Roberto Spurio

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.

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.

Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions.

The most characteristic event of cold‐shock activation in Escherichia coli is believed to be the de novo synthesis of CspA. We demonstrate, however, that the cellular concentration of this protein is ≥50 μM during early exponential growth at 37°C; therefore, its designation as a major cold‐shock protein is a misnomer. The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid‐to‐late exponential growth phase while the CspA level declines, although always remaining clearly detectable. A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium. The extent of cold‐shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre‐existing level of CspA which suggests feedback autorepression by this protein. Both transcriptional and post‐transcriptional controls regulate cspA expression under non‐stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA‐binding proteins Fis and H‐NS both in vivo and in vitro, while its decreased half‐life with increasing cell density contributes to its rapid disappearance. The cspA mRNA instability is due to its 5′ untranslated leader and is counteracted in vivo by the cold‐shock DeaD box RNA helicase (CsdA).

Translation initiation factor IF3: two domains, five functions, one mechanism?

Initiation factor IF3 contains two domains separated by a flexible linker. While the isolated N‐domain displayed neither affinity for ribosomes nor a detectable function, the isolated C‐domain, added in amounts compensating for its reduced affinity for 30S subunits, performed all activities of intact IF3, namely: (i) dissociation of 70S ribosomes; (ii) shift of 30S‐bound mRNA from ‘stand‐by’ to ‘P‐decoding’ site; (iii) dissociation of 30S–poly(U)–NacPhe‐tRNA pseudo‐ initiation complexes; (iv) dissociation of fMet‐tRNA from initiation complexes containing mRNA with the non‐canonical initiation triplet AUU (AUUmRNA); (v) stimulation of mRNA translation regardless of its start codon and inhibition of AUUmRNA translation at high IF3C/ribosome ratios. These results indicate that while IF3 performs all its functions through a C‐domain–30S interaction, the N‐domain function is to provide additional binding energy so that its fluctuating interaction with the 30S subunit can modulate the thermodynamic stability of the 30S–IF3 complex and IF3 recycling. The localization of IF3C far away from the decoding site and anticodon stem–loop of P‐site‐bound tRNA indicates that the IF3 fidelity function does not entail its direct contact with these structures.

Late events of translation initiation in bacteria: a kinetic analysis

Binding of the 50S ribosomal subunit to the 30S initiation complex and the subsequent transition from the initiation to the elongation phase up to the synthesis of the first peptide bond represent crucial steps in the translation pathway. The reactions that characterize these transitions were analyzed by quench‐flow and fluorescence stopped‐flow kinetic techniques. IF2‐dependent GTP hydrolysis was fast (30/s) followed by slow Pi release from the complex (1.5/s). The latter step was rate limiting for subsequent A‐site binding of EF‐Tu·GTP·Phe‐tRNAPhe ternary complex. Most of the elemental rate constants of A‐site binding were similar to those measured on poly(U), with the notable exception of the formation of the first peptide bond which occurred at a rate of 0.2/s. Omission of GTP or its replacement with GDP had no effect, indicating that neither the adjustment of fMet‐tRNAfMet in the P site nor the release of IF2 from the ribosome required GTP hydrolysis.

Expression of the gene encoding the major bacterial nucleoid protein H‐NS is subject to transcriptional auto‐repression

Expression of a promoterless cat gene fused to a DNA fragment of approximately 400 bp, beginning at –313 of Escherichia coli hns, was significantly repressed in E. coli and Salmonella typhimurium strains with wild‐type hns but not in mutants carrying hns alleles. CAT expression from fusions containing a shorter (110 bp) segment of hns was essentially unaffected in the same genetic backgrounds. The stage of growth was found to influence the extent of repression which was maximum (approximately 75%) in mid‐log cultures and negligible in cells entering the stationary phase. The level of repression in early‐log phase was lower than in mid‐log phase cultures, probably because of the presence of high levels of Fis protein, which counteracts the H‐NS inhibition by stimulating hns transcription. The effects observed in vivo were mirrored by similar results obtained in vitro upon addition of purified H‐NS and Fis protein to transcriptional systems programmed with the same hns caf fusions. Electrophoretic gel shift assays, DNase I footprinting and cyclic permutation get analyses revealed that H‐NS binds preferentially to the upstream region of its own gene recognizing two rather extended segments of DNA on both sides of a bend centred around –150. When these sites are filled by H‐NS, an additional site between approximately –20 and –65, which partly overlaps the promoter, is also occupied. Binding of H‐NS to this site is probably the ultimate cause of transcriptional auto‐repression.

Mapping the fMet-tRNAfMet binding site of initiation factor IF2

The interaction between fMet‐tRNAfMet and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the specificity of this interaction are localized within the acceptor hexanucleotide fMet‐3′ACCAAC of the initiator tRNA and a fairly small area at the surface of the β‐barrel structure of the 90‐amino acid C‐terminal domain of IF2 (IF2 C‐2). A weak but specific interaction between IF2 C‐2 and formyl‐methionyl was also demonstrated. The surface of IF2 C‐2 interacting with fMet‐tRNAfMet has been mapped using two independent approaches, site‐ directed mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side‐chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNAfMet, and the surface lined with residues K702‐S660, along which the acceptor arm of the initiator tRNA spans in the direction 3′ to 5′.

Structure of the fMet‐tRNAfMet‐binding domain of B.stearothermophilus initiation factor IF2

The three‐dimensional structure of the fMet‐tRNAfMet‐binding domain of translation initiation factor IF2 from Bacillus stearothermophilus has been determined by heteronuclear NMR spectroscopy. Its structure consists of six antiparallel β‐strands, connected via loops, and forms a closed β‐barrel similar to domain II of elongation factors EF‐Tu and EF‐G, despite low sequence homology. Two structures of the ternary complexes of the EF‐Tu·aminoacyl‐tRNA· GDP analogue have been reported and were used to propose and discuss the possible fMet‐tRNAfMet‐binding site of IF2.

Nature and mechanism of the in vivo oligomerization of nucleoid protein H-NS

Two types of two‐hybrid systems demonstrate that the transcriptional repressor, nucleoid‐associated protein H‐NS (histone‐like, nucleoid structuring protein) forms dimers and tetramers in vivo, the latter being the active form of the protein. The H‐NS ‘protein oligomerization’ domain (N‐domain) is unable to oligomerize in the absence of the intradomain linker while the ‘DNA‐binding’ C‐domain clearly displays a protein–protein interaction capacity, which contributes to H‐NS tetramerization and which is lost following Pro115 mutation. Linker deletion or substitution with KorB linker abolishes H‐NS oligomerization. A model describing H‐NS dimerization and tetramerization based on all available data and suggesting the existence in the tetramer of a bundle of four α‐helices, each contributed by an H‐NS monomer, is presented.