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Query: UNIPROT:P01350 (gastrin)
 9,683 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli tRNA-guanine transglycosylase is an enzyme which catalyzes replacement of guanine (G34) of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr) by free guanine or free preQ1 base by a base exchange reaction in the biosynthesis of queuosine (Q) (Okada, N., and Nishimura, S. (1979) J. Biol. Chem. 254, 3061-3066). The gene encoding for this enzyme was amplified from the E. coli genome by polymerase chain reaction and inserted into an overexpression vector, pJLA503. The enzyme was overexpressed by heat induction in E. coli transformed by this recombinant plasmid and purified to homogeneity by two column chromatographies. The sequence requirement in tRNA for recognition by this enzyme was investigated using minihelices corresponding to the anti-codon arm of E. coli tRNA(His). Two uridine residues (U33, U35) were found to be prerequisite for such recognition by this enzyme. Position 32 required pyrimidines, because the enzyme activity toward the minihelices was markedly reduced or entirely lost when this residue was replaced by purines or was deleted. Adenosine at position 37 and the G30-C40 base pair were not essential despite their conservation. Our results suggest that the enzyme recognizes the U33-G34-U35 sequence in the anti-codon loop and not the tertiary structure of tRNA itself.
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PMID:A UGU sequence in the anticodon loop is a minimum requirement for recognition by Escherichia coli tRNA-guanine transglycosylase. 752 9

A series of 5-substituted 2-aminopyrrolo[2,3-d]pyrimidin-4(3H)-ones have been synthesized in order to study the substrate specificity of the tRNA-guanine transglycosylase (TGT) from Escherichia coli. A number of these compounds were initially examined as inhibitors of radiolabeled guanine incorporation into tRNA catalyzed by TGT [Hoops, G. C., Garcia, G. A., & Townsend, L. B. (1992) 204th National Meeting of the American Chemical Society, Washington, DC, August 23-28, 1992, Division of Medicinal Chemistry, Abstract 113]. The kinetic parameters of these analogues as substrates in the TGT reaction have been determined by monitoring the loss of radiolabeled guanine from 8-[14C]G34-tRNA. This study reveals that the tRNA-guanine transglycosylase from E. coli will tolerate a wide variety of substituents at the 5-position. The role of the 5-substituent appears to be entirely in binding/recognition with no apparent effects upon catalysis. A correlation between N7 pKa and Vmax suggests the deprotonation of N7 during the reaction, which must occur prior to subsequent glycosidic bond formation, appears to be partially rate-determining for the natural substrate. Comparison of the Kis of 7-methyl-substituted competitive inhibitors to the Kms of their corresponding substrates suggests that some substrates (including preQ1) are kinetically "sticky" (i.e., Km is equivalent to Kd) and other substrates have Kms that reflect catalytic rates as well as binding.
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PMID:tRNA-guanine transglycosylase from Escherichia coli: structure-activity studies investigating the role of the aminomethyl substituent of the heterocyclic substrate PreQ1. 757 54

In vitro selection was used to isolate active Escherichia coli tRNA(Phe) variants from randomized libraries. Functional tRNAs were first selected by multiple rounds of binding to Escherichia coli phenylalanyl-tRNA synthetase. These variants were then aminoacylated and selected for affinity to elongation factor-Tu. By randomizing potential recognition nucleotides, the importance of residues U20, G34, A35, A36 and U59, previously identified to be required for specific recognition by E. coli phenylalanyl-tRNA synthetase (FRS), was confirmed. However, the sequences of several active variants imply that the wild-type tertiary interactions G10-C25-U45 and A26-G44 are not required for recognition, as previously suggested. Selection of functional tRNAs from a second library randomized at positions normally involved in conserved tertiary interactions revealed new combinations of nucleotides at these positions, suggesting the presence of novel tertiary interactions. In both libraries, active sequences containing deletions were isolated. Taken together, it is clear that FRS is active with substrates having an unexpectedly broad sequence diversity. Finally, the potency of this method is illustrated by the identification of a second class of variants that was isolated by virtue of the presence of an impurity in the FRS preparation.
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PMID:Selection for active E. coli tRNA(Phe) variants from a randomized library using two proteins. 768 42

Serine tRNA gene derivatives with altered anticodons were introduced to the temperature-sensitive serT42 mutant, whose tRNA(1Ser) shows a base substitution of A10 for wild type G10. When a low copy number vector-system was used, the growth and beta-galactosidase synthetic activity of the serT42 mutant were restored by complementation with the tRNA(5Ser) (T34) gene or the tRNA(1Ser) (G34) gene as well as the tRNA(1Ser) (wt) gene, but not with tRNA(5Ser) (wt), tRNA(1Ser) (A34) or tRNA(1Ser) (C34) genes at 42 degrees C. When multicopy vectors were used, the transformation even with tRNA(1Ser) (A10) gene restored the growth and beta-galactosidase synthetic activity at 42 degrees C. The tRNA(1Ser) (A10) showed no thermosensitivity in serine acceptor activity by in vitro assay. At 42 degrees C, the amount of tRNA(1Ser) (A10) in the serT42 mutant was almost the same as those in the wild type. The nucleotides in the tRNA(1Ser) (A10) were found to be fully modified like those in the wild type tRNA(1Ser). Both of the tRNAs transcribed from tRNA(5Ser) (T34) and tRNA(1Ser) (G34) genes showed serine acceptor activity. Modified nucleosides of these tRNAs were also analyzed.
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PMID:Suppression of the serT42 mutation with modified tRNA(1Ser) and tRNA(5Ser) genes. 806 26

Molecular recognition of Escherichia coli tRNA(Ile) by the cognate isoleucyl-tRNA synthetase (IleRS) was studied by analyses of chemical footprinting with N-nitroso-N-ethylurea and aminoacylation kinetics of variant tRNA(Ile) transcripts prepared with bacteriophage T7 RNA polymerase. IleRS binds to the acceptor, dihydrouridine (D), and anticodon stems as well as to the anticodon loop. The "complete set" of determinants for the tRNA(Ile) identity consists of most of the nucleotides in the anticodon loop (G34, A35, U36, t6A37 and A38), the discriminator nucleotide (A73), and the base-pairs in the middle of the anticodon, D and acceptor stems (C29.G41, U12.A23 and C4.G69, respectively). As for the tertiary base-pairs, two are indispensable for the isoleucylation activity, whereas the others are dispensable. Correspondingly, some of the phosphate groups of these dispensable tertiary base-pair residues were shown to be exposed to N-nitroso-N-ethylurea when tRNA(Ile) was bound with IleRS. Furthermore, deletion of the T psi C-arm only slightly impaired the tRNA(Ile) activity. Thus, it is proposed that the recognition by IleRS of all the widely distributed identity determinants is coupled with a global conformational change that involves the loosening of a particular set of tertiary base-pairs of tRNA(Ile).
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PMID:Molecular recognition of the identity-determinant set of isoleucine transfer RNA from Escherichia coli. 811 89

Escherichia coli tRNA(Asp) was overproduced in E. coli up to 15-fold from a synthetic tRNA(Asp) gene placed in a plasmid under the dependence of an isopropyl-beta,D-thiogalactopyranoside-inducible promoter. Purification to nearly homogeneity (95%) was achieved after two HPLC DEAE-cellulose columns. E. coli tRNA(Asp)[G34] (having guanine instead of queuine at position 34) was obtained by the same procedure except that it was overproduced in a strain lacking the enzyme responsible for queuine modification. Nucleoside analysis showed that, except for the replacement of Q34 by G34 in mutant-derived tRNA(Asp), the base modification levels of both tRNAs are the same as those in wild-type E. coli tRNA(Asp). Kinetic properties of tRNA(Asp)[Q34] and [G34] with yeast AspRS compared to those in the homologous reactions in yeast and E. coli clearly indicate that the major identity elements are the same in both organisms: the conserved discriminant base and the anticodon triplet. In connection with this, we explored by site-directed mutagenesis the functional role of the interactions which, as revealed by the crystallographic structure, occur between the wobble base of yeast tRNA(Asp) and two residues of yeast AspRS. Their absence strongly affected aspartylation and the kd of tRNA(Asp). Each contact individually restores almost completely the wild-type acylation properties of the enzyme; thus, wobble base recognition in yeast appears to be more protected against mutational events than in E. coli, where only one contact is thought to occur at position 34.
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PMID:Overproduction and purification of native and queuine-lacking Escherichia coli tRNA(Asp). Role of the wobble base in tRNA(Asp) acylation. 826 43

We have investigated the functional relationship between nucleotides in yeast tRNAAsp that are important for aspartylation by yeast aspartyl-tRNA synthetase. Transcripts of tRNAAsp with two or more mutations at identity positions G73, G34, U35, C36 and base pair G10-U25 have been prepared and the steady-state kinetics of their aspartylation were measured. Multiple mutations affect the catalytic activities of the synthetase mainly at the level of the catalytic constant, kcat. Kinetic data were expressed as free energy variation at transition state of these multiple mutants and comparison of experimental values with those calculated from results on single mutants defined three types of relationships between the identity nucleotides of this tRNA. Nucleotides located far apart in the three-dimensional structure of the tRNA act cooperatively whereas nucleotides of the anticodon triplet act either additively or anti-cooperatively. These results are related to the specific interactions of functional groups on identity nucleotides with amino acids in the protein as revealed by the crystal structure of the tRNAAsp/aspartyl-tRNA synthetase complex. These relationships between identity nucleotides may play an important role in the biological function of tRNAs.
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PMID:Additive, cooperative and anti-cooperative effects between identity nucleotides of a tRNA. 833 8

A gel shift assay that distinguishes the aminoacylated form from the deacylated form of tRNAs was used to study the requirements for aminoacylation of Escherichia coli tRNA(Asn) in vivo. tRNA(Asn) derivatives containing single base changes in their anticodons or discriminator bases were constructed, and the extent of in vivo aminoacylation was determined directly. Substitution of U35 with C35 or U36 with C36 abolished aminoacylation of the tRNA. Substitution of G34 with C34 converted tRNA(Asn) into a lysine acceptor. Thus, each of the anticodon nucleotides are important for aminoacylation of tRNA(Asn). Substitution of discriminator base G73 with A73 affected the extent of aminoacylation in vivo indicating that the discriminator base also contributes to aminoacylation of tRNA(Asn).
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PMID:The anticodon and discriminator base are important for aminoacylation of Escherichia coli tRNA(Asn). 834 9

A single-strand-specific nuclease from wheat chloroplasts (ChS nuclease) was tested as a tool for RNA secondary and tertiary structure investigations, using yeast tRNA(Phe) and yeast tRNA(Asp) as models. In tRNA(Phe) the nuclease introduced main primary cleavages at positions U33, A35 and A36 in the anticodon-loop and G18 and G19 in the D-loop. In tRNA(Asp) the main primary cleavages occurred at positions U33, G34 and U35 in the anticodon-loop and the lower one at position C20:1 in the D-loop. No primary cleavages were observed within the double-stranded stems. Because ChS nuclease has (i) a low molecular weight, (ii) a wide pH range of action (5.0 to 7.5) (iii) no divalent cation requirement in the reaction mixture and (iv) can be obtained as a pure protein in rather large quantities it appeared to be a very good tool for secondary and tertiary structural studies of RNAs.
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PMID:Structural specificity of nuclease from wheat chloroplasts stroma. 864 43

The aspartate identity of tRNA for AspRS from Thermus thermophilus has been investigated by kinetic analysis of the aspartylation reaction of different tRNA molecules and their variants as well as of tRNAPhe variants with transplanted aspartate identity elements. It is shown that G10, G34, U35, C36, C38, and G73 determine recognition and aspartylation of yeast and T.thermophilus tRNA(Asp) by the thermophilic AspRS. This set of nucleotides specifies also tRNA aspartylation in the homologous yeast and Escherichia coli systems. Structural considerations indicate that the major aspartate identity elements interact with amino acids conserved in all AspRSs. It follows that the structural features of tRNA and synthetase specifying aspartylation are mainly conserved in various structural contexts and in organisms adapted to different life conditions. Mutations of tRNA identity elements provoke drastic losses of charging in the heterologous system involving yeast tRNA(Asp) and T. thermophilus AspRS. In the homologous systems, the mutational effects are less pronounced. However, effects in E. coli and T. thermophilus exceed those in yeast which are particularly moderate, indicating variations in the individual contributions of identity elements for aspartylation in prokaryotes and eukaryotes. Analysis of multiple tRNA mutants reveals cooperativity between the cluster of determinants of the anticodon loop and the additional determinants G10 and G73 for efficient aspartylation in the thermophilic system, suggesting that conformational changes trigger formation of the functional tRNA/synthetase complex.
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PMID:Identity of prokaryotic and eukaryotic tRNA(Asp) for aminoacylation by aspartyl-tRNA synthetase from Thermus thermophilus. 865 22


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