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R H Buckingham

Publications and source records attributed to R H Buckingham.

16 recordsLinked to original sources
PubMed1991

The nucleotide sequence of rpsL and its flanking regions in Salmonella typhimurium.

The ribosomal protein (r-protein)-encoding gene, rpsL, and regions flanking it, from Salmonella typhimurium, have been sequenced directly from polymerase chain reaction-amplified chromosomal DNA. The deduced amino acid sequence is identical to that of the Escherichia coli rpsL encoded r-protein. At the nucleotide level, the similarity is 98%, suggesting a strong pressure for the conservation of this important protein. More surprisingly, the noncoding sequences surrounding the gene are also conserved at the 98% level, suggesting that they too are functionally important.

Amino Acid Sequence↗
PubMed1991

Suppression of a double missense mutation by a mutant tRNA(Phe) in Escherichia coli.

We report here the isolation of a mutant tRNAPhe that suppresses a double missense auxotrophic mutation in trpA of Escherichia coli, trpA218. The doubly mutant protein product differs from wild-type TrpA by the replacements of Phe22 by Leu and Gly211 by Ser. A partial revertant TrpA phenotype can be obtained from trpA218 by changing either Leu22 back to Phe or Ser211 back to Gly. Translational suppressors were previously obtained that act at codon 211, replacing the Ser211 in the TrpA218 protein, presumably with Gly. In the present study, we selected for trpA218 suppressors caused by mutation of a cloned tRNAPhe gene, pheV. DNA sequence analysis of the suppressor isolated reveals a singular structural alteration, changing the anticodon from 5'-GAA-3' to 5'-GAG-3'. Sequencing of trpA218 confirmed the likely identity of Leu22 as CUC. The new missense suppressor, designated pheV(SuCUC), is lethal to the cell when highly expressed, as from a high copy number plasmid. This may be due to efficient replacement of Leu by Phe at CUC (and, probably, CUU) codons throughout the genome. We anticipate that pheV(SuCUC) will prove, like other missense suppressors, to be extremely useful in studies on the specificity and accuracy of decoding.

Base Sequence↗
PubMed1991

Peptidyl-tRNA hydrolase is involved in lambda inhibition of host protein synthesis.

Escherichia coli rap mutants do not support vegetative growth of bacteriophage lambda and die upon transcription of lambda DNA bar sites. Bacteria harbouring a pth(ts) mutation synthesize thermosensitive peptidyl-tRNA hydrolase (Pth) and die at 42 degrees C from a defect in protein synthesis. We present evidence that both rap and pth(ts) mutations affect the same gene: (i) peptidyl-tRNA hydrolase activity was found to be defective in rap mutants; (ii) at a threshold temperature, pth cells, like rap mutants, prevented lambda growth and were killed by transcription of cloned bar sites; (iii) sequencing a 1600 bp DNA fragment comprising both loci revealed an ORF located within the limits set by a complementation analysis and encoding a putative polypeptide of 21 kDa; (iv) cloning and sequencing of rap and pth(ts) mutant DNAs both revealed single nucleotide transitions from the wild type ORF sequence, resulting in Arg134 to His and Gly101 to Asp changes respectively. Analysis of plasmid-directed proteins identified a polypeptide of approximately 21 kDa; the N-terminal sequence, amino acid composition and isoelectric point of this protein match those expected from the ORF nucleotide sequence. We propose that Pth activity, directly or indirectly, is the target for lambda bar RNA leading to rap cell death.

Bacteriophage lambda↗
PubMed1991

Missense substitutions lethal to essential functions of EF-Tu.

We have used a simple selection and screening method to isolate function defective mutants of EF-Tu. From 28 mutants tested, 12 different missense substitutions, individually lethal to some essential function of EF-Tu, were identified by sequencing. In addition we found a new non-lethal missense mutation. The frequency of isolation of unique mutations suggests that this method can be used to easily isolate many more. The lethal mutations occur in all three structural domains of EF-Tu, but most are in domain II. We aim to use these mutants to define functional domains on EF-Tu.

Base Sequence↗
PubMed1991

Mutant ribosomes can generate dominant kirromycin resistance.

Mutations in the two genes for EF-Tu in Salmonella typhimurium and Escherichia coli, tufA and tufB, can confer resistance to the antibiotic kirromycin. Kirromycin resistance is a recessive phenotype expressed when both tuf genes are mutant. We describe a new kirromycin-resistant phenotype dominant to the effect of wild-type EF-Tu. Strains carrying a single kirromycin-resistant tuf mutation and an error-restrictive, streptomycin-resistant rpsL mutation are resistant to high levels of kirromycin, even when the other tuf gene is wild type. This phenotype is dependent on error-restrictive mutations and is not expressed with nonrestrictive streptomycin-resistant mutations. Kirromycin resistance is also expressed at a low level in the absence of any mutant EF-Tu. These novel phenotypes exist as a result of differences in the interactions of mutant and wild-type EF-Tu with the mutant ribosomes. The restrictive ribosomes have a relatively poor interaction with wild-type EF-Tu and are thus more easily saturated with mutant kirromycin-resistant EF-Tu. In addition, the mutant ribosomes are inherently kirromycin resistant and support a significantly faster EF-Tu cycle time in the presence of the antibiotic than do wild-type ribosomes. A second phenotype associated with combinations of rpsL and error-prone tuf mutations is a reduction in the level of resistance to streptomycin.

Base Sequence↗
PubMed1990

Codon context.

The analysis of coding sequences reveals nonrandomness in the context of both sense and stop codons. Part of this is related to nucleotide doublet preference, seen also in non-coding sequences and thought to arise from the dependence of mutational events on surrounding sequence. Another nonrandom context element, relating the wobble nucleotides of successive codons, is observed even when doublet preference, codon usage and bias in amino acid doublets are all allowed for. Several phenomena related to protein synthesis have been shown in vivo to be affected by the nucleotide sequence around codons. Thus, nonsense and missense suppression, elongation rate, precision of tRNA selection and polypeptide chain termination are all affected by codon context. At present, it remains unclear how these phenomena may influence the evolution of nonrandomness in the context of codons in natural sequences.

Codon↗
PubMed1990

Mutants of pheV in Escherichia coli affecting control by attenuation of the pheS, T and pheA operons. Two distinct mechanisms for de-attenuation.

Two mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, were previously isolated because they affect attenuator control of the pheS, T operon when the mutant pheV genes are carried by the plasmid pBR322. We show that the two mutants (A44 and A46) affect attenuator control by different mechanisms. The effect of mutant A44 on pheS, T expression can be progressively decreased by overproduction of Phe-tRNA synthetase, consistent with the mutant tRNA acting as a competitive inhibitor of the enzyme. By contrast, the effect on attenuation of mutant A46 increases with overproduction of Phe-tRNA synthetase, indicating that the mutant must be charged to affect attenuation; we propose that this mutant affects translation directly and causes derepression by competing with wild-type tRNA in translation of the attenuator region leader peptide. Mutant A46 but not mutant A44 leads to further de-attenuation in a miaA background. The presence of two different mechanisms for de-attenuation is further indicated by the finding that a second attenuator controlled by Phe codon translation, from the pheA operon, is affected quite differently by the mutant tRNAs. Finally, experiments involving the introduction of the mutations A44 and A46 into an amber suppressor derived from tRNA(Phe) suggest that both species can function in protein synthesis but with reduced efficiency; mutant A46 is less efficient than mutant A44, consistent with a defect in elongation.

Amino Acid Sequence↗
PubMed1990

Third position base changes in codons 5' and 3' adjacent UGA codons affect UGA suppression in vivo.

The base sequence around nonsense codons affects the efficiency of nonsense codon suppression. Published data, comparing different nonsense sites in a mRNA, implicate the two bases downstream of the nonsense codon as major determinants of suppression efficiency. However, the results we report here indicate that the nature of the contiguous upstream codon can also affect nonsense suppression, as can the third (wobble) base of the contiguous downstream codon. These conclusions are drawn from experiments in which the two Ser codons UCU233 and UCG235 in a nonsense mutant form (UGA234) of the trpA gene in Escherichia coli have been replaced with other Ser codons by site-directed mutagenesis. Suppression of these trpA mutants has been studied in the presence of a UGA nonsense suppressor derived from glyT. We speculate that the non-site-specific effects of the two adjacent downstream bases may be largely at the level of the termination process, whereas more site-specific or codon-specific effects may operate primarily on the activity of the suppressor tRNA.

Base Sequence↗
PubMed1990

A radioactive assay for the physiological activity of the tryptophan synthetase alpha subunit in crude extracts of Escherichia coli.

A modified assay has been devised for the physiological reaction, indole-3-glycerol phosphate to Trp, of the enzyme tryptophan synthetase. The assay may be applied to crude bacterial extracts, and is based on the measurement of incorporation of radioactivity from [3H]Ser into Trp. Comparison with previous colorimetric assays indicates an improvement in sensitivity of about 30-fold, and advantages in terms of sample economy and simplified manipulation.

Colorimetry↗
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↗