Claude Chiaruttini

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.

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. Both MicA and GcvB bind phoPQ mRNA in vivo and in vitro around the translation initiation region of phoP. Binding of either small RNA is sufficient to inhibit ribosome binding and induce mRNA degradation. Surprisingly, however, MicA and GcvB have different effects on the levels of the PhoP protein and therefore on the expression of the PhoP regulon. These results highlight the complex connections between small RNAs and transcriptional regulation networks in bacteria.

Messenger RNA secondary structure and translational coupling in the Escherichia coli operon encoding translation initiation factor IF3 and the ribosomal proteins, L35 and L20☆

The Escherichia coli infC-rpmI-rplT operon encodes translation initiation factor IF3 and the ribosomal proteins, L35 and L20, respectively. The expression of the last cistron (rplT) has been shown to be negatively regulated at a post-transcriptional level by its own product, L20, which acts at an internal operator located within infC. The present work shows that L20 directly represses the expression of rpmI, and indirectly that of rplT, via translational coupling with rpmI. Deletions and an inversion of the coding region of rpmI, suggest an mRNA secondary structure forming between sequences within rpmI and the translation initiation site of rplT. To verify the existence of this structure, detailed analyses were performed using chemical and enzymatic probes. Also, mutants that uncoupled rplT expression from that of rpmI, were isolated. The mutations fall at positions that would base-pair in the secondary structure. Our model is that L20 binds to its operator within infC and represses the translation of rpmI. When the rpmI mRNA is not translated, it can base-pair with the ribosomal binding site of rplT, sequestering it, and abolishing rplT expression. If the rpmI mRNA is translated, i.e. covered by ribosomes, the inhibitory structure cannot form leaving the translation initiation site of rplT free for ribosomal binding and for full expression. Although translational coupling in ribosomal protein operons has been suspected to be due to the formation of secondary structures that sequester internal ribosomal binding sites, this is the first time that such a structure has been shown to exist.

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. The first site is a pseudoknot formed by a base‐pairing interaction between nucleotide sequences separated by more than 280 nucleotides, whereas the second site is an irregular helix formed by base‐pairing between neighbouring nucleotide sequences. Despite these differences, the mRNA folds in such a way that both L20 binding sites share secondary structure similarity with the L20 binding site located at the junction between helices H40 and H41 in 23S rRNA. Using a set of genetic, biochemical, biophysical, and structural experiments, we show here that all three sites are recognized similarly by L20.

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. However, little is know about the molecular mechanisms of this process. It is thought that the long basic r-protein extensions that penetrate deeply into the subunit cores play a key role through disorder-order transitions and/or co-folding mechanisms. A current view is that such structural transitions may facilitate the proper rRNA folding. In this paper, the structures of the proteins L3, L4, L13, L20, L22 and L24 that have been experimentally found to be essential for the first steps of ribosome assembly have been compared. On the basis of their structural and dynamics properties, three categories of extensions have been identified. Each of them seems to play a distinct function. Among them, only the coil-helix transition that occurs in a phylogenetically conserved cluster of basic residues of the L20 extension appears to be strictly required for the large subunit assembly in eubacteria. The role of α helix-coil transitions in 23 S RNA folding is discussed in the light of the calcium binding protein calmodulin that shares many structural and dynamics properties with L20.

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. Here, we present the X‐ray structure of the unbound ribosomal protein L20, the long basic amino‐terminal extension of which has been previously interpreted as fully disordered in the absence of RNA. This study provides the first detailed picture of two protein folding states trapped together in a crystal and indicates that unfolding occurs in discrete regions of the whole protein, corresponding mainly to RNA‐binding residues. The electrostatic destabilization of the long α‐helix and a structural communication between the two L20 domains are reminiscent of those observed in calmodulin. The detailed comparison of the two conformations observed in the crystal provides new insights into the role of unfolded extensions in ribosomal assembly.

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. It is composed of an unstructured N-terminal domain comprising residues 1-58 and a C-terminal alpha-helical domain. This is in contrast with what is observed in the bacterial 50S subunit, where the N-terminal region folds as an elongated alpha-helical region. The solution structure of the C-terminal domain shows that several solvent-accessible, conserved residues are clustered on the surface of the molecule and are probably involved in RNA recognition. In vivo studies show that this domain is sufficient to repress the expression of the cistrons encoding L35 and L20 in the IF3 operon. The ability of L20 C-terminal domain to specifically recognise RNA suggests an assembly mechanism for L20 into the ribosome. The pre-folded C-terminal domain would make a primary interaction with a specific site on the 23S rRNA. The N-terminal domain would then fold within the ribosome, participating in its correct 3D assembly.

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. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites—i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations—i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by “one-side” clamping.

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. coli synthesizes two Msr, A and B, which exhibit substrate diastereospecificity. The bacterial iron-responsive small RNA (sRNA) RyhB controls iron metabolism by modulating intracellular iron usage. We show in this paper that RyhB is a direct regulator of the msrB gene that encodes the MsrB enzyme. RyhB down-regulates msrB transcripts along with Hfq and RNaseE proteins since mutations in the ryhB, fur, hfq, or RNaseE-encoded genes resulted in iron-insensitive expression of msrB. Our results show that RyhB binds to two sequences within the short 5′UTR of msrB mRNA as identified by reverse transcriptase and RNase and lead (II) protection assays. Toeprinting analysis shows that RyhB pairing to msrB mRNA prevents efficient ribosome binding and thereby inhibits translation initiation. In vivo site directed-mutagenesis experiments in the msrB 5′UTR region indicate that both RyhB-pairing sites are required to decrease msrB expression. Thus, this study suggests a novel mechanism of translational regulation where a same sRNA can basepair to two different locations within the same mRNA species. In contrast, expression of msrA is not influenced by changes in iron levels.

Escherichia coli ribosomal protein L20 binds as a single monomer to its own mRNA bearing two potential binding sites

Ribosomal protein L20 is crucial for the assembly of the large ribosomal subunit and represses the translation of its own mRNA. L20 mRNA carries two L20-binding sites, the first folding into a pseudoknot and the second into an imperfect stem and loop. These two sites and the L20-binding site on 23S ribosomal RNA are recognized similarly using a single RNA-binding site located on one face of L20. In this work, using gel filtration and fluorescence cross-correlation spectroscopy (FCCS) experiments, we first exclude the possibility that L20 forms a dimer, which would allow each monomer to bind one site of the mRNA. Secondly we show, using affinity purification and FCCS experiments, that only one molecule of L20 binds to the L20 mRNA despite the presence of two potential binding sites. Thirdly, using RNA chemical probing, we show that the two L20-binding sites are in interaction. This interaction provides an explanation for the single occupancy of the mRNA. The two interacting sites could form a single hybrid site or the binding of L20 to a first site may inhibit binding to the second. Models of regulation compatible with our data are discussed.