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.

Late events in translation initiation. Adjustment of fMet-tRNA in the ribosomal P-site.

The requirements for the adjustment of fMet-tRNA in the ribosomal P-site have been analyzed by studying the formation of fMet-puromycin in a Bacillus stearothermophilus system. The binding of fMet-tRNA to the 30 S ribosomal subunit is not drastically affected by the omission of GTP, mRNA, mRNA and GTP, or by replacing GTP with GTP analogues. The adjustment of fMet-tRNA in the P site has stricter requirements and fMet-puromycin formation occurred at its maximum rate and extent when fMet-tRNA was bound to 30 S subunits programmed with the AUG triplet or with an mRNA in the presence of GTP. Neither GTP nor the mRNA, however, were found to be essential. Omission of GTP caused only a slight reduction in the rate of fMet-puromycin formation without a significant change of the activation energy, while omission of the template resulted in a requirement for a higher activation energy. In the absence of both GTP and template, however, essentially no fMet-puromycin was formed, indicating that these components cooperate in the adjustment of the initiator tRNA in the P-site. The contribution of various structural elements of the mRNA in determining this adjustment was investigated. It was found that the codon-anticodon interaction and the filling of the ribosomal mRNA channel with a polyribonucleotide are necessary (but not sufficient singly) for the correct orientation of the initiator tRNA in the absence of GTP. The nature of the initiation triplet and the occurrence and/or the strength of the Shine-Dalgarno interaction were also found to contribute to the orientation of the bound fMet-tRNA.

Structure and function of bacterial initiation factors.

Bacteria require three initiation factors, IF1, IF2 and IF3, to start protein synthesis. In the last few years the elucidation of both structural and mechanistic aspects pertaining to these proteins has made substantial progress. In this article we outline the translation initiation process in bacteria and review these recent developments giving a summary of the main features of the structure and function of the initiation factors.

Ribosomal localization of the mRNA in the 30S initiation complex as revealed by UV crosslinking.

Translation initiation complexes consisting of 30S ribosomal subunits, 32P-labelled mRNA (002 mRNA), fMet-tRNA and the three initiation factors were subjected to UV-crosslinking to determine the protein and rRNA neighbors of the bound mRNA by immunochemical methods and by nucleic acid hybridization techniques. The mRNA was found to be crosslinked to a specific region of the 16S rRNA spanning from nucleotide 418 to 615 and to ribosomal proteins S1 and S21 (the main targets), S3, S10, S12 and S14; a low level of crosslinking was also detected with S2, S7, S13, S18 and S19.

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 exercises 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 Vmax of 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 NH2-terminal cleavage of Escherichia coli translational initiation factor IF3. A mechanism to control the intracellular level of the factor?

A short form of Escherichia coli translational initiation factor IF3, repeatedly found both in vivo and in vitro, lacking the positively charged N-terminal hexapeptide has been produced by mild trypsinization. The properties of this short form of IF3 have been studied. Compared to the long native form of the factor, the shortened IF3 displays a markedly decreased thermal stability and affinity for the 30 S ribosomal subunit, as well as a reduced biological activity in protein synthesis. Following the loss of the N-terminal hexapeptide, a second peptide bond (Lys-90-Val-91) becomes easily accessible to proteolytic attack suggesting that formation of the short IF3 may be the first step in the physiological degradation of the factor.

Structure-function relationship in Escherichia coli translational initiation factors. Characterization of IF1 by high-resolution 1H-NMR spectroscopy.

Escherichia coli translational initiation factor IF1 was studied by 1H-NMR spectroscopy at 400 MHz. IF1 displays a very well resolved spectrum in both aromatic and aliphatic regions. Other spectral characteristics include relatively narrow resonance lines and lack of relevant cross-relaxation phenomena. The resonances of the aromatic residues, in particular of the two His and two Tyr, were assigned by selective chemical modifications and spectroscopic techniques to individual residues in the protein sequence. The relative mobility of various residues of IF1 has been evaluated on the basis of the spin-lattice relaxation times which are rather short and homogeneous. Overall the factor appears to have a complex secondary and tertiary structure and to be a flexible protein whose residues have a high degree of internal mobility.

Mechanism of translational initiation in prokaryotes. Evidence for a direct effect of IF2 on the activity of the 30 S ribosomal subunit.

Initiation factor IF2 from either Escherichia coli or Bacillus stearothermophilus was found to possess the previously undetected property of stimulating the template-dependent ribosomal binding of aminoacyl-tRNAs with free alpha-NH2 groups. IF1, which had no detectable activity alone, was found to stimulate the activity of E. coli IF2 and, to a lesser extent, that of B. stearothermophilus IF2. Since in the absence of ribosomes not even a weak interaction between the two IF2 molecules and the aminoacyl-tRNAs was detected, the present findings indicate that IF2 can act at the ribosomal level stimulating aminoacyl-tRNA binding without prior formation of a binary complex with the aminoacyl-tRNA. IF2 does not appear to open or strengthen a weak A-site binding, but rather to enhance aminoacyl-tRNA binding to a 30 S site equivalent to the P-site by slowing down the rate of aminoacyl-tRNA dissociation from ribosomes.

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

Translation initiation factor IF2 from Bacillus stearothermophilus (741 amino acids, Mr 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 of fMet-tRNA, long-distance tertiary interactions between the IF2 domains may take place.

Proteolysis of Bacillus stearothermophilus IF2 and specific protection by GTP.

Translation initiation factor IF2 from Bacillus stearothermophilus (741 amino acids, Mr = 82,043) was subjected to trypsinolysis alone or in the presence of GTP. Following electroblotting and automated amino acid sequencing of the resulting peptides, the location and the sequential order of the main cleavage sites were identified. Trypsinolysis of IF2 ultimately generates two compact domains: a 24.5 kDa C-terminal fragment and a 40 kDa G-fragment which is obtained only in the presence of GTP which strongly protects a cleavage site within the GTP binding domain.

Modulation of ribosomal recruitment to 5'-terminal start codons by translation initiation factors IF2 and IF3.

Sequence determinants and structural features of the RNA govern mRNA-ribosome interaction in bacteria. However, ribosomal recruitment to leaderless mRNAs, which start directly with the AUG start codon and do not bear a Shine-Dalgarno sequence like canonical mRNAs, does not appear to rely on 16S rRNA-mRNA interactions. Here, we have studied the effects of translation initiation factors IF2 and IF3 on 30S initiation at a 5'-terminal AUG and at a competing downstream canonical ribosome binding site. We show that IF2 affects the forward kinetics of 30S initiation complex formation at the 5'-terminal AUG as well as the stability of these complexes. Moreover, the IF2:IF3 molar ratio was found to play a decisive role in translation initiation of a leaderless mRNA both in vitro and in vivo indicating that the translational efficiency of an mRNA is not only intrinsically determined but can be altered depending on the availability of components of the translational machinery.

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 x SSC binds DNA with an apparent Ka approximately equal to 1.1 x 10(4) 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.

The fMet-tRNA binding domain of translational initiation factor IF2: role and environment of its two Cys residues.

Mutations of the cysteines (positions 668 and 714) were generated in the IF2 C domain of Bacillus stearothermophilus translation initiation factor IF2. The corresponding proteins were characterized functionally and structurally. Most (yet not all) amino acid replacements at both positions resulted in severe reduction of the fMet-tRNA binding activity of IF2 C without grossly altering its structure. Our work demonstrates that: (a) both Cys residues are buried within an hydrophobic core and not accessible to protonation or chemical substitution, (b) neither Cys is functionally essential and (c) both Cys residues are located near the active site, probably without participating directly in fMet-tRNA binding.

Interaction of fMet-tRNA(fMet) with the C-terminal domain of translational initiation factor IF2 from Bacillus stearothermophilus.

Analytical ultracentrifugation studies indicated that the C-terminal domains of IF2 comprising amino acid residues 520-741 (IF2 C) and 632-741 (IF2 C-2) bind fMet-tRNA with similar affinities (K(d) at 25 degrees C equal to 0.27 and 0.23 microM, respectively). Complex formation between fMet-tRNA(fMet) and IF2 C or IF2 C-2 is accompanied by barely detectable spectral changes as demonstrated by a comparison of the Raman spectra of the complexes with the calculated sum of the spectra of the individual components. These results and the temperature dependence of the K(d) of the protein-RNA complexes indicate that complex formation is not accompanied by obvious conformational changes of the components, and possibly depends on a rather small binding site comprising only a few interacting residues of both components.

Ribosome-mRNA contact sites at different stages of translation initiation as revealed by cross-linking of model mRNAs.

Two model mRNAs, one with and one without the Shine-Dalgarno (SD) sequence, were bound to Escherichia coli 30S ribosomal subunits in the presence and absence of initiation factors and initiator tRNA and then cross-linked by diepoxybutane. The distribution of the cross-linked mRNA among rRNA and ribosomal proteins (r-proteins) and the extent to which individual r-proteins react was found to be affected by the presence or absence of the SD sequence and by the initiation factors and initiator tRNA. The results are consistent with the hypothesis that the position of the 30S-bound mRNA is shifted under the influence of the initiation factors and fMet-tRNA from a stand-by position towards a second site where the decoding of the initiation triplet by the initiator tRNA occurs.

Interaction of the main cold shock protein CS7.4 (CspA) of Escherichia coli with the promoter region of hns.

Escherichia coli protein CS7.4 (CspA), homologous to the class of eukaryotic Y-box DNA-binding proteins, is a cold shock transcriptional activator of at least two genes, hns and gyrA. It was demonstrated that all or nearly all the elements necessary for the stimulation of hns transcription by CS7.4 protein are located in the proximal 110 bp DNA fragment of this gene with no additional elements being present in a longer fragment (660 bp) extending further upstream from the hns promoter. Protein CS7.4 bound strongly to the 110 bp segment of the hns promoter in crude extracts of cold shocked cells, but the purified protein displayed a weak interaction with the same DNA fragment. Purified CS7.4 protein also caused increased or decreased accessibility to DNase I at different sites of the 110 bp fragment of hns but the majority of these effects was seen only in the presence of RNA polymerase. Since gel shift experiments showed that protein CS7.4 stimulated the binding of RNA polymerase to the promoter of hns and since it is known that there are similarities between CS7.4 and ssDNA-binding proteins, we suggest that formation of the open complex by the RNA polymerase or protein-protein contacts between CS7.4 and the RNA polymerase are prerequisites for and/or the effects of the interaction of CS7.4 with its DNA target. The presence of a conserved CCAAT element in the hns promoter region, on the other hand, was found not to be stringently required for cold shock activation since expression of E coli of an hns-cat fusion containing the Proteus vulgaris hns promoter lacking a CCAAT box increased over four-fold after cold shock.

Mutagenesis of the downstream region of the Escherichia coli hns promoter.

The promoter of hns, the structural gene for the abundant nucleoid-associated protein H-NS of Escherichia coli, contains, downstream of the initiation site, two four bp-long 'CG clamps', one of which overlaps the potential target sequence (CCAAT) of CspA, the cold-shock transcriptional enhancer of this gene. To establish the role of these potential regulatory signals during the cold-shock activation of hns, the CCCCAAT sequence has been subjected to mutagenesis, weakening the strength of the CG clamp and scrambling or inverting the CCAAT sequence. The resulting mutated hns promoters were placed in front of a reporter gene (cat) and their activity was studied in cells subjected to cold-shock under conditions where the increase in the concentration of CspA is either large or small. Our results allow us to conclude that although not essential, the CCCCAAT sequence, mainly due to the presence of the CG clamp, may play an important role in the CspA-mediated regulation of hns expression at both transcriptional and translational levels.

Relationship between size of mRNA ribosomal binding site and initiation factor function.

The rate and the extent of the binding of initiator fMet-tRNA(fMet) to 30S ribosomal subunits in the presence of IF1, IF2 and GTP is either inhibited or slightly stimulated by the presence of IF3 depending on whether the initiation triplet AUG or the polynucleotide poly(AUG) is used as template. To determine the length of the template required for the transition from the AUG- to the poly(AUG)-type of behavior in the presence of IF3, the ribosomal binding of fMet-tRNA was studied in response to AUG triplets extended on either the 5'- or the 3'-side by stretches of homo-oligonucleotides of different lengths. When the binding of fMet-tRNA was studied at equilibrium it was found that IF3 no longer inhibits the amount of ternary complex formed if AUG is extended either 10 nucleotides on the 5'- or 35-40 nucleotides on the 3'-side. When the initial rate of ternary complex formation is considered, shorter extensions (4 nucleotides on the 5'-side or 20-30 nucleotides on the 3'-side) are sufficient to elicit a substantial stimulation by IF3. These results are discussed in relation to the mechanism of action of the initiation factors in the selection of the initiation region of the mRNA by ribosomes.

Escherichia coli DNA-binding protein H-NS is localized in the nucleoid.

Electron microscope localization of the 15.4-kDa DNA-binding protein H-NS was carried out in Escherichia coli cells subjected to cryosubstitution followed by immuno-labelling with the protein A/gold technique. Three types of E. coli cells were used: (1) "normal" cells growing exponentially at 37 degrees C; (2) "cold-shocked" cells two hours after the shift from 37 degrees C to 10 degrees C; and (3) cells in which an expression vector had been induced to overproduce H-NS. The results clearly indicate that in all 3 cases, the vast majority of the molecules reacting with anti-H-NS antibodies are localized within the nucleoid and at the border between the nucleoid and the ribosome-rich cytoplasm, which supports the premise that H-NS is implicated in the condensation of the nucleoid.

Mutagenesis of the cyanobacterium Spirulina platensis by UV and nitrosoguanidine treatment.

The production of Spirulina platensis cells resistant to 8-azaguanine or beta-(2-thienyl)-DL-alanine following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation is described. The conditions for the mutagenesis were determined by monitoring cell viability and the appearance of the two types of mutants as a function of the stage of growth of the tricomes and the length and the conditions of the treatment. The optimal conditions for UV and MNNG mutagenesis were found to be 1-3 min irradiation and 30 min incubation with 50 micrograms MNNG/ml of tricomes derived from cultures entering stationary phase sonicated for 10 s and 5 s respectively. Under these conditions beta-(2-thienyl)-DL-alanine-resistant mutants appeared at a frequency greater than or equal to 10(-4) and greater than or equal to 10(-5) following UV- and MNNG-mutagenesis, respectively. Mutants resistant to 8-azaguanine were found at a frequency approx. 10(-5) only after MNNG mutagenesis. A few chlorate-resistant mutants were also obtained following UV treatment.