Mathias Springer

The structure of threonyl-tRNA synthetase-tRNA(Thr) complex enlightens its repressor activity and reveals an essential zinc ion in the active site

E. coli threonyl-tRNA synthetase (ThrRS) is a class II enzyme that represses the translation of its own mRNA. We report the crystal structure at 2.9 A resolution of the complex between tRNA(Thr) and ThrRS, whose structural features reveal novel strategies for providing specificity in tRNA selection. These include an amino-terminal domain containing a novel protein fold that makes minor groove contacts with the tRNA acceptor stem. The enzyme induces a large deformation of the anticodon loop, resulting in an interaction between two adjacent anticodon bases, which accounts for their prominent role in tRNA identity and translational regulation. A zinc ion found in the active site is implicated in amino acid recognition/discrimination.

Transfer RNA-mediated editing in threonyl-tRNA synthetase. The class II solution to the double discrimination problem.

Threonyl-tRNA synthetase, a class II synthetase, uses a unique zinc ion to discriminate against the isosteric valine at the activation step. The crystal structure of the enzyme with an analog of seryl adenylate shows that the noncognate serine cannot be fully discriminated at that step. We show that hydrolysis of the incorrectly formed ser-tRNA(Thr) is performed at a specific site in the N-terminal domain of the enzyme. The present study suggests that both classes of synthetases use effectively the ability of the CCA end of tRNA to switch between a hairpin and a helical conformation for aminoacylation and editing. As a consequence, the editing mechanism of both classes of synthetases can be described as mirror images, as already seen for tRNA binding and amino acid activation.

The role of the AUU initiation codon in the negative feedback regulation of the gene for translation initiation factor IF3 in Escherichia coli

The expression of the infC gene encoding translation initiation factor IF3 is negatively autoregulated at the level of translation, i.e. the expression of the gene is derepressed in a mutant infC background where the IF3 activity is lower than that of the wild type. The special initiation codon of infC, AUU, has previously been shown to be essential for derepression in vivo. In the present work, we provide evidence that the AUU initiation codon causes derepression by itself, because if the initiation codon of the thrS gene, encoding threonyl‐tRNA synthetase, is changed from AUG to AUU, its expression is also derepressed in an infC mutant background. The same result was obtained with the rpsO gene encoding ribosomal protein S15. We also show that derepression of infCthrS, and rpsO is obtained with other ‘abnormal’ initiation codons such as AUA, AUC, and CUG which initiate with the same low efficiency as AUU, and also with ACG which initiates with an even lower efficiency. Under conditions of IF3 excess, the expression of infC is repressed in the presence of the AUU or other ‘abnormal’ initiation codons. Under the same conditions and with the same set of ‘abnormal’ initiation codons, the repression of thrS and rpsO expression is weaker. This result suggests that the infC message has specific features that render its expression particularly sensitive to excess of IF3. We also studied another peculiarity of the infC message, namely the role of a GC‐rich sequence located immediately downstream of the initiation codon and conserved through evolution. This sequence was proposed to interact with a conserved region in 16S RNA and enhance translation initiation. Unexpectedly, mutating this GC‐rich sequence increases infC expression, indicating that this sequence has no enhancing role. Chemical and enzymatic probing of infC RNA synthesized in vitro indicates that this GC‐rich sequence might pair with another region of the mRNA. On the basis of our in vivo results we propose, as suspected from earlier in vitro results, that IF3 regulates the expression of its own gene by using its ability to differentiate between ‘normal’ and ‘abnormal’ initiation codons.

Autogenous control of Escherichia coli threonyl-tRNA synthetase expression in Vivo

The regulation of the expression of thrS, the structural gene for threonyl-tRNA synthetase, was studied using several thrS-lac fusions cloned in lambda and integrated as single copies at att lambda. It is first shown that the level of beta-galactosidase synthesized from a thrS-lac protein fusion is increased when the chromosomal copy of thrS is mutated. It is also shown that the level of beta-galactosidase synthesized from the same protein fusion is decreased if wild-type threonyl-tRNA synthetase is overproduced from a thrS-carrying plasmid. These results strongly indicate that threonyl-tRNA synthetase controls the expression of its own gene. Consistent with this hypothesis it is shown that some thrS mutants overproduce a modified form of threonyl-tRNA synthetase. When the thrS-lac protein fusion is replaced by several types of thrS-lac operon fusions no effect of the chromosomal thrS allele on beta-galactosidase synthesis is observed. It is also shown that beta-galactosidase synthesis from a promoter-proximal thrS-lac operon fusion is not repressed by threonyl-tRNA synthetase overproduction. The fact that regulation is seen with a thrS-lac protein fusion and not with operon fusions indicates that thrS expression is autoregulated at the translational level. This is confirmed by hybridization experiments which show that under conditions where beta-galactosidase synthesis from a thrS-lac protein fusion is derepressed three- to fivefold, lac messenger RNA is only slightly increased.

MicA sRNA links the PhoP regulon to cell envelope stress.

Numerous small RNAs regulators of gene expression exist in bacteria. A large class of them binds to the RNA chaperone Hfq and act by base pairing interactions with their target mRNA, thereby affecting their translation and/or stability. They often have multiple direct targets, some of which may be regulators themselves, and production of a single sRNA can therefore affect the expression of dozens of genes. We show in this study that the synthesis of the Escherichia coli pleiotropic PhoPQ two‐component system is repressed by MicA, a σE‐dependent sRNA regulator of porin biogenesis. MicA directly pairs with phoPQ mRNA in the translation initiation region of phoP and presumably inhibits translation by competing with ribosome binding. Consequently, MicA downregulates several members of the PhoPQ regulon. By linking PhoPQ to σE, our findings suggest that major cellular processes such as Mg2+ transport, virulence, LPS modification or resistance to antimicrobial peptides are modulated in response to envelope stress. In addition, we found that Hfq strongly affects the expression of phoP independently of MicA, raising the possibility that even more sRNAs, which remain to be identified, could regulate PhoPQ synthesis.

Bacterial translational control at atomic resolution.

Translational regulation allows rapid adaptation of protein synthesis to environmental conditions. In prokaryotes, the synthesis of many RNA-binding proteins is regulated by a translational feedback mechanism involving a competition between their natural substrate and their binding site on mRNA, which are often thought to resemble each other. This article describes the case of threonyl-tRNA synthetase, which represses the translation of its own mRNA. Recent data provide the first opportunity to describe at the atomic level both the extent and the limit of mimicry between the way this enzyme recognizes tRNA(Thr) and its regulatory site in mRNA. The data also give some clues about how the binding of the synthetase to its mRNA inhibits translation.

Escherichia coli protein synthesis initiation factor IF3 controls its own gene expression at the translational level in vivo.

Measurements of the relative synthesis rates of mRNAs transcribed from the gene (thrS) for threonyl-tRNA synthetase and the adjacent gene (infC) for initiation factor IF3 show four- to fivefold more infC mRNA than thrS mRNA in vivo, suggesting that infC expression can be controlled independently of thrS expression. S1 mapping experiments reveal the existence of two transcription initiation sites for infC mRNAs internal to the thrS structural gene. Both the mRNA measurements and the S1 mapping experiments indicate that the majority of infC transcription initiates at the infC proximal promoter. In agreement with these results, the deletion of the infC distal promoter from infC-lacZ gene fusions does not affect the expression of these gene fusions in vivo. Measurements of the relative synthesis rate of infC mRNA in vivo in infC- strains overproducing IF3 shows that infC mRNA levels are normal in these strains, thus suggesting that IF3 regulates the translation of infC mRNAs in vivo. Extension of these experiments using infC-lacZ gene fusions carried on lambda bacteriophage and integrated at the lambda att site on the Escherichia coli chromosome shows that the expression of infC-lacZ protein fusions, but not infC-lacZ operon fusions, is derepressed in two infC- strains. A cellular excess of IF3 represses the expression of an infC-lacZ protein fusion but not an infC-lacZ operon fusion. Measurements of the relative mRNA synthesis rates of hybrid infC-lacZ mRNA synthesized from an infC-lacZ protein fusion under conditions of a fourfold derepression or a threefold repression of hybrid IF3-beta-galactosidase expression shows that the hybrid infC-lacZ mRNA levels remain unchanged. These results indicate that the cellular levels of IF3 negatively regulate the expression of its own gene, infC, at the translational level in vivo.

Structural basis of translational control by Escherichia coli threonyl tRNA synthetase.

Escherichia coli threonyl-tRNA synthetase (ThrRS) represses the translation of its own messenger RNA by binding to an operator located upstream of the initiation codon. The crystal structure of the complex between the core of ThrRS and the essential domain of the operator shows that the mRNA uses the recognition mode of the tRNA anticodon loop to initiate binding. The final positioning of the operator, upon which the control mechanism is based, relies on a characteristic RNA motif adapted to the enzyme surface. The finding of other thrS operators that have this conserved motif leads to a generalization of this regulatory mechanism to a subset of Gram-negative bacteria.

Post-transcriptional control by global regulators of gene expression in bacteria

Several authentic or potential global regulators have recently been shown to act at the post-transcriptional level. This is the case for Hfq (HF-1), which is involved in the regulation of an increasing number of genes in Escherichia coli, and CsrA (RsmA) responsible for controlling the expression of genes for extracellular enzymes and secondary metabolism in Gram-negative bacteria. The cold-shock proteins of the CspA family are able to destabilise mRNA secondary structures at low temperature and, therefore, also seem to act post-transcriptionally. These findings illustrate a more general aspect of post-transcriptional control which, in the past, was generally restricted to regulators acting at a single target. The expression of several global transcriptional regulators, such as the stationary phase and heat-shock sigma factors and H-NS, have also recently been shown to be themselves under post-transcriptional control. These examples underline the importance of this type of control in bacterial gene regulation.

Activation of the Kss1 invasive-filamentous growth pathway induces Ty1 transcription and retrotransposition in Saccharomyces cerevisiae.

ABSTRACT Using a set of genomic TY1A-lacZ fusions, we show that Ste12 and Tec1, two transcription factors of the Kss1 mitogen-activated protein kinase (MAPK) cascade activate Ty1 transcription inSaccharomyces cerevisiae. This result strongly suggests that the invasive-filamentous pathway regulates Ty1 transcription. Since this pathway is active in diploid cells, we suspected that Ty1 transposition might occur in this cell type, despite the fact that this event has been never reported before (unless activated by heterologous promoters such as that of GAL1). We demonstrate here that constitutive activation of the invasive-filamentous pathway by theSTE11-4 allele or by growth in low-nitrogen medium induces Ty1 transcription and retrotransposition in diploid cells. We show that Ty1 retrotransposition can be activated by STE11-4 in haploid cells as well. Our findings provide the first evidence that Ty1 retrotransposition can be activated by environmental signals that affect differentiation. Activation of the Kss1 MAPK cascade by stress is known to cause filament formation that permits the search for nutrients away from the colonization site. We propose that activation of Ty1 retrotransposition by this cascade could play a role in adaptive mutagenesis in response to stress.