scholarly journals The effect of hypermethylation on the functional properties of transfer ribonucleic acid. Ribosome-binding and polypeptide synthesis

1970 ◽  
Vol 119 (3) ◽  
pp. 587-593 ◽  
Author(s):  
J. Hay ◽  
D. J. Pillinger ◽  
E. Borek
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Binding Assay ◽  
Covalent Bond ◽  
Methyl Groups ◽  
Ribosome Binding ◽  
Phosphodiester Bonds ◽  
Polypeptide Synthesis ◽  
The Stability ◽  
Escherichia Coli B

1. Phenylalanyl-tRNA formed after chemical hypermethylation of Escherichia coli B tRNA was able to bind to ribosomes with the same efficiency as normal phenylalanyl-tRNA. 2. Under incubation conditions used in the ribosome-binding assay, hypermethylation of tRNA did not measurably decrease the stability of either inter-nucleotide phosphodiester bonds or the covalent bond between amino acid and tRNA in phenylalanyl-tRNA. 3. The ability of hypermethylated tRNA to take part in polyphenylalanine synthesis was inhibited progressively as the degree of hypermethylation increased. 4. Hypermethylation of tRNA affected polyphenylalanine synthesis at the stage of amino acid recognition and at a further point in the synthesis but not at the level of codon–anticodon recognition. 5. The formation of polylysine was more seriously affected by hypermethylation of tRNA than would be accounted for by inhibition of amino acid acceptance alone. 6. Polyproline formation was completely inhibited by the presence of 7mol% excess of methyl groups in tRNA. 7. The possibility of a link between amino acid acceptance and ribosome-binding was suggested for phenylalanyl-tRNA, but not for lysyl- or prolyl-tRNA.

scholarly journals The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acids in relaxed and stringent amino acid auxotrophs of Escherichia coli

1972 ◽  
Vol 128 (5) ◽  
pp. 1007-1020 ◽  
Author(s):  
W. J. H. Gray ◽  
J. E. M. Midgley
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Heterogeneous Material ◽  
Acid Synthesis ◽  
Sucrose Density Gradient ◽  
23S Rrna ◽  
Ribonucleic Acids ◽  
Mrna Content ◽  
High Concentrations ◽  
The Stability

The biosynthesis and stability of various RNA fractions was studied in RCstr and RCrel multiple amino acid auxotrophs of Escherichia coli. In conditions of amino acid deprivation, RCstr mutants were labelled with exogenous nucleotide bases at less than 1% of the rate found in cultures growing normally in supplemented media. Studies by DNA–RNA hybridization and by other methods showed that, during a period of amino acid withdrawal, not more than 60–70% of the labelled RNA formed in RCstr mutants had the characteristics of mRNA. Evidence was obtained for some degradation of newly formed 16S and 23S rRNA species to heterogeneous material of lower molecular weight. This led to overestimations of the mRNA content of rapidly labelled RNA from such methods as simple examination of sucrose-density-gradient profiles. In RCrel strains the absolute and relative rates of synthesis of the various RNA fractions were not greatly affected. However, the stability of about half of the mRNA fraction was increased in RCrel strains during amino acid starvation, giving kinetics of mRNA labelling and turnover that were identical with those found in either RCstr or RCrel strains inhibited by high concentrations of chloramphenicol. Coincidence hybridization techniques showed that the mRNA content of amino acid-starved RCstr auxotrophs was unchanged from that found in normally growing cells. In contrast, RCrel strains deprived of amino acids increased their mRNA content about threefold. In such cultures the mRNA content of accumulating newly formed RNA was a constant 16% by wt.

scholarly journals The pseudoazurin gene from Thiosphaera pantotropha: analysis of upstream putative regulatory sequences and overexpression in Escherichia coli

1997 ◽  
Vol 321 (3) ◽  
pp. 699-705 ◽  
Author(s):  
Yun-Chung LEUNG ◽  
Christopher CHAN ◽  
John S. READER ◽  
Anthony C. WILLIS ◽  
Rob J. M. van SPANNING ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Binding Site ◽  
Paracoccus Denitrificans ◽  
Regulatory System ◽  
Ribosome Binding Site ◽  
Regulatory Sequences ◽  
Ribosome Binding ◽  
Thiosphaera Pantotropha

The pseudoazurin gene from Thiosphaera pantotropha has been cloned and sequenced. The deduced amino acid sequence showed that the protein contains an unusually alanine-rich signal peptide, 22 amino acid residues in length, which targets the protein to the periplasm. This pseudoazurin was expressed in large amounts in the periplasm of Escherichia coli when the gene with its native ribosome-binding site was placed downstream of the lac promoter. Removal of a putative hairpin-forming structure upstream of the ribosome-binding site increased the yield of the purified protein to ∼80 mg/l. The recombinant protein is indistinguishable from that purified from its natural host. A primer extension study indicated that the pseudoazurin structural gene (pazS) is under the control of the Fnr/Nnr regulatory system, but no promoter-binding sequence could be recognized. The amino acid sequence of pseudoazurin from Paracoccus denitrificans is also reported.

scholarly journals Involvement of the N Terminus of Ribosomal Protein L11 in Regulation of the RelA Protein of Escherichia coli

2001 ◽  
Vol 183 (22) ◽  
pp. 6532-6537 ◽  
Author(s):  
Xiaoming Yang ◽  
Edward E. Ishiguro
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Ribosomal Protein ◽  
Stringent Response ◽  
Direct Interaction ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Ribosome Binding ◽  
N Terminus ◽  
Ribosomal Protein L11

ABSTRACT Amino acid-deprived rplK (previously known asrelC) mutants of Escherichia coli cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. To investigate the role of L11 in the stringent response, a derivative ofrplK encoding L11 lacking the N-terminal 36 amino acids (designated ′L11) was constructed. Bacteria overexpressing ′L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The overexpressed ′L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA. By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11. To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis. The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype. These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA.

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