Aminoacyl-tRNA Synthetases in the Bacterial World.

Aminoacyl-tRNA synthetases (aaRSs) are modular enzymes globally conserved in the three kingdoms of life. All catalyze the same two-step reaction, i.e.

Escherichia coli ribosomal protein S1 unfolds structured mRNAs onto the ribosome for active translation initiation.

Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5' untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step.

The sRNA RyhB regulates the synthesis of the Escherichia coli methionine sulfoxide reductase MsrB but not MsrA.

Controlling iron homeostasis is crucial for all aerobically grown living cells that are exposed to oxidative damage by reactive oxygen species (ROS), as free iron increases the production of ROS. Methionine sulfoxide reductases (Msr) are key enzymes in repairing ROS-mediated damage to proteins, as they reduce oxidized methionine (MetSO) residues to methionine. E.

Post-transcriptional control of the Escherichia coli PhoQ-PhoP two-component system by multiple sRNAs involves a novel pairing region of GcvB.

PhoQ/PhoP is a central two-component system involved in magnesium homeostasis, pathogenicity, cell envelope composition, and acid resistance in several bacterial species. The small RNA GcvB is identified here as a novel direct regulator of the synthesis of PhoQ/PhoP in Escherichia coli, and this control relies on a novel pairing region of GcvB. After MicA, this is the second Hfq-dependent small RNA that represses expression of the phoPQ operon.

Aminoacyl-tRNA Synthetases in the Bacterial World.

Aminoacyl-tRNAsynthetases (aaRSs) are modular enzymesglobally conserved in the three kingdoms of life. All catalyze the same two-step reaction, i.e.

Probing ribosomal protein-RNA interactions with an external force.

Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA.

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.

The role of disordered ribosomal protein extensions in the early steps of eubacterial 50 S ribosomal subunit assembly.

Although during the past decade research has shown the functional importance of disorder in proteins, many of the structural and dynamics properties of intrinsically unstructured proteins (IUPs) remain to be elucidated. This review is focused on the role of the extensions of the ribosomal proteins in the early steps of the assembly of the eubacterial 50 S subunit. The recent crystallographic structures of the ribosomal particles have revealed the picture of a complex assembly pathway that condenses the rRNA and the ribosomal proteins into active ribosomes.

Structural probing of RNA thermosensors.

Chemical probing of RNA structure has become one of the most popular approaches to map the conformation of RNA molecules of various sizes under well-defined experimental conditions. The method monitors the sensitivity of each nucleotide to various chemicals, which reflects its hydrogen-bonding environment within the RNA molecule. The goal of this chapter is to provide the reader with an experimental guide to mapping the secondary structure of RNA thermosensors in vitro with the most suitable chemical probes.

A competition mechanism regulates the translation of the Escherichia coli operon encoding ribosomal proteins L35 and L20.

Escherichia coli ribosomal protein (r-protein) L20 is essential for the assembly of the 50S ribosomal subunit and is also a translational regulator of its own rpmI-rplT operon, encoding r-proteins L35 and L20 in that order. L20 directly represses the translation of the first cistron and, through translational coupling, that of its own gene. The translational operator of the operon is 450 nt in length and includes a long-range pseudoknot interaction between two RNA sequences separated by 280 nt.

Mutations in residues involved in zinc binding in the catalytic site of Escherichia coli threonyl-tRNA synthetase confer a dominant lethal phenotype.

Escherichia coli threonyl-tRNA synthetase is a homodimeric protein that acts as both an enzyme and a regulator of gene expression: the protein aminoacylates tRNA(Thr) isoacceptors and binds to its own mRNA, inhibiting its translation. The enzyme contains a zinc atom in its active site, which is essential for the recognition of threonine. Mutations in any of the three amino acids forming the zinc-binding site inactivate the enzyme and have a dominant negative effect on growth if the corresponding genes are placed on a multicopy plasmid.

Coexistence of two protein folding states in the crystal structure of ribosomal protein L20.

The recent finding of intrinsically unstructured proteins defies the classical structure-function paradigm. However, owing to their flexibility, intrinsically unstructured proteins generally escape detailed structural investigations. Consequently little is known about the extent of conformational disorder and its role in biological functions.

Severe adenine starvation activates Ty1 transcription and retrotransposition in Saccharomyces cerevisiae.

Ty1 retrotransposons of the yeast Saccharomyces cerevisiae are activated by different kinds of stress. Here we show that Ty1 transcription is stimulated under severe adenine starvation conditions. The Bas1 transcriptional activator, responsible for the induction of genes of the de novo AMP biosynthesis pathway (ADE) in the absence of adenine, is not involved in this response.

Characterization of the molecular mechanisms involved in the differential production of erythrose-4-phosphate dehydrogenase, 3-phosphoglycerate kinase and class II fructose-1,6-bisphosphate aldolase in Escherichia coli.

A gapA-pgk gene tandem coding the glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase, is most frequently found in bacteria. However, in Enterobacteriaceae, gapA is replaced by an epd open reading frame (ORF) coding an erythrose-4-phosphate dehydrogenase and an fbaA ORF coding the class II fructose-1,6-bisphosphate aldolase follows pgk. Although epd expression is very low in Escherichia coli, we show that, in the presence of glucose, the 3 epd, pgk and fbaA ORFs are efficiently cotranscribed from promoter epd P0.

Double molecular mimicry in Escherichia coli: binding of ribosomal protein L20 to its two sites in mRNA is similar to its binding to 23S rRNA.

Escherichia coli ribosomal L20 is one of five proteins essential for the first reconstitution step of the 50S ribosomal subunit in vitro. It is purely an assembly protein, because it can be withdrawn from the mature subunit without effect on ribosome activity. In addition, L20 represses the translation of its own gene by binding to two sites in its mRNA.

Impact of ionizing radiation on the life cycle of Saccharomyces cerevisiae Ty1 retrotransposon.

Ty1 elements, LTR-retrotransposons of Saccharomyces cerevisiae, are known to be activated by genetic and environmental stress. Several DNA-damaging agents have been shown to increase both Ty1 transcription and retrotransposition. To explore further the relationship between Ty1 mobility and DNA damage, we have studied the impact of ionizing radiation at different steps of the Ty1 life cycle.

The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control.

The Escherichia coli autoregulatory ribosomal protein L20 consists of two structurally distinct domains. The C-terminal domain is globular and sits on the surface of the large ribosomal subunit whereas the N-terminal domain has an extended shape and penetrates deep into the RNA-rich core of the subunit. Many other ribosomal proteins have analogous internal or terminal extensions.

Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding.

The ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA.

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.

The modular structure of Escherichia coli threonyl-tRNA synthetase as both an enzyme and a regulator of gene expression.

In addition to its role in tRNA aminoacylation, Escherichia coli threonyl-tRNA synthetase is a regulatory protein which binds a site, called the operator, located in the leader of its own mRNA and inhibits translational initiation by competing with ribosome binding. This work shows that the two essential steps of regulation, operator recognition and inhibition of ribosome binding, are performed by different domains of the protein. The catalytic and the C-terminal domain of the protein are involved in binding the two anticodon arm-like structures in the operator whereas the N-terminal domain of the enzyme is responsible for the competition with the ribosome.

NMR structure of bacterial ribosomal protein l20: implications for ribosome assembly and translational control.

L20 is a specific protein of the bacterial ribosome, which is involved in the early assembly steps of the 50S subunit and in the feedback control of the expression of its own gene. This dual function involves specific interactions with either the 23S rRNA or its messenger RNA. The solution structure of the free Aquifex aeolicus L20 has been solved.

An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes.

In Listeria monocytogenes, virulence genes are maximally expressed at 37 degrees C, almost silent at 30 degrees C and controlled by PrfA, a transcriptional activator whose expression is thermoregulated. Here, we show that the untranslated mRNA (UTR) preceding prfA, forms a secondary structure, which masks the ribosome binding region. Mutations predicted to destabilize this structure led to virulence gene expression and invasion of mammalian cells at 30 degrees C.

Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: a possible case of ribosomal RNA-messenger RNA molecular mimicry.

In addition to being a component of the large ribosomal subunit, ribosomal protein L20 of Escherichia coli also acts as a translational repressor. L20 is synthesized from the IF3 operon that contains three cistrons coding for IF3, and ribosomal proteins L35 and L20. L20 directly represses the expression of the gene encoding L35 and the expression of its own gene by translational coupling.

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.