Claudio Gualerzi

Identification, cloning, nucleotide sequence and chromosomal map location of hns, the structural gene for Escherichia coli DNA-binding protein H-NS

SummaryBeginning with a synthetic oligonucleotide probe derived from its amino acid sequence, we have identified, cloned and sequenced the hns gene encoding H-NS, an abundant Escherichia coli 15 kDa DNA-binding protein with a possible histone-like function. The amino acid sequence of the protein deduced from the nucleotide sequence is in full agreement with that determined for H-NS. By comparison of the restriction map of the cloned gene and of its neighboring regions with the physical map of E. coli K12 as well as by hybridization of the hns gene with restriction fragments derived from the total chromosome, we have located the hns gene oriented counterclockwise at 6.1 min on the E. coli chromosome, just before an IS30 insertion element.

Proteins from the prokaryotic nucleoid: The interaction between protein NS and DNA involves the oligomeric form of the protein and at least one Arg residue

The interaction of the prokaryotic DNA‐scaffolding proteins NS (from Escherichia coli and BS‐NS (from Bacillus stearothermophilus) with DNA has been investigated. Upon binding of NS to DNA, the resolution of its 400 MHz 1H‐NMR spectrum is lost, due to the broadening of all resonance lines. These effects are reversed by increasing the ionic strength. Since with increasing amounts of DNA added all resonances broaden progressively and simultaneously without prior selective loss of the spectral features due to the tertiary and quarternary structure of the protein, it is suggested that NS binds to DNA in the aggregated form (octameric?) and without gross alteration of its tertiary structure. By selective chemical modification of BS‐NS it was found that at least one Arg residue, located in the major hydrophilic, positively charged, conserved peptide of the protein (positions 51–70) is necessary for the interaction of BS‐NS with DNA.

Proteins from the prokaryotic nucleoid Interaction of nucleic acids with the 15 kDa Escherichia coli histone-like protein H-NS

The interaction between nucleic acids and Escherichia coli H‐NS, an abundant 15 kDa histone‐like protein, has been studied by affinity chromatography, nitrocellulose filtration and fluorescence spectroscopy. Intrinsic fluorescence studies showed that the single Trp residue of H‐NS (position 108) has a restricted mobility and is located within an hydrophobic region inaccessible to both anionic and cationic quenchers. Binding of H‐NS to nucleic acids, however, results in a change of the microenvironment of the Trp residue and fluorescence quenching; from the titration curves obtained with addition of increasing amounts of poly(dA)‐poly(dT) and poly(dC)‐poly(dG) it can be estimated that an H‐NS dimer in 1.5 × SSC binds DNA with an apparent K a−1.1 × 104 M−1· bp−1. H‐NS binds to double‐stranded DNA with a higher affinity than the more abundant histone‐like protein NS(HU) and, unlike NS, prefers double‐stranded to single‐stranded DNA and DNA to RNA; both monovalent and divalent cations are required for optimal binding.

Mechanism of protein biosynthesis in prokaryotic cells: Effect of initiation factor IF1 on the initial rate of 30 S initiation complex formation

To define the step at which translational initiation factor IF1 excercises its stimulation, initial rate kinetic analyses of 30 S initiation complex formation were carried out in the presence and absence of this factor. It was shown that, without affecting the affinity of the ribosomes either for the initiator tRNA or for the poly(AUG) used as template, IF1 increases approximately 2.5‐fold the limiting V maxof the ‘pre‐ternary complex’ ← ternary complex transition which represents the rate‐limiting step in 30 S initiation complex formation. This kinetic effect titrates with the 30 S ribosomal subunit which must therefore represent the target of IF1 action.To define the step at which translational initiation factor IF1 excercises its stimulation, initial rate kinetic analyses of 30 S initiation complex formation were carried out in the presence and absence of this factor. It was shown that, without affecting the affinity of the ribosomes either for the initiator tRNA or for the poly(AUG) used as template, IF1 increases approximately 2.5-fold the limiting Vmaxof the ‘pre-ternary complex’ ← ternary complex transition which represents the rate-limiting step in 30 S initiation complex formation. This kinetic effect titrates with the 30 S ribosomal subunit which must therefore represent the target of IF1 action.

The topographical localization of IF3 on Escherichia coli 30 S ribosomal subunits as a clue to its way of functioning

A problem of considerable interest concerns the topographical localization of the translational initiation factors on the ribosome. This is essential to improve our knowledge of the ribosomal topography and to gain a better insight into the physical and mechanistic aspects of the interactions between the factors and the ribosome. Several protein-RNA and protein-protein crosslinking studies dealt so far with this subject (reviews [ 1,2]). Here, we present results obtained on the protein neighborhood of IF3 by use of very mild reaction conditions. Taking into account the functional and structural properties of IF3, its topographical localization on the ribosome and its effect on the properties of other ribosomal proteins, we also present an hypothetical model which attempts to explain how the factor might function. some experiments 70 S ‘tight couples’ and ribosomal subunits derived from them were prepared essentially as in [4]. Initiation factor IF3 was purified [5], and labelled in vitro by either reaction with N-[ 3H] ethylmaleimide (NEM) or by reductive methylation with [ 14C] formaldehyde as detailed in [ 61. Poly(U)-dependent polyphenylalanine synthetic activity test for 30 S ribosomal subunits was performed as in [7].

Molecular cloning and sequence of theBacillus stearothermophilus translational initiation factor IF2 gene

SummaryThe structural gene for theBacillus stearothermophilus initiation factor IF2 was localized to a 6 kbHindIII restriction fragment by cross-hybridization with theSstI-SmaI fragment of theEscherichia coli infB gene. This fragment corresponds to the central region of the molecule containing the GTP-binding domain which is homologous inE. coli IF2, EF-Tu, EF-G and the humanras1 oncogene protein. After cloning into pACYC177, theHindIII fragment was further analysed by restriction mapping and cross-hybridization. A smaller (2.2 kb)SphI-HindIII fragment, which showed cross-hybridization, was subcloned into M13 phage and sequenced by the dideoxy chain-terminating method. This fragment was found to contain the entire IF2 gene except for the region coding for the N-terminus. This remaining region, coding for 45 amino acids, was located by homologous hybridization on an overlappingClaI-SstI fragment which was also subcloned and sequenced. Overall, theB. stearothermophilus IF2 gene codes for a protein of 742 amino acids (Mr=82,043) whose primary sequence displays extensive homology with the C-terminal two-thirds (but little or no homology with the N-terminal one-third) of the correspondingE. coli IF2 molecule. When cloned into an expression vector under the control of the λPL promoter, theB. stearothermophilus IF2 gene, reconstituted by ligation of the two separately cloned pieces, could be expressed at high levels inE. coli cells.

Escherichia coli 30S mutants lacking protein S20 are defective in translation initiation

The 30S ribosomal subunits derived from Escherichia coli TA114, a a temperature-sensitive mutant lacking ribosomal protein S20, were shown to be defective in two ways: (a) they have a reduced capacity for association with the 50S ribosomal subunit which results in the impairment of most of the functions requiring a coordinated interaction between the two subunits; (b) they are defective in functions which do not require their interaction with the large subunit (i.e., the formation of ternary complexes with aminocyl-tRNAs and templates, including the formation of 30S initiation complexes with fMet-tRNA and mRNA). The 30S (-S20) subunits seem to interact normally with both template and aminoacyl-tRNA individually, but appear to be impaired in the rate-limiting isomerization step leading to the formation of a codon-anticodon interaction in the P site.

Structure-function relationships in Escherichia coliinitiation factors: II. Elucidation of the primary structure of initiation factor IF-1

Initiation factor IF-1 is the smallest of the three initiation factors [l] and also the one whose functional role remains so far least clear [2,3]. In fact, while no specific function has so far been attributed to this factor, IF-1 seems to stimulate more or less all the activities of the other two initiation factors [2,3] through mechanism(s) which remain obscure. It has been reported that IF-1 binds to both 30 S ribosomal subunits, preferentially in the presence of IF-2 and IF-3 [4-61 and to 70 S ribosomes [4]. Binding to the 30 S subunit produces an increase in the affinity of these particles for initiation factor IF-2 [7]. IF-1 also stimulates the ribosome dissociation activity of IF-3 by increasing both ‘on’ and ‘off rate constants for the dissociation of 70 S ribosomes [8]. The reports that neither Bacillus stearothermophik [9] nor Caulobacter crescentus [lo] contain IF-l-like activities are puzzling. However, the most intriguing aspect of the IF-1 functioning concerns the mechanism by which it supposedly helps the recycling of IF-2 at the 70 S level [ 1 l-131 and the mechanism and the timing of IF-l binding and release from the ribosomes. Finally, although a number of ribosomal proteins neighbouring IF-1 on the 30 S ribosomal subunits have been identified by use of proteinprotein crosslinking reagents [5], nothing is known about the molecular nature of the interaction between IF-1 and ribosomes. In this paper, as the first step toward a better understanding of the function and structure-function

Structure-function relationship in Escherichia coli initiation factors: Environment of the Cys residue and evidence for a hydrophobic region in initiation factor IF3 by fluorescence and ESR spectroscopy

Abstract Escherichia coli translational initiation factor IF3 contains a single Cys residue at position 66 of its primary structure. The environment and the relevance of this residue for the biological activity of the factor has been investigated by the use of several -SH group reagents, some of them carrying ESR or fluorescent probes. It was found that: (a) The Cys residue in itself is not essential for activity since its modification with N -ethylmaleimide, iodoacetamide, fluorescein maleimide, and the spin-label tetramethylpiperidinoxyl maleimide resulted in little or no loss of biological activity. (b) The Cys residue is exposed and reactive in native unbound protein as well as in 30-S-bound IF3, although the rate, but not the extent, of reaction is somewhat affected by the state of the protein (faster in urea-denatured IF3 and slower in 30-S-bound IF3). (c) ESR spectroscopy of spin-labeled IF3 showed that the SH-bound probe is weakly immobilized and located in a hydrophilic environment in the native-free protein; upon binding to 30-S ribosomal subunits, however, the probe becomes more immobilized. High concentrations of salt reverse this effect, which is not observed upon binding of IF3 to the synthetic polynucleotide poly(AUG) or trinucleotide (AUG). (d) Formation of an adduct between the Cys residue and four different maleimide derivatives bearing hydrophobic fluorescent probes (e.g., anilinonaphthyl maleimide) results in the inactivation of the factor. Molecular dimensions and spectral properties suggest that the fluorescent probe reaches and becomes immobilized in a hydrophobic region (patch or pocket) situated within approximately 8 A of the SH group; energy transfer from Tyr residues also suggests that the hydrophobic region contains or is close to at least one Tyr residue, (e) The same hydrophobic region probably constitutes the primary binding region where the hydrophobic probe anilinonaphthylsulfonate (the chromophore of anilinonaphthyl maleimide) binds noncovalently to the IF3 molecule, producing an increase in fluorescence quantum yield and an inactivation similar to that caused by anilinonaphthyl maleimide. Taken together, we interpret these results to indicate that although the Cys residue is found in a hydrophilic environment and is not essential for ribosomal binding of the factor, it is located near the edge of the binding site, within a short distance of a hydrophobic region essential for activity.

Proteins from the Prokaryotic Nucleoid. Structural and Functional Characterization of the Escherichia coli DNA-Binding Proteins NS (HU) and H-NS

In spite of the commonplace notion that prokaryotes do not contain histones and that their DNA is not organized in chromatin, evidence is accumulating that bacterial DNA is indeed organized in nucleosomelike structures by histonelike proteins. Since the problem of the physical packaging and confinement of the genetic material can be regarded as a fundamental problem in all biological systems and in light of the underlying architectural unity of all living cells, it appears unlikely that completely different strategies have evolved to meet the same basic need. If the general strategies are the same, or at least, very similar, then it is possible that the differences existing between the pro- and eukaryotic systems may merely reflect differences in the tactics that each system must have perfected in order to harmonize its DNA-packaging mechanism with its specific physiological requirements.