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Receiver domains are the dominant molecular switches in bacterial signalling. Although several structures of non-phosphorylated receiver domains have been reported, a detailed structural understanding of the activation arising from phosphorylation has been impeded by the very short half-lives of the aspartylphosphate linkages. Here we present the first structure of a receiver domain in its active state, the phosphorylated receiver domain of the bacterial enhancer-binding protein NtrC (nitrogen regulatory protein C). Nuclear magnetic resonance spectra were taken during steady-state autophosphorylation/dephosphorylation, and three-dimensional spectra from multiple samples were combined. Phosphorylation induces a large conformational change involving a displacement of beta-strands 4 and 5 and alpha-helices 3 and 4 away from the active site, a register shift and an axial rotation in helix 4. This creates an exposed hydrophobic surface that is likely to transmit the signal to the transcriptional activation domain.

Bacterial Proteins↗

PubMedDate not supplied

[Recognition between tRNAs and aminoacyl-tRNA-synthetases of different specificities].

A few examples of incorrect interactions between aminoacyl-tRNA-synthetases and tRNAs extracted from the same organism have already been demonstrated. These interactions can lead, in most cases, to incorrect aminoacylations. The lack of specificity of the aminoacyl-tRNA suggests that incorrect interactions could be a general phenomenon. The aim of this study is to check whether incorrect interactions are a general feature, i.e. whether every aminoacyl-tTNA-synthetase is able to interact with homologous non-cognate tRNAs. In that case, it is interesting to know whether a given aminoacyl-tRNA-synthetase is able to recognize any tRNA or only a particular group of tRNAs. The existence of such groups would lead to the concept of tRNA families. For that, we estimated the affinities of non-cognate homologous tRNA species for yeast valyl-tRNA-synthetase by using competition experiments. The measured affinities varied, in standard aminoacylation conditions, between 1:100 to 1:1000 of that of the non-cognate tRNA. In the absence of Mg2+ ions or in the presence of low concentration of this cation, the affinities were higher and could reach 1:3 of the affinity of the cognate tRNA. On the other hand, we determined the inhibitory effect of a high concentration of tRNAVal toward the aminoacylation of tRNAs specific for 13 amino acids. In order to compare the effects, we determined approximate Km/Ki values. These values ranged from 0.07 for methionyl tRNA synthetase to 0.002 for leucyl tRNA synthetase. For some aminoacyl-tRNA-synthetases, the inhibition was too low to be detected by this technique. Two conclusions arise from this study. First, it seems that non-specific recognitions are quite a general phenomenon. Secondly, if one classifies tTNAs according to their affinities for valyl-tRNA-synthetase, it does not appear any well cut group of tRNAs. This result is not conflicting with the fact that on the basis of aminoacylation criteria several authors have found tRNA and aminoacyl-tRNA-synthetase families since we have already shown that discrimination depends rather on the maximal velocity of the reaction than on the affinity between the tRNA and the aminoacyl-tRNA-synthetases. Finally, the non-existence of clear-cut recognition families of tRNAs casts some doubts on the approach consisting in the characterisation of recognition sites of the tRNAs by the aminoacyl-tRNA-synthetases by comparing the sequences of tRNAs which are amonoacylated by a given aminoacyl-tRNA-synthetase.

Binding Sites↗