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Biomedical subjects

R H Buckingham

Publications and source records attributed to R H Buckingham.

At least 55 records · Page 3Linked to original sources
PubMed1984

The effect of point mutations affecting Escherichia coli tryptophan tRNA on anticodon-anticodon interactions and on UGA suppression.

tRNA species in Escherichia coli that translate codons starting with U contain 2-methyl-thio-N6-isopentenyl-adenosine in position 37, 3' adjacent to the anticodon. The role of this hypermodification in protein synthesis and trp operon attenuation has been investigated. Temperature-jump relaxation methods have been applied to study the interaction between E. coli tRNAPro, with anticodon VGG (V is uridine-5-oxyacetic acid) complementary to that of tRNATrp, and three species of E. coli tRNATrp: wild type tRNATrp (with ms2i6A37 and G24), UGA suppressor tRNATrp (with ms2i6A37 and A24 in the dihydrouridine stem but the same anticodon CCA), and the same suppressor molecule but ms2i6A-deficient as a result of the mutation miaA. Complex formation between tRNAPro and ms2i6A-containing tRNATrp shows thermodynamic parameters close to those found for several other pairs of tRNA with complementary anticodons. However, ms2i6A-deficient tRNATrp makes less stable complexes with tRNAPro, which dissociate eightfold faster. No effect on the complementary anticodon interaction of the mutation in the dihydrouridine stem can be detected. When the tRNA analogous to the opal codon, E. coli tRNASerIV (anticodon VGA) replaces tRNAPro in similar experiments, very weak complexes are observed with both normally hypermodified species of tRNATrp, the wild type and UGA suppressor; these show a lifetime about 50-fold shorter than with tRNAPro, but are again similar. No complex formation is detectable with the ms2i6A-deficient species. This may explain why the hypermodification is necessary for the efficient suppression of the UGA terminator of Q beta coat protein in vitro. The data on complexes with tRNAPro suggest that deficiency in ms2i6A may also reduce the efficiency of UGG reading. Thus, miaA may affect trp operon attenuation by slowing translation of the tandem UGG codons in the leader sequence. Temperature-jump differential spectra suggest that ms2i6 stabilizes the anticodon interaction by improved stacking of base 37.

Anticodon↗
PubMed1984

An effect of codon context on the mistranslation of UGU codons in vitro.

Effects of codon context on nonsense codon suppression may act either through release factor recognition of termination codons or aminoacyl-tRNA selection by the ribosome. The latter hypothesis has been studied by comparing misreading by Escherichia coli UGA suppressor tryptophan tRNA of UGU (cysteine) codons in two synthetic polymers, poly(U-G) and poly( U5 , G), which differ in sequence around the UGU codons. In vitro translation of these polymers in a cell-free system from E. coli yielded selection errors of 4 X 10(-3) and 1.75 X 10(-2) for UGU codons in poly(U-G) and poly( U5 , G), respectively. This difference suggests that codon context may significantly affect misincorporation of amino acids into protein.

Codon↗
PubMed1984

Construction of a UGA suppressor tRNA by modification in vitro of yeast tRNACys.

In this paper we describe the construction of a yeast tRNACys UGA suppressor. After specific hydrolysis of the parent molecule, the first base of the anticodon GCA was replaced by a uracil. The resulting molecule, harboring a UCA anticodon, was injected into Xenopus laevis oocytes in order to test its biological activities. The level of aminoacylation was similar to that of the parent molecule. Readthrough of the UGA termination codon in beta-globin mRNA, coinjected with the tRNA, indicated suppressor activity; however, tRNACys (anticodon UCA) was a much less efficient suppressor than others tested under the same conditions. We see no post-transcriptional modification of the uracil in the anticodon wobble position after injection into oocytes. This may be related to the low suppressor activity; however, it is also possible that other features of tRNACys structure may be unadapted to efficient UCA anticodon function.

Animals↗
PubMed1984

Does codon composition influence ribosome function?

Escherichia coli ribosomes pre-initiated with N-acetyl-Val-tRNAVal elongate strictly alternating poly(U-G) at a rate between eight and 12 peptide bonds per second per ribosome in vitro. Comparisons with poly(U)-primed poly(Phe) synthesis show that these systems function with the same rates which are close to those of protein synthesis in vivo. This indicates that, at least in vitro, codon composition has no marked influence on the speed of elongation when the concentration of ternary complex is saturating. Furthermore, the missense frequencies for the two polymers are within the same range: the missense substitution of Trp for Cys is 10(-4) and that of Met for Val is 10(-3) in the poly(U-G)-primed system. These data argue against models that explain the codon preference of certain gene families by postulating effects of high or low GC content of codons on the performance characteristics of ribosomes.

Codon↗
PubMed1983

Identification of clones carrying an E. coli tRNAPhe gene by suppression of phenylalanyl-tRNA synthetase thermosensitive mutants.

Two libraries of cloned E. coli DNA were screened for plasmids which complemented thermosensitive phenylalanyl-tRNA synthetase mutants. Four plasmids were isolated which complemented pheS and pheT thermosensitive mutations but which do not carry pheS or pheT, the structural genes for phenylalanyl-tRNA synthetase. All these plasmids increased the intracellular tRNAPhe concentration. Three plasmids were shown to carry the structural gene for tRNAPhe which we call pheU. By restriction enzyme analysis, DNA blotting and DNA:tRNA hybridization, pheU was localised to a 280 bp fragment within a 5.6 kb PstI restriction fragment of E.coli DNA.

Amino Acyl-tRNA Synthetases↗
PubMed1981

Photocrosslinking of thiolated aminoacyl-tRNA to ribosomal RNA and proteins.

tRNA has been converted to a form that can be photoactivated by chemical modification of some of the exposed cytidine residues to thio-4-uridine A certain percentage of the modified molecules can be charged and bound to the ribosome; thiolated fMet-tRNAfMet is bound to the P-site as shown by puromycin reactivity. Near the UV irradiation produces covalent crosslinks between total thiolated AA-tRNA or fMet-tRNAfMet and the ribosome. AA-tRNA becomes crosslinked to both 30S and 50S subunits but fMet-tRNAfMet to 50S subunits alone. In each case, crosslinking of tRNA was found to be not only to ribosomal proteins, but also to rRNA. The covalent complexes appear sufficiently stable to allow identification of the proteins or rRNA sequences involved.

Escherichia coli↗
PubMed1981

Effect of oligonucleotide AGAGGAGGU on protein synthesis in vitro.

The oligonucleotide AGAGGAGGU, complementary to the 3' end of 16S RNA has been shown to inhibit 70S initiation complex formation on E. coli ribosomes (Taniguchi, T. and Weissmann, C., 1978. Nature, 275, 770-772). We have prepared this nonanucleotide in larger quantities by a combination of DEAE cellulose-urea chromatography and reverse phase (RPC 5) chromatography. The inhibitory effect of AGAGGAGGU on initiation complex formation has been confirmed. Furthermore, when added to a complete system for in vitro protein synthesis, the translation of Q beta RNA was inhibited by the nonanucleotide. No selectivity was observed in the inhibition of the coat protein and replicase protein synthesis. When both Q beta RNA and pAUG were present, some stimulation of pAUG binding to 70S ribosomes was observed on addition of AGAGGAGGU, as previously reported (Taniguchi and Weissmann, ibid). No effect was observed in the absence of Q beta RNA. This observation is discussed.

Escherichia coli↗
PubMed1981

Tritium exchange on transfer RNA: slowly exchanging protons sensitive to a change in the dihydrouridine stem.

Measurements of tritium exchange on tRNA were made for periods from 0.5 min to 8 hr after separation from labeled solvent. The exchange curve was analysed in terms of three kinetic classes of exchanging protons with half-lives of 5 hr (12 protons), 0.54 hr (37 protons) and about 3.5 min (58 protons) at 0 degrees C in 0.14 M K+/10 mM Mg2+. The behaviour under varying ionic conditions of protons in the slowest exchange class and of some protons in the intermediate class suggests that they are dependent on the tertiary structure of the molecule. Moreover, in the same range of exchange times characteristic of these latter protons, about 9 more protons were observed in the case of a mutant form of tRNA Trp, the UGA-suppressor species, than in the wild-type tRNATrp. These two species differ only in base 24 in the dihydrouridine stem. This dynamic difference between the wild-type and suppressor species may be related to a functionally important difference in coupling between the conformation of the molecule and interactions at the anticodon.

Escherichia coli↗
PubMed1980

Protection against non-enzymatic deacylation of Met-tRNA Met m by the ribosome : an approach to the measurement of aminoacyl-tRNA : ribosome affinity constants.

When bound to E. coli 70S ribosomes in the presence of poly (A, U, G) at 37 degrees C, Met-tRNA Met m is hydrolysed 20 times slower than when free in solution under the same conditions (lifetimes of 8.3 h and 22 min respectively). It is shown how this large difference in rate of hydrolysis may be used to study the equilibrium of aminoacyl-tRNA binding to ribosomes.

Escherichia coli↗
PubMed1979

Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E. coli: a potential application for the purification of the enzyme.

E. coli tryptophanyl-tRNA synthetase can form a complex with Blue-dextran Sepharose, in the presence or in the absence of Mg++. In its absence, the complex is dissociated by either ATP or cognate tRNATrp. However, in the presence of Mg++, only tRNATrp can dissociate the complex whereas ATP has no effect. E. coli total tRNA or tRNAMet, at the same concentration, cannot displace the synthetase from the complex. It is suggested that the Blue-dextran binds to the synthetase through its tRNA binding domain. This hypothesis is supported by previous findings with polynucleotide phosphorylase showing that Blue-dextran Sepharose can be used in affinity chromatography to recognize a polynucleotide binding site of the protein. The selective elution by its cognate tRNA of Trp-tRNA synthetase bound to Blue-dextran Sepharose provides a rapid and efficient purification of the enzyme. Examples of other synthetases and nucleotidyl transferases are also discussed.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Recognition of tRNA Trp by initiation factors from Escherichia coli.

Binding of acetyl or formyltryptophanyl-tRNA Trp from Escherichia coli or beef liver to E. coli ribosomes is strongly stimulated by E. coli initiation factors and requires GTP. The N-acylated tryptophan is puromycin reactive. Polypeptide chain initiation with acetyltryptophan dependent on poly(U,G) has been demonstrated and is highly dependent on added initiation factors. tRNA Trp appears, therefore, to share some structural features with tRNAfMet of significance to the process of polypeptide chain initiation.

Escherichia coli↗
PubMed1977

Codon specificity of UGA suppressor tRNATrp from Escherichia coli.

A synthetic polyribonucleotide, poly(U5,G), was used to study the codon specificity of wild-type and UGA suppressor tRNATrp from Escherichia coli. Phe (UUU) incorporation directed by this synthetic messenger is reduced somewhat by omission from the incubation mixtures of Val (GUU), Leu (UUG), or Cys (UGU). In contrast, omission of Cys stimulates Trp incorporation, and this effect is much more pronounced with the UGA suppressor tRNATrp than with wild-type tRNA. The apparent replacement of Cys by Trp is specific, because the omission of Val or Leu slightly inhibits Trp incorporation. These data suggest that the UGA suppressor tRNATrp can translate codons of the form UGN (N is any ribonucleotide). In other words, the suppressor tRNATrp translates codons that properly match two out of the three anticodon nucleotides.

Base Sequence↗
PubMed1977

Aminoacylation of tRNA Trp from beef liver, yeast and E. coli by beef pancrease tryptophan-tRNA ligase. Stoichiometry of tRNATrp binding.

The Michaelis constants and the maximum velocities in the aminoacylation reaction of tRNATrp from beef liver, yeast and E. coli by pure beef pancreas tryptophan-tRNA ligase show that this mammalian enzyme recognizes and charges the two eucaryotic tRNAs with the same efficiency. The rate of aminoacylation of the procaryotic tRNATrp by the enzyme is three orders of magnitude lower. The pH optimum of aminoacylation is 8 for both eucaryotic tRNAs. The optimum magnesium concentration is different. The rate is maximum when magnesium concentration is stoichiometric to ATP concentration for tRNATrp from beef liver and 10 mM above ATP concentration for tRNATrp from yeast. The number of binding sites on the enzyme for the two eucaryotic tRNAs has been measured by equilibrium filtration on Sephadex G-100 and found equal to two.

Adenosine Triphosphate↗
PubMed1976

Anticodon conformation and accessibility in wild-type and suppressor tryptophan tRNA from E. coli.

The association between Trp-tRNA and Pro-tRNA, which have complementary anticodon sequences, has been used as a probe of anticodon conformation. It is unaffected, however, by the base change in the D-stem present in UGA-suppressor Trp-tRNA. This does not support the hypothesis that UGA suppression depends upon a conformational change induced in the anticodon. The stable denatured form of wild-type Trp-tRNA no longer interacts with Pro-tRNA; the structure of the anticodon region must therefore be quite different in the denatured form.

Anticodon↗
PubMed1975

No correlation between native and denatured forms of tRNA(Trp) form Escherichia coli and the resistant and sensitive molecules characterised by phosphorolysis. Two classes of conformation characterised by phosphorolysis in both native and denatured tRNA(Trp).

Some tRNA molecules in solution are sensitive to attack by polynucleotide phosphorylase while others are resistant, even with pure species of tRNA. Further analysis of this behaviour has revealed an underlying microheterogeneity in tRNA structure. In order to clarify the relation between the sensitive and resistant classes of tRNA, and the native and denatured forms with respect to amino acid acceptance, the phosphorolysis of tRNATrp from Escherichia coli has been investigated. Native tRNATrp is similar to species examined previously: resistant and sensitive classes are observed and the sensitive proportion increases with temperature. At 20 degrees C both native and denatured tRNATrp are stable under phosphorolysis conditions, and denaturated tRNATrp is found also to possess resistant and sensitive classes. About 10% of both native and denatured tRNATrp is rapidly phosphorolysed at 20 degrees C, but the rate of conversion of resistant denatured tRNATrp to the sensitive class is about twice as fact as for the native form. Thus it can be concluded that the sensitive molecules of tRNATrp attacked by polynucleotide phosphorylase are not due to denaturation.

Drug Stability↗