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M Springer

Publications and source records attributed to M Springer.

At least 55 records · Page 3Linked to original sources
PubMed1990

Escherichia coli threonyl-tRNA synthetase and tRNA(Thr) modulate the binding of the ribosome to the translational initiation site of the thrS mRNA.

Escherichia coli threonyl-tRNA synthetase binds to the leader region of its own mRNA at two major sites: the first shares some analogy with the anticodon arm of several tRNA(Thr) isoacceptors and the second corresponds to a stable stem-loop structure upstream from the first one. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. The enzyme is still able to bind to derepressed mRNA mutants resulting from single substitutions in the anticodon-like arm. This binding is restricted to the stem-loop structure of the second site. However, the interaction of the enzyme with this site fails to occlude ribosome binding. tRNA(Thr) is able to displace the wild-type mRNA from the enzyme at both sites and suppresses the inhibitory effect of the synthetase on the formation of the translational initiation complex. Our results show that tRNA(Thr) acts as an antirepressor on the synthesis of its cognate aminoacyl-tRNA synthetase. This repression/derepression double control allows precise adjustment of the rate of synthesis of threonyl-tRNA synthetase to the tRNA level in the cell.

Anticodon↗
PubMed1990

The translational regulation of threonyl-tRNA synthetase. Functional relationship between the enzyme, the cognate tRNA and the ribosome.

The E. coli threonyl-tRNA synthetase gene is negatively autoregulated at the translational level by a direct binding of the enzyme to the leader region of the thrS mRNA. This region folds in four well-defined domains. The enzyme binds to the leader at two major sites: the first is a stem-loop structure located in domain II upstream of the translational initiation site (domain I) which shares structural analogies with the anticodon arm of several tRNA(Thr) isoacceptors. The second site corresponds to a stable stem-loop structure located in domain IV. Both sites are separated by a large unpaired region (domain III). In vivo and in vitro experiments show that the structural integrity of both sites is required for the regulatory process. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. tRNA(Thr) suppresses this inhibitory effect by displacing the mRNA from the enzyme at both the upstream stem-loop structure and the tRNA-like anticodon arm.

Anticodon↗
PubMed1990

Translated translational operator in Escherichia coli. Auto-regulation in the infC-rpmI-rplT operon.

The genes coding for translation initiation factor IF3 (infC) and for the ribosomal proteins L35 (rpmI) and L20 (rplT) are transcribed in that order from a promoter in front of infC. The last two cistrons of the operon (rpmI and rplT) can be transcribed from a weak secondary promoter situated within the first cistron (infC). Previous experiments have shown that the expression of infC, the first cistron of the operon, is negatively autoregulated at the translational level and that the abnormal AUU initiation codon of infC is responsible for the control. We show that the expression of the last cistron (rplT) is also autoregulated at the posttranscriptional level. The L20 concentration regulates the level of rplT expression by acting in trans at a site located within the first cistron (infC) and thus different from that at which IF3 is known to act. This regulatory site, several hundred nucleotides upstream from the target gene (rplT), was identified through deletions, insertions and a point mutation. Thus, the expression of the operon is controlled in trans by the products of two different cistrons acting at two different sites. The localization within an open reading frame (infC) of a regulatory site acting in cis on the translation of a downstream gene (rplT) is new and was unforeseen since ribosomes translating through the regulatory site might be expected to impair either the binding of L20 or the mRNA secondary structure change caused by the binding. The possible competition between translation of the regions acting in cis and the regulation of the expression of the target gene is discussed.

Bacterial Proteins↗
PubMed1989

[The treatment of acute gastroenteritis in children in The Netherlands].

A national survey on the treatment of acute gastroenteritis in children was conducted among 10% of the Dutch general practitioners and 10% of the paediatricians. Oral Rehydration Solution was used by less than 40% of the general practitioners, while 95% of the paediatricians used it (nearly) always. About a quarter of the general practitioners used antidiarrhoeal medication in contrast to the paediatricians who hardly or never used these drugs. The refeeding period after the acute rehydration therapy showed great difference. The need for a uniform dietary advice was felt by nearly half the general practitioners. It is concluded that the internationally accepted primary treatment with O.R.S. has insufficient impact on the Dutch general practitioners. Greater knowledge on the unique features of this method is needed. The dietary advice after the primary treatment needs more uniformity.

Acute Disease↗
PubMed1989

tRNA-like structures and gene regulation at the translational level: a case of molecular mimicry in Escherichia coli.

Escherichia coli threonyl-tRNA synthetase regulates the translation of its own mRNA by binding to it in a region, called the operator, located in front of the ribosomal binding site. The primary and secondary structures of the operator resemble those of the anticodon arm of several tRNA(Thr) isoacceptor species. We reasoned that if the interaction between the synthetase and its two partially analogous ligands, the tRNA and the mRNA, had some common features, single mutations in the enzyme should affect both interactions in a very similar way. We thus isolated synthetase mutants (called super-repressors) that repress the translation of their mRNA in trans to an extreme level, and other mutants that are completely unable to perform any repression. The super-repressors, which are suspected to bind their mRNA with high affinity, are shown to bind the tRNA with an increased affinity. The non-repressing mutants, which are suspected to have lost their capacity to bind the mRNA, are shown to bind their tRNA with less affinity. The binding properties of the mutant enzymes for the other substrates, ATP and threonine, are unchanged. The observed correlation between regulatory and aminoacylation defects strongly suggests that the synthetase recognizes the similar parts of its two RNA ligands--the anticodon-like arm of the mRNA and the true anticodon arm of the tRNA--in an analogous way.

Amino Acid Sequence↗
PubMed1989

DNA hybridization in animal taxonomy: a critique from first principles.

DNA hybridization is a "distance method" for phylogenetic reconstruction and, as such, shares a set of assumptions, advantages, and problems with other techniques that do not directly employ character data. The technique purports to measure the average percent mismatch of homologous nucleotide sequences between the single-copy genomes of species. This measurement, as any other, is subject to considerations of accuracy and precision. While replicate measurements and technical modifications can improve precision, the accuracy of such measurements is limited by the equivalence of genomes under comparison. Such routine events in genome evolution as gene duplication and deletion may complicate the interpretation of DNA hybridization distances. Beyond measurement limitations, the most serious potential distortions of distances are due to biased sequence sampling and homoplasy. These problems, however, do not necessarily preclude phylogenetic reconstruction, and their effects may be mitigated by numerical corrections. Homoplasy, in particular, is a difficulty faced by all methods of phylogenetic inference. If such distortions can be eliminated, mitigated by correction, or shown to be trivial, pairwise tree-construction strategies should provide reliable estimates of phylogeny.

Animals↗
PubMed1988

Tertiary structure of Escherichia coli tRNA(3Thr) in solution and interaction of this tRNA with the cognate threonyl-tRNA synthetase.

The solution structure of Escherichia coli tRNA(3Thr) (anticodon GGU) and the residues of this tRNA in contact with the alpha 2 dimeric threonyl-tRNA synthetase were studied by chemical and enzymatic footprinting experiments. Alkylation of phosphodiester bonds by ethylnitrosourea and of N-7 positions in guanosines and N-3 positions in cytidines by dimethyl sulphate as well as carbethoxylation of N-7 positions in adenosines by diethyl pyrocarbonate were conducted on different conformers of tRNA(3Thr). The enzymatic structural probes were nuclease S1 and the cobra venom ribonuclease. Results will be compared to those of three other tRNAs, tRNA(Asp), tRNA(Phe) and tRNA(Trp), already mapped with these probes. The reactivity of phosphates towards ethylnitrosourea of the unfolded tRNA was compared to that of the native molecule. The alkylation pattern of tRNA(3Thr) shows some similarities to that of yeast tRNA(Phe) and mammalian tRNA(Trp), especially in the D-arm (positions 19 and 24) and with tRNA(Trp), at position 50, the junction between the variable region and the T-stem. In the T-loop, tRNA(3Thr), similarly to the three other tRNAs, shows protections against alkylation at phosphates 59 and 60. However, tRNA(3Thr) is unique as far as very strong protections are also found for phosphates 55 to 58 in the T-loop. Compared with yeast tRNA(Asp), the main differences in reactivity concern phosphates 19, 24 and 50. Mapping of bases with dimethyl sulphate and diethyl pyrocarbonate reveal conformational similarities with yeast tRNA(Phe). A striking conformational feature of tRNA(3Thr) is found in the 3'-side of its anticodon stem, where G40, surrounded by two G residues, is alkylated under native conditions, in contrast to other G residues in stem regions of tRNAs which are unreactive when sandwiched between two purines. This data is indicative of a perturbed helical conformation in the anticodon stem at the level of the 30-40 base pairs. Footprinting experiments, with chemical and enzymatic probes, on the tRNA complexed with its cognate threonyl-tRNA synthetase indicate significant protections in the anticodon stem and loop region, in the extra-loop, and in the amino acid accepting region. The involvement of the anticodon of tRNA(3Thr) in the recognition process with threonyl-tRNA synthetase was demonstrated by nuclease S1 mapping and by the protection of G34 and G35 against alkylation by dimethyl sulphate. These data are discussed in the light of the tRNA/synthetase recognition problem and of the structural and functional properties of the tRNA-like structure present in the operator region of the thrS mRNA.

Affinity Labels↗
PubMed1988

Translational control in E. coli: the case of threonyl-tRNA synthetase.

Genetic studies have shown that expression of the E. coli threonyl-tRNA synthetase (thrS) gene is negatively auto-regulated at the translational level. A region called the operator, located 110 nucleotides downstream of the 5' end of the mRNA and between 10 and 50 bp upstream of the translational initiation codon in the thrS gene, is directly involved in that control. The conformation of an in vitro RNA fragment extending over the thrS regulatory region has been investigated with chemical and enzymatic probes. The operator locus displays structural similarities to the anti-codon arm of threonyl tRNA. The conformation of 3 constituent mutants containing single base changes in the operator region shows that replacement of a base in the anti-codon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in regulation. However mutation in or close to the anti-codon-like stem results in a partial or complete rearrangement of the structure of the operator region. Further experiments indicate that there is a clear correlation between the way the synthetase recognises each operator, causing translational repression, and threonyl-tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1988

Messenger RNA structure and gene regulation at the translational level in Escherichia coli: the case of threonine:tRNAThr ligase.

Previous work showed that the expression of the Escherichia coli threonine:tRNAThr ligase (EC 6.1.1.3)-encoding gene (thrS) is negatively autoregulated at the translational level and that a region called the operator that is located between 10 and 50 base pairs upstream of the translation initiation codon of the thrS gene is directly involved in that control. The conformation of an in vitro synthesized RNA fragment extending over the thrS regulatory region has been investigated using chemical and enzymatic probes. This study shows that the RNA folds into four well-defined secondary-structure domains, one of them displaying structural similarities to the anticodon arm of tRNAThr. The conformation of three constitutive mutants containing single base changes in the operator region leading to the loss of the regulatory control was also investigated. The replacement of a base in the anticodon-like loop does not induce any conformational change, suggesting that the residue concerned is directly involved in the regulatory process. However, single mutations in or close to the anticodon-like stem result in a partial or complete reorganization of the structure of the operator region. These rearrangements should affect the binding of the ligase to the operator, leading to loss of the regulatory process.

Amino Acyl-tRNA Synthetases↗