Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:Q96FX7 (tRNA)
 26,753 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl-tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The RNAPhe was cross-linked to the enzyme by non-specific ultraviolet irradiation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4-thiouridine (S4U) residues introduced in the tRNA molecule, or by Schiff's base formation between periodate-oxidized tRNAPhe (tRNAPheox) and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation. The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA. The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the beta subunit (Mr 63000). The phenylalanine residue, either free or joined to the adenylate, could be cross-linked with equal efficiency to eigher type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits. The Schiff's base formation between tRNAPheox and the enzyme shows the existence of a lysyl group close to the binding site for the 3'-terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast phenylalanyl-tRNA synthetase with pyridoxal phosphate and the 2',3'-dialdehyde derivative of ATP, oATP.
 ...
PMID:Yeast phenylalanyl-tRNA synthetase. Affinity and photoaffinity labelling of the stereospecific binding sites. 38 Sep 96

The selAB operon codes for the proteins selenocysteine synthase and SELB which catalyse the synthesis and cotranslational insertion of selenocysteine into protein. This communication deals with the biochemical characterisation of these proteins and in particular with their specific interaction with the selenocysteine-incorporating tRNA(Sec). Selenocysteine synthase catalyses the synthesis of selenocysteyl-tRNA(Sec) from seryl-tRNA(Sec) in a pyridoxal phosphate-dependent reaction mechanism. The enzyme specifically recognizes the tRNA(Sec) molecule; a cooperative interaction between the tRNA binding site and the catalytically active pyridoxal phosphate site is suggested. SELB is an EF-Tu-like protein which specifically complexes selenocysteyl-tRNA(Sec). Interaction with the selenol group of the side chain of the aminoacylated residue is a prerequisite for the formation of a stable SELB.tRNA complex. Mechanistically, this provides the biochemical basis for the exclusive selection of selenocysteyl-tRNA(Sec) in the decoding step of a selenocysteine-specific UGA triplet.
 ...
PMID:The function of selenocysteine synthase and SELB in the synthesis and incorporation of selenocysteine. 183 7

The nucleotide sequence of the selA gene from Escherichia coli whose product is involved in the conversion of seryl-tRNA(Sec UCA) into selenocysteyl-tRNA(Sec UCA) was determined. selA codes for a polypeptide of a calculated Mr of 50,667; a protein of appropriate size was synthesized in vivo in a T7 promoter/polymerase system. An assay for SELA activity was devised which is based on the seryl-tRNA(Sec UCA)-dependent incorporation of [75Se] selenium into acid-insoluble material. It was used to follow SELA purification from cells that overproduced the protein from a phage T7 promoter plasmid. Purified native SELA protein migrates in gel filtration experiments with a native Mr of about 600,000. SELA contains 1 mol of bound pyridoxal 5-phosphate/mol of 50-kDa subunit. Evidence is presented that the overall conversion of seryl-tRNA(Sec UCA) to selenocysteyl-tRNA(Sec UCA) occurs at the SELA protein. SELA, therefore, has the function of a selenocysteine synthase.
 ...
PMID:Selenocysteine synthase from Escherichia coli. Nucleotide sequence of the gene (selA) and purification of the protein. 200 84

The product of the selA gene, selenocysteine synthase, is a pyridoxal 5-phosphate-containing enzyme which catalyzes the conversion of seryl-tRNA(Sec UCA) into selenocysteyl-tRNA(Sec UCA). Reduction of the aldimine group of pyridoxal 5-phosphate inactivates the enzyme. When reacted with seryl-tRNA(Sec UCA) as sole substrate, pyruvate (and possibly also ammonia) is released; in the presence of a high concentration of potassium borohydride, alanyl-tRNA(Sec UCA) is formed from seryl-tRNA(Sec UCA). These results support the notion that the formyl group of pyridoxal phosphate forms a Schiff base with the alpha-amino group of L-serine with the subsequent 2,3-elimination of a water molecule and the generation of an aminoacrylyl-tRNA(Sec UCA) intermediate. ATP is not required for this reaction step, but it is necessary for the conversion of aminoacrylyl-tRNA into selenocysteyl-tRNA(Sec UCA) which, in addition, requires the SELD protein and reduced selenium. Selenocysteine synthase forms a stable complex with seryl-tRNA(Sec UCA) with one tRNA molecule bound per two 50-kDa monomers. The enzyme does not interact with serine-inserting tRNA species. Taken together, the results show that biosynthesis of selenocysteine takes place in the enzyme-bound state and involves the dehydration of L-serine esterified to tRNA in a first step formally followed by the 2,3-addition of HSe- which is provided by the SELD protein in an ATP-dependent reaction in the form of a reactive selenium donor molecule.
 ...
PMID:Selenocysteine synthase from Escherichia coli. Analysis of the reaction sequence. 200 85

A hemA mutant of Escherichia coli containing a multicopy plasmid which complemented the mutation excreted 5-aminolevulinic acid (ALA) into the medium. [1-14C]glutamate was substantially incorporated into ALA by this strain, whereas [2-14C]glycine was not. Periodate degradation of labeled ALA showed that C-5 of ALA was derived from C-1 of glutamate. The synthesis of ALA by two sonicate fractions which had been processed by gel filtration and dialysis, respectively, was dependent on glutamate, ATP, NADPH, tRNA(Glu), and pyridoxal phosphate. tRNA(Glu) stimulated ALA synthesis in a concentration-dependent manner. Pretreatment with RNase reduced this stimulation. The amino acid sequence of the cloned insert, derived from the nucleotide sequence (J.-M. Li, C. S. Russell, and S. D. Cosloy, J. Cell Biol. 107:617a, 1988), showed no homology with any ALA synthase sequenced to date. These results suggest that E. coli synthesizes ALA by the C5 pathway from the intact five-carbon chain of glutamate.
 ...
PMID:5-Aminolevulinic acid synthesis in Escherichia coli. 265 7

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, delta-aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO2. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA(Glu), ATP, Mg2+, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[3H]glutamate or 1-[14C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[14C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the alpha subgroup of purple bacteria.
 ...
PMID:Distribution of delta-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups. 278 25

The interaction of aminoacyl-tRNA synthetase with RNA and polyanions was studied. The inhibition of the enzymes by polyU, polyI and heparin was demonstrated. It was found that this interaction is of limited specificity and is typical of single-stranded RNAs which possess no orderly secondary structure as well as of other polyanions possessing similar polyelectrolytic properties. Data from kinetic analysis and lysyl-tRNA synthetase modification by pyridoxal phosphate are suggestive of participation of the tRNA binding site in the enzyme interaction with polyanions.
 ...
PMID:[Interaction of amino acyl-tRNA-synthetases from the rabbit liver with RNA and polyanions]. 316 23

epsilon-Amino groups of lysines of 30 S ribosomal subunits with affinity for phosphate groups were selectively modified in situ by reaction with pyridoxal phosphate and reduction of the Schiff base with nonradioactive or radioactive sodium borohydride. This reaction modified only a limited number of ribosomal proteins and resulted in the loss of only some 30 S activities. The modified proteins were identified and the extent of their modification determined. The main targets of the reaction were S3 greater than S1 greater than S6. The activity most severely affected by the pyridoxal phosphate reaction was mRNA-dependent aminoacyl-tRNA binding. Some inhibition of poly(U) binding was also observed, while neither binding of initiation factors nor association with 50 S subunits was inhibited. The inhibition of aminoacyl-tRNA binding showed distinct selectivity: the inhibition was far greater with NAcPhe-tRNA than with fMet-tRNA and with "A" site than with "P" site binding. In addition, initiation complex formation with some mRNAs (e.g. MS2 RNA) was affected more than with others (e.g. T7 early mRNA). Ribosome reconstitution experiments showed that the modification of protein S3 was the primary cause of the inhibition; a role was also played by ribosomal proteins S1, S2, and S21. Substrate protection experiments showed that the 30 S activity can be protected from pyridoxal phosphate inactivation upon formation of a ternary complex with poly(U) and tRNAPhe or NAcPhe-tRNAPhe. Accordingly, the extent of modification of ribosomal protein S3 was reduced in the ternary complex while modification of S1 was reduced in the presence of poly(U) alone.
 ...
PMID:Chemical modification in situ of Escherichia coli 30 S ribosomal proteins by the site-specific reagent pyridoxal phosphate. Inactivation of the aminoacyl-tRNA and mRNA binding sites. 633 45

Higher plants, algae, cyanobacteria and several other photosynthetic and non-photosynthetic bacteria synthesize 5-aminolaevulinate by a tRNA(Glu)-mediated pathway. Glutamate is activated at the alpha-carboxyl by ligation to tRNA(Glu) with an aminoacyl-tRNA synthetase. An NADPH-dependent reductase converts glutamyl-tRNA(Glu) to glutamate 1-semialdehyde, which is finally converted to 5-aminolaevulinate by an aminotransferase. These components are soluble and in plants and algae are located in the chloroplast stroma. In plants and algae the tRNA(Glu) is encoded in chloroplast DNA whereas the enzymes are encoded in nuclear DNA. The tRNA(Glu) has a hypermodified 5-methylaminomethyl-2-thiouridine-pseudouridine-C anticodon and probably plays a role in the light-dark regulation of 5-aminolaevulinate synthesis. Ligation of glutamate to tRNA(Glu) requires ATP and Mg2+ and proceeds via a ternary intermediate. Glutamyl-tRNA(Glu) reduction appears to involve formation of a complex. Glutamate 1-semialdehyde non-enzymically synthesized by reductive ozonolysis from gamma-vinyl GABA is used as substrate by the last enzyme. Glutamate-1-semialdehyde aminotransferase contains pyridoxal phosphate as a prosthetic group. The enzyme is converted to spectrally different forms by treatment with 4,5-diaminovalerate or 4,5-dioxovalerate. The pyridoxamine 5'-phosphate form of the enzyme converts (S)-glutamate 1-semialdehyde to 5-aminolaevulinate via 4,5-diaminovalerate through a bi-bi ping-pong mechanism.
 ...
PMID:Enzymic and mechanistic studies on the conversion of glutamate to 5-aminolaevulinate. 784 60

We have improved the in vitro assay for 4-thiouridine (s(4)U) biosynthesis in Escherichia coli tRNA by substituting an unmodified tRNA transcript as substrate and including recombinant ThiI protein, a known factor required for s(4)U synthesis. Using this assay, we have purified an enzyme from wild-type E. coli that is able to provide sulfur for s(4)U synthesis in vitro. The purified protein has a molecular weight of 45 kDa and contains pyridoxal phosphate as a cofactor. This protein catalyzes sulfur transfer from cysteine to tRNA and is analogous to factor C previously reported (Lipsett, M. N. (1972) J. Biol. Chem. 247, 1458-1461). UV spectroscopy and HPLC analysis of thiolated tRNA and its digests confirm that the product of the in vitro reaction is s(4)U. N-Terminal sequence analysis of the purified protein identifies it as IscS, a recently characterized NifS-like cysteine desulfurase that mobilizes sulfur for the synthesis of [Fe-S] clusters. We have cloned and overexpressed iscS and show that the recombinant protein displayed tRNA sulfurtransferase activity equal to that of the native protein. We also show that, of the multiple proteins in E. coli with cysteine desulfurase activity as observed by native gel staining, only IscS is able to mobilize the sulfur for transfer to tRNA. Our identification of IscS as a tRNA sulfurtransferase provides support for this activity in vivo and further expands the role for NifS proteins as versatile sulfur-carrying enzymes.
 ...
PMID:IscS is a sulfurtransferase for the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA. 1060 Jan 18


1 2 Next >>