Anna La Teana

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.

Antagonistic involvement of FIS and H-NS proteins in the transcriptional control of hns expression

Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inversion stimulation) protein binds to at least seven sites in the promoter region of hns. These sites extend from −282 to +25 with two sites, closely flanking the DNA bend located at −150 from the transcriptional startpoint, partly overlapping the H‐NS binding sites involved in the transcriptional autorepression of hns. The interplay between FIS, H‐NS and the hns promoter region were studied by examining the effects of FIS and H‐NS on in vitro transcription of hns–cat fusions, as well as looking at the effect of FIS on preformed complexes containing H‐NS and a DNA fragment derived from the hns promoter region. Taken together, our data suggest that in the cell, FIS and H‐NS interact with the promoter region of hns and influence their respective interactions (possibly competing for the same binding site), eliciting antagonistic effects so that an interplay between these proteins might contribute to the transcriptional control of hns

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.

Initiation factor IF 2 binds to the alpha-sarcin loop and helix 89 of Escherichia coli 23S ribosomal RNA.

During initiation of protein synthesis in bacteria, translation initiation factor IF2 is responsible for the recognition of the initiator tRNA (fMet-tRNA). To perform this function, IF2 binds to the ribosome interacting with both 30S and 50S ribosomal subunits. Here we report the topographical localization of translation initiation factor IF2 on the 70S ribosome determined by base-specific chemical probing. Our results indicate that IF2 specifically protects from chemical modification two sites in domain V of 23S rRNA, namely A2476 and A2478, and residues around position 2660 in domain VI, the so-called sarcin-ricin loop. These footprints are generated by IF2 regardless of the presence of fMet-tRNA, GTP, mRNA, and IF1. IF2 causes no specific protection of 16S rRNA. We observe a decreased reactivity of residues A1418 and A1483, which is an indication that the initiation factor has a tightening effect on the association of ribosomal subunits. This result, confirmed by sucrose density gradient analysis, seems to be a universally conserved property of IF2.

Mapping the Active Sites of Bacterial Translation Initiation Factor IF3

IF3C is the C-terminal domain of Escherichia coli translation initiation factor 3 (IF3) and is responsible for all functions of this translation initiation factor but for its ribosomal recycling. To map the number and nature of the active sites of IF3 and to identify the essential Arg residue(s) chemically modified with 2,3-butanedione, the eight arginine residues of IF3C were substituted by Lys, His, Ser and Leu, generating 32 variants that were tested in vitro for all known IF3 activities. The IF3-30S subunit interaction was inhibited strongly by substitutions of Arg99, Arg112, Arg116, Arg147 and Arg168, the positive charges being important at positions 116 and 147. The 70S ribosome dissociation was affected by mutations of Arg112, Arg147 and, to a lesser extent, of Arg99 and Arg116. Pseudo-initiation complex dissociation was impaired by substitution of Arg99 and Arg112 (whose positive charges are important) and, to a lesser extent, of Arg116, Arg129, Arg133 and Arg147, while the dissociation of non-canonical 30S initiation complexes was preserved at wild-type levels in all 32 mutants. Stimulation of mRNA translation was reduced by mutations of Arg116, Arg129 and, to a lesser extent, of Arg99, Arg112 and Arg131 whereas inhibition of non-canonical mRNA translation was affected by substitutions of Arg99, Arg112, Arg168 and, to a lesser extent, Arg116, Arg129 and Arg131. Finally, repositioning the mRNA on the 30S subunit was affected weakly by mutations of Arg133, Arg131, Arg168, Arg147 and Arg129. Overall, the results define two active surfaces in IF3C, and indicate that the different functions of IF3 rely on different molecular mechanisms involving separate active sites.

Novel Structural and Functional Aspects of Translational Initiation Factor IF2

IF2 is the largest of the proteins involved in translation interacting directly with the ribosome and, along with the elongation factors EF-Tu and EF-G, belongs to the growing family of the GTP/GDP binding proteins which are involved in a large number of cellular regulatory functions. From the structural point of view, the best characterized examples of this group of proteins are H-ras p21 oncogene protein and the above-mentioned EF-Tu for which a refined crystal structure is available at 1.35 A (Pai et al., 1990) and at 2.6 A (Clark et al., 1990) resolution, respectively. The primary structure of IF2 revealed large sequence homologies with EF-Tu; however, these are restricted to the GTP/GDP binding motifs (Sacerdot et al., 1984) situated in the middle and in the N-terminal portion of the molecule of IF2 and EF-Tu, respectively.

Proteolysis of Bacillus stearothermophilus IF2 and specific protection by fMet-tRNA

Translation initiation factor IF2 from Bacillus steaiothermophilus (741 amino acids, M r 82,043) was subjected to trypsinolysis alone or in the presence of fMet‐tRNA. The initiator tRNA was found to protect very efficiently the Arg308‐Ala309 bond within the GTP binding site of IF2 and, more weakly, three bonds (Lys146‐Gln147, Lys154‐Glu155 and Arg519‐Ser520). The first two are located at the border between the non‐conserved, dispensable (for translation) N‐terminal portion and the conserved G‐domain of the protein, the third is located at the border between the G‐ and C‐domains. Since IF2 is known to interact with fMet‐tRNA through its protease‐resistant C‐ (carboxyl terminus) domain, the observed protection suggests that, upon binding or fMet‐tRNA, long‐distance tertiary interactions between the IF2 domains may take place.

The Function of Initiation Factors in Relation to mRNA—Ribosome Interaction and Regulation of Gene Expression

Initiation of translation in prokaryotes begins, in ≧90% of the cases, with an AUG triplet which offers the best basepairing with the anticodon CAU of the initiator tRNA; the wobbling triplets GUG, UUG and AUU are found more rarely with the latter being found in a single case (i.e. the infC gene which encodes translation initiation factor IF3) (Gren 1984). The reason for this degeneracy of the initiation triplet and its influence on the level of translational expression remain intriguing. In some cases changing the rare initiation triplet into the more common AUG results in moderate (Reddy et al. 1985; Khudyakov et al. 1988) to large (Brombach and Pon 1987) increases of expression. Inspection of the catalogue of genes starting with the rare triplets, however, seems to argue against the idea that these codons are used to attain a substantial reduction in the level of translation since this catalogue includes some of the most abundantly expressed genes in E. coli such as tufA (EF-Tu), hupB (HU1) as well as some ribosomal protein (r-protein) genes; furthermore, hupB is expressed at approximately the same level as hupA (HU2) and the r-protein genes beginning with GUG or UUG are expressed in amounts stoichiometrically equivalent to those of r-protein genes beginning with AUG.