. The complexes show a conformational change in the active site compared to the unliganded structure. Surprising discoveries, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculation about the regulatory role of the auxiliary site in formation of the PurLSQ complex as well as the evolutionary relationship of PurLs from different organisms. (+info)
Reversible compartmentalization of de novo purine biosynthetic complexes in living cells.
(15/39)
Carbocyclic substrates for de novo purine biosynthesis.
(16/39)
The carbocyclic analogues of phosphoribosylamine, glycinamide ribonucleotide, and formylglycinamide ribonucleotide have been prepared as the racemates. Carbocyclic phosphoribosylamine was utilized as a substrate by the monofunctional glycinamide ribonucleotide synthetase from Escherichia coli as well as the glycinamide ribonucleotide synthetase activity of the eucaryotic trifunctional enzyme of de novo purine biosynthesis. Furthermore, carbocyclic glycinamide ribonucleotide was processed in the reverse reaction catalyzed by these enzymes. In addition, carbocyclic formylglycinamide ribonucleotide was converted, by E. coli formylglycinamide ribonucleotide synthetase, to carbocyclic formylglycinamidine ribonucleotide, which was accepted as a substrate by the aminoimidazole ribonucleotide synthetase activity of the trifunctional enzyme. This study has afforded carbocyclic substrate analogues, in particular for the chemically labile phosphoribosyl amine, for the initial steps of de novo purine biosynthesis. (+info)