Claudio O. Gualerzi

Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS

The expression of plasmid‐borne virF of Shigella encoding a transcriptional regulator of the AraC family, is required to initiate a cascade of events resulting in activation of several operons encoding invasion functions. H‐NS, one of the main nucleoid‐associated proteins, controls the temperature‐dependent expression of the virulence genes by repressing the in vivo transcription of virF only below a critical temperature (∼32°C). This temperature‐dependent transcriptional regulation has been reproduced in vitro and the targets of H‐NS on the virF promoter were identified as two sites centred around −250 and −1 separated by an intrinsic DNA curvature. H‐NS bound cooperatively to these two sites below 32°C, but not at 37°C. DNA supercoiling within the virF promoter region did not influence H‐NS binding but was necessary for the H‐NS‐mediated transcriptional repression. Electrophoretic analysis between 4 and 60°C showed that the virF promoter fragment, comprising the two H‐NS sites, undergoes a specific and temperature‐dependent conformational transition at ∼32°C. Our results suggest that this modification of the DNA target may modulate a cooperative interaction between H‐NS molecules bound at two distant sites in the virF promoter region and thus represents the physical basis for the H‐NS‐dependent thermoregulation of virulence gene expression.

Translational control of prokaryotic gene expression

Awareness of the importance of post-transcriptional control of gene expression in prokaryotes has grown enormously over the past ten years. In particular, translation features as a step where both control over constitutive rates of gene expression, as well as cis and trans regulation are exercised. Recent research has provided us with new insights into the molecular basis of these phenomena.

Structure of the 30S translation initiation complex

Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct messenger RNA start site and the reading frame are selected when, with the help of initiation factors IF1, IF2 and IF3, the initiation codon is decoded in the peptidyl site of the 30S ribosomal subunit by the fMet-tRNAfMet anticodon. This yields a 30S initiation complex (30SIC) that is an intermediate in the formation of the 70S initiation complex (70SIC) that occurs on joining of the 50S ribosomal subunit to the 30SIC and release of the initiation factors. The localization of IF2 in the 30SIC has proved to be difficult so far using biochemical approaches, but could now be addressed using cryo-electron microscopy and advanced particle separation techniques on the basis of three-dimensional statistical analysis. Here we report the direct visualization of a 30SIC containing mRNA, fMet-tRNAfMet and initiation factors IF1 and GTP-bound IF2. We demonstrate that the fMet-tRNAfMet is held in a characteristic and precise position and conformation by two interactions that contribute to the formation of a stable complex: one involves the transfer RNA decoding stem which is buried in the 30S peptidyl site, and the other occurs between the carboxy-terminal domain of IF2 and the tRNA acceptor end. The structure provides insights into the mechanism of 70SIC assembly and rationalizes the rapid activation of GTP hydrolysis triggered on 30SIC–50S joining by showing that the GTP-binding domain of IF2 would directly face the GTPase-activated centre of the 50S subunit.

Leaderless mRNAs in bacteria: surprises in ribosomal recruitment and translational control.

It is commonly believed that the translational efficiency of prokaryotic mRNAs is intrinsically determined by both primary and secondary structures of their translational initiation regions. However, for leaderless mRNAs starting with the AUG initiating codon occurring in bacteria, archaea and eukaryotes, there is no evidence for ribosomal recruitment signals downstream of the 5′‐terminal AUG that seems to be the only necessary and constant element. Studies in Escherichia coli have brought to light that the ratio of initiation factors IF2 and IF3 plays a decisive role in translation initiation of leaderless mRNA, indicating that the translational efficiency of this mRNA class can be modulated depending on the availability of components of the translational machinery. Recent data suggested that the start codon of bacterial leaderless mRNAs is recognized by a ribosome‐IF2‐fMet‐tRNA complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes, which points to a conceptual similarity in all initiation pathways. In fact, the faithful translation of lead‐erless mRNAs in heterologous systems shows that the ability to translate leaderless mRNAs is an evo‐lutionarily conserved function of the translational apparatus.

The oligomeric structure of nucleoid protein H‐NS is necessary for recognition of intrinsically curved DNA and for DNA bending

Escherichia coli hns, encoding the abundant nucleoid protein H‐NS, was subjected to site‐directed mutagenesis either to delete Pro115 or to replace it with alanine. Unlike the wild‐type protein, hyperproduction of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less drastic compaction of the nucleoid. Gel shift and ligase‐mediated circularization tests demonstrated that the mutant proteins retained almost normal affinity for non‐curved DNA, but lost the wild‐type capacity to recognize preferentially curved DNA and to actively bend non‐curved DNA, a property of wild‐type H‐NS demonstrated here for the first time. DNase I footprinting and in vitro transcription experiments showed that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required for H‐NS transcription autorepression and to inhibit transcription from the same promoter. The failure of the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity for non‐curved DNA can be attributed to a defect in protein–protein interaction resulting in a reduced capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by H‐NS of the oligomerization domain (C‐domain) of bacteriophage λ cI repressor.

Post-transcriptional regulation of CspA expression in Escherichia coli

The Escherichia coli cspA gene, encoding the major cold‐shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible λ PL promoter. After induction of transcription by thermal inactivation of the λ ts repressor, abundant expression of the product (CspA) was obtained if the cells were subsequently incubated at 10°C, but poor expression was obtained if the cells were incubated at 37°C or 30°C. The reason for this differential temperature‐dependent expression was investigated and it was found that: (i) the CspA content of the cells decreased more rapidly at 37°C compared to 10°C, regardless of whether transcription was turned off by addition of rifampicin; (ii) both the chemical and functional half‐lives of the cspA transcript were substantially longer at 10°C compared to 37°C; (iii) S30 extracts as well as 70S ribosomes prepared from cold‐shocked cells translated CspA mRNA (but not phage MS2 RNA) more efficiently than equivalent extracts or ribosomes obtained from control cells grown at 37°C; and (iv) purified CspA stimulated CspA mRNA translation. Overall, these results indicate that a selective modification of the cold‐shocked translational apparatus favouring translation of CspA mRNA, and an increased stability of this mRNA at low temperature, may play an important role in the induction of cspA expression during cold shock.

Specific protection of 16 S rRNA by translationalinitiation factors

The binding of initiation factors to 30 S ribosomal subunits protects specific sets of nucleotides in 16 S rRNA from base-specific chemical probes. Initiation factor 3 (IF-3) protects residues G700, G703 and G791 from attack by kethoxal. These protected bases are close to those in 16 S rRNA that are protected by 50 S subunits, providing a structural basis for the subunit dissociation activity of IF-3. The IF-3-dependent protections also flank bases that are protected by P-site-bound tRNA, in keeping with the possibility that IF-3 may interact with initiator tRNA, or influence the properties of the 30 S P site during initiation. IF-1 protects G530, A1492 and A1493 and causes enhanced reactivity of A1408. These bases are precisely the ones that are protected by the binding of tRNA to the ribosomal A site. This suggests that IF-1 mimics A-site-bound tRNA, and could serve to prevent premature binding of aminoacyl tRNA by blocking the 30 S A site. We were unable to detect any effect of IF-2 on the reactivity pattern of 16 S rRNA, suggesting that this factor may interact primarily through protein-protein interactions.

Lethal overproduction of the Escherichia coli nucleoid protein H-NS: ultramicroscopic and molecular autopsy.

SummaryThe Escherichia coli hns gene, which encodes the nucleoid protein H-NS, was deprived of its natural promoter and placed under the control of the inducible lambda PL promoter. An hns mutant yielding a protein (H-NSΔ12) with a deletion of four amino acids (Gly112-Arg-Thr-Pro115) was also obtained. Overproduction of wild-type (wt) H-NS, but not of H-NSΔ12, resulted in a drastic loss of cell viability. The molecular events and the morphological alterations eventually leading to cell death were investigated. A strong and nearly immediate inhibition of both RNA and protein synthesis were among the main effects of overproduction of wt H-NS, while synthesis of DNA and cell wall material was inhibited to a lesser extent and at a later time. Upon cryofixation of the cells, part of the overproduced protein was found in inclusion bodies, while the rest was localized by immunoelectron microscopy to the nucleoids. The nucleoids appeared condensed in cells expressing both forms of H-NS, but the morphological alterations were particularly dramatic in those overproducing wt H-NS; their nucleoids appeared very dense, compact and almost perfectly spherical. These results provide direct evidence for involvement of H-NS in control of the organization and compaction of the bacterial nucleoid in vivo and suggest that it may function, either directly or indirectly, as transcriptional repressor and translational inhibitor.

The structure of the translational initiation factor IF1 from E.coli contains an oligomer‐binding motif

The structure of the translational initiation factor IF1 from Escherichia coli has been determined with multidimensional NMR spectroscopy. Using 1041 distance and 78 dihedral constraints, 40 distance geometry structures were calculated, which were refined by restrained molecular dynamics. From this set, 19 structures were selected, having low constraint energy and few constraint violations. The ensemble of 19 structures displays a root‐mean‐square deviation versus the average of 0.49 Å for the backbone atoms and 1.12 Å for all atoms for residues 6–36 and 46–67. The structure of IF1 is characterized by a five‐stranded β‐barrel. The loop connecting strands three and four contains a short 310 helix but this region shows considerably higher flexibility than the β‐barrel. The fold of IF1 is very similar to that found in the bacterial cold shock proteins CspA and CspB, the N‐terminal domain of aspartyl‐tRNA synthetase and the staphylococcal nuclease, and can be identified as the oligomer‐binding motif. Several proteins of this family are nucleic acid‐binding proteins. This suggests that IF1 plays its role in the initiation of protein synthesis by nucleic acid interactions. Specific changes of NMR signals of IF1 upon titration with 30S ribosomal subunit identifies several residues that are involved in the interaction with ribosomes.

The virF promoter in Shigella: more than just a curved DNA stretch

In the human enteropathogen Shigella transcription of virF, the primary regulator of the invasion functions, is strictly temperature‐dependent and is antagonistically mediated by H‐NS and FIS, which bind to specific sites on the virF promoter. Here we report on the relevance of DNA geometry to the themoregulation of virF and demonstrate that the virF promoter hosts a major DNA bend halfway between two H‐NS sites. The bent region has been mutagenized in vitro to mimic temperature‐induced changes of DNA curvature. Functional analysis of curvature mutants and of promoter constructs in which the two H‐NS sites are phased‐out by a half–helix turn reveals that modifying the spatial relationships between these sites severely affects the interaction of H‐NS with the virF promoter, as well as its in vivo and in vitro temperature‐dependent activity. The role of promoter curvature as thermosensor is also compatible with the present observation that, with increasing temperature, the virF bending centre moves downstream at a rate having its maximum around the transition temperature, abruptly unmasking a binding site for the transcriptional activator FIS.