The hns (27 min) gene encoding the 15.4-kDa nucleoid protein H-NS was shown to belong to the cold shock regulon ofEscherichia coli, its expression being enhanced 3-to 4-fold during the growth lag that follows a shift from 37C to 100C. A 110-base-pair (bp) DNA fragment containing the promoter of hns fused to a promoterless cat gene (hns-cat fusion) conferred a similar cold shock response to the expression of chloramphenicol acetyltransferase (CAT) activity in vivo and in coupled transcription-translation systems prepared with extracts of cold-shocked cells. Extracts of the same cells produce a specific gel shift ofthe 110-bp DNA fragment and this fragment, immobilized on a solid support, specifically retains a single 7-kDa protein present only in cold-shocked cells that was found to be identical to F10.6 (CS7.4), the product of espA. This purified protein, which is homologous to human DNAbinding protein YB-1, recognizes some feature of the 110-bp promoter region of hns and acts as a cold shock transcriptional activator of this gene since it stimulates the expression of CAT activity and of cat transcription in in vitro systems programmed with plasmid DNA carrying the hns-cat fusion.Several bacterial proteins with DNA-binding property have been implicated in condensation of the chromosome and in organization of the prokaryotic nucleoid. The most abundant and best characterized of these are HU (NS) and H-NS (Hla) proteins (for reviews, see refs. 1-4). H-NS (136 residues) is a neutral, heat-stable, dimeric protein (5) that displays high affinity for curved DNA (6) and has been localized primarily in the nucleoid by immunoelectron microscopy (7). H-NS is encoded by hns, a gene that has been cloned and characterized in Escherichia coli (8) as well as in other Enterobacteriaceae (9) and that has been ultimately mapped at 27 min on the E. coli chromosome (4). Mutations in hns were found to increase bacteriophage Mu-specific transcription and to increase dramatically the mini-Mu transposition rate (10). Several mutations causing a number of apparently unrelated phenotypes have been found to be allelic with hns. These are bglY, which activates expression of the cryptic bgl operon (11) and causes large chromosomal deletions (12); pilG, which greatly increases the site-specific DNA inversion responsible for fimbrial phase variation (13); drdX, which induces expression of the pilus adhesin (pap) genes at low temperature in uropathogenic strains (14); cur-], causing a conditional uracil requirement (15); osmZ, altering the osmoregulated expression of proU operon (16, 17); and virR, which affects the temperature-regulated expression of plasmid-borne virulence genes in Shigella flexneri (18).A common basis for these pleiotropic effects could be an altered compaction and fluidity of the genome (12) leading to (or coupled with) a transcriptional derepression of some genes.An important role of H-NS in controlling the compaction of the nucleoid is also suggested by the observation that the nucleoids undergo a dramatic conden...

Translational initiation factor 2 (IF2) is a guanine nucleotidebinding protein that can bind guanosine 3 ,5 -(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands. ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis.fast kinetics ͉ GTP ͉ translation regulation ͉ nutritional stress I nitiation factor 2 (IF2) is the only initiation factor that is ribosome-bound throughout the entire translation initiation pathway, participating initially in the formation of the 30S initiation complex (30SIC) and subsequently in the assembly of the 70S initiation complex (70SIC), a process that ultimately results in formation of the first peptide bond (initiation dipeptide) and generates the first ribosomal pretranslocation complex (for reviews, see refs. 1-5). Thus, it could be predicted that IF2 functions are accompanied͞modulated by conformational changes that could be either consequence or cause of the interactions of IF2 with its various ligands (30S and 50S ribosomal subunits, fMet-tRNA, GTP, GDP⅐Pi, and GDP). Crystallographic (6) and NMR (7) studies have, in fact, shown that, depending upon the nature of their ligand (i.e., GTP or GDP), aIF5B, the archaeal homologue of bacterial IF2, as well as isolated IF2G2, the G domain of IF2, undergo large structural changes. On the other hand, chemical probing (8) and cryo-EM (9, 10) have clearly shown that several conformational changes of the factor occur during the early, middle, and late events of translation initiation.Bacterial cells growing under optimal nutritional conditions contain a high (Ͼ1 mM) concentration of GTP and a vanishingly low level of GDP. Thus, IF2 is expected to exist and to bind the 30S subunit almost exclusively in the GTP form, because it displays similar affinity for GTP and GDP, both K d s being in the 10-to 100-M range (11). The IF2⅐GTP was shown to have a higher affinity for the 30S ribosomal subunit than either IF2⅐GDP or free IF2 (11). The adjustment of fMet-tRNA in the P site (ref. 12 and refs. therein) and the release of IF2 from 70SIC (13-15) have been attributed to the IF2-dependent GTP hydrolysis, which is very rapidly triggered by th...

Initiation factor IF3 contains two domains separated by a¯exible linker. While the isolated N-domain displayed neither af®nity for ribosomes nor a detectable function, the isolated C-domain, added in amounts compensating for its reduced af®nity 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 pseudoinitiation 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¯uctu-ating 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 ®delity function does not entail its direct contact with these structures.

The 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-NS delta 12) 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-NS delta 12, 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.

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