Claude Lazdunski

Translation is a non-uniform process: Effect of tRNA availability on the rate of elongation of nascent polypeptide chains☆

We reported elsewhere (Varenne et al., 1982) that, during synthesis of a number of colicins in Escherichia coli, intermediate nascent chains of discrete sizes accumulated, suggesting a variable rate of translation. In this paper, a detailed analysis provides arguments that this phenomenon, at least for the proteins under study, is not related to aspects of messenger RNA such as secondary structure. It is linked to the difference in transfer RNA availability for the various codons. Experimental analysis of translation of other proteins in E. coli confirms that the main origin for the discontinuous translation in the polypeptide elongation cycle is the following. For a given codon, the stochastic search of the cognate ternary complex (aminoacyl-tRNA-EF-Tu-GTP) is the rate-limiting step in the elongation cycle: transpeptidation and translocation steps are much faster. The degree of slackening in ribosome movement is almost proportional to the inverse of tRNA concentrations. The verification of this model and its possible physiological significance are discussed.

The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity?

The outer membrane of gram-negative bacteria acts as a barrier against harmful lipophilic compounds and larger molecules unable to diffuse freely through the porins. However, outer membrane proteins together with the Tol-Pal and TonB systems have been exploited for the entry of macromolecules such as bacteriocins and phage DNA through the Escherichia coli cell envelope. The TonB system is involved in the active transport of iron siderophores and vitamin B12, while no more precise physiological role of the Tol-Pal system has yet been defined than its requirement for cell envelope integrity. These two systems, containing an energized inner membrane protein interacting with outer membrane proteins, share similarities.

Bacteriocins, microcins and lantibiotics

to the Microcin Session.- Escherichia coli genes regulating the production of microcins MCCB17 and MCCC7.- Uptake and mode of action of the peptide antibiotic microcin B17.- The structure and maturation pathway of microcin B17.- Bacteriocins of Gram-positive bacteria: an opinion regarding their nature, nomenclature and numbers.- Molecular properties of Lactobacillus bacteriocins.- Lactococcal bacteriocins: genetics and mode of action.- to the Lantibiotics session.- Lantibiotics : An overview and conformational studies on Gallidermin and Pep5.- Biosynthesis of the lantibiotic Pep5 and mode of action of type A lantibiotics.- Identification of genes involved in lantibiotic synthesis.- Pore forming bacteriocins.- In vivo properties of colicin A: channel activity and translocation.- Site-directed fluorescence spectroscopy as a tool to study the membrane insertion of colicin A.- Structure-function of the colicin E1 ion channel: voltage-driven translocation and gating of a tetra- (or hexa-) helix channel.- Voltage-dependent gating of colicin E1 channels in planar bilayers.- Immunity to colicins.- Immunity protein to pore forming colicins.- Specificity determinants for the interaction of colicin E9 with its immunity protein.- Structural studies on colicin E3 and its immunity protein.- Study of the import mechanisms of colicins through protein engineering and K+ efflux kinetics.- Import and export of colicin M.- TolA: structure, location and role in the uptake of colicins.- Domains of the Escherichia coli BtuB protein involved in outer membrane association and interaction with colicin translocation components.- A structure-function analysis of BtuB, the E.coli vitamin B12 outer membrane transport protein.- General introduction to the secretion of bacteriocins.- Functioning of the pCloDF13 encoded BRP.- Structure/function relationships in the signal sequence of the colicin A lysis protein.- The secretion of colicin V.- to the session on the evolution of bacteriocins.- Molecular evolution of E colicin plasmids with emphasis on the endonuclease types.- Immunity specificity and evolution of the nuclease-type E colicins.- Replicon evolution of ColE2-related plasmids.- Manuscripts of poster presentations.- Genetic determinants for microcin H47, an Escherichia coli chromosome-encoded antibiotic.- BLIS production in the genus Streptococcus.- A new Leuconostoc bacteriocin, Mesentericin Y105, bactericidal to Listeria monocytogenes.- Cloning and characterisation of a lysin gene from a Listeria bacteriophage.- Transformation of Enterococcus faecalis OGIX with the plasmid pMB2 encoding for the peptide antibiotic AS-48, by protoplast fusion and regeneration on calcium alginate.- NMR studies of lantibiotics: the three-dimensional structure of nisin in aqueous solution.- Localization and phenotypic expression of genes involved in the biosynthesis of the Lactococcus lactis subsp. lactis lantibiotic nisin.- Expression of nisin in Bacillus subtllis.- Tn5301, a Lactococcal transposon encoding genes for nisin biosynthesis.- Development of yeast inhibitory compounds for incorporation into silage inoculants.- The excC and excD genes of Escherichia coli K-12 encode the peptidoglycan-associated lipoprotein (PAL) and the TolQ protein, respectively.- Resistance and tolerance of bacteria to E colicins.- Construction and characterization of chimeric proteins between pyocins and colicin E3.- Colicins as anti-tumour drugs.- List of Participants.

Complete nucleotide sequence of the structural gene for colicin A, a gene translated at non-uniform rate*

The complete nucleotide sequence of the structural gene for colicin A has been established. This sequence consists of 1776 base-pairs. According to the predicted amino acid sequence, the colicin A polypeptide chain comprises 592 amino acids and has a molecular weight of 62,989. The amino-terminal part is rich in proline and glycine and accordingly secondary structure prediction indicates that this region (1 to 185) is beta-structured. The rest of the molecule (residues 186 to 592) is very rich in alpha-helix. An uncharged amino acid sequence of 48 residues is located in the C-terminal part of the molecule, which is involved in the membrane depolarization caused by colicin A. A similar region has been found in colicin E1, which has the same mode of action as colicin A. Three peptides of these bacteriocins were found to be homologous, but a comparison of the bacteriocin genes did not reveal any significant homology out of the corresponding regions. The codon usage of both genes, however, exhibits some similarity and is quite different from that of genes coding for highly or weakly expressed proteins of Escherichia coli.

The Tol/Pal system function requires an interaction between the C‐terminal domain of TolA and the N‐terminal domain of TolB

The Tol/Pal system of Escherichia coli is composed of the YbgC, TolQ, TolA, TolR, TolB, Pal and YbgF proteins. It is involved in maintaining the integrity of the outer membrane, and is required for the uptake of group A colicins and DNA of filamentous bacteriophages. To identify new interactions between the components of the Tol/Pal system and gain insight into the mechanism of colicin import, we performed a yeast two‐hybrid screen using the different components of the Tol/Pal system and colicin A. Using this system, we confirmed the already known interactions and identified several new interactions. TolB dimerizes and the periplasmic domain of TolA interacts with YbgF and TolB. Our results indicate that the central domain of TolA (TolAII) is sufficient to interact with YbgF, that the C‐terminal domain of TolA (TolAIII) is sufficient to interact with TolB, and that the amino terminal domain of TolB (D1) is sufficient to bind TolAIII. The TolA/TolB interaction was confirmed by cross‐linking experiments on purified proteins. Moreover, we show that the interaction between TolA and TolB is required for the uptake of colicin A and for the membrane integrity. These results demonstrate that the TolA/TolB interaction allows the formation of a trans‐envelope complex that brings the inner and outer membranes in close proximity.

Functional domains of colicin A

A large number of mutations which introduce deletions in colicin A have been constructed. The partially deleted colicin A proteins were purified and their activity in vivo (on sensitive cells) and in vitro (in planar lipid bilayers) was assayed. The receptor‐binding properties of each protein were also analysed. From these results, we suggest that the NH2‐terminal region of colicin A (residues 1 to 172) is involved in the translocation step through the outer membrane. The central region of colicin A (residues 173 to 336) contains the receptor‐binding domain. The COOH‐terminal domain (residues 389 to 592) carries the pore‐forming activity.

Distinct regions of the colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli

Group A colicins need proteins of the Escherichia coli envelope Tol complex (TolA, TolB, TolQ and TolR) to reach their cellular target. The N‐terminal domain of colicins is involved in the import process. The N‐terminal domains of colicins A and E1 have been shown to interact with TolA, and the N‐terminal domain of colicin E3 has been shown to interact with TolB. We found that a pentapeptide conserved in the N‐terminal domain of all group A colicins, the ‘TolA box’, was important for colicin A import but was not involved in the colicin A–TolA interaction. It was, however, involved in the colicin A–TolB interaction. The interactions of colicin A N‐terminal domain deletion mutants with TolA and TolB were investigated. Random mutagenesis was performed on a construct allowing the colicin A N‐terminal domain to be exported in the bacteria periplasm. This enabled us to select mutant protein domains unable to compete with the wild‐type domain of the entire colicin A for import into the cells. Our results demonstrate that different regions of the colicin A N‐terminal domain interact with TolA and TolB. The colicin A N‐terminal domain was also shown to form a trimeric complex with TolA and TolB.

Individual domains of colicins confer specificity in colicin uptake, in pore-properties and in immunity requirement☆

Six different hybrid colicins were constructed by recombining various domains of the two pore-forming colicins A and E1. These hybrid colicins were purified and their properties were studied. All of them were active against sensitive cells, although to varying degrees. From the results, one can conclude that: (1) the binding site of OmpF is located in the N-terminal domain of colicin A; (2) the OmpF, TolB and TolR dependence for translocation is also located in this domain; (3) the TolC dependence for colicin E1 is located in the N-terminal domain of colicin E1; (4) the 183 N-terminal amino acid residues of colicin E1 are sufficient to promote E1AA uptake and thus probably colicin E1 uptake; (5) there is an interaction between the central domain and C-terminal domain of colicin A; (6) the individual functioning of different domains in various hybrids suggests that domain interactions can be reconstituted in hybrids that are fully active, whereas in others that are much less active, non-proper domain interactions may interfere with translocation; (7) there is a specific recognition of the C-terminal domains of colicin A and colicin E1 by their respective immunity proteins.

Effect of distribution of unfavourable codons on the maximum rate of gene expression by an heterologous organism

We have analysed theoretically the effect of the relative position of unfavourable codons on the maximum level of synthesis of foreign proteins in E. coli. We predict that the occurrence of such codons scattered in the corresponding genes has little effect. In contrast, clustering (in our terminology indicating directly adjacent codons) of unfavourable codons is predicted to dramatically reduce the maximum level of protein synthesis. The context effect would explain the reduction of expression level for a chloramphenicol acetyl transferase gene modified by Robinson et al. (1984), which contains 4 contiguous unfavourable codons. As an example, we predict that due to the different downstream contexts of unfavourable codons in the alpha 1 and beta interferon genes, the maximum level of synthesis in E. coli for these proteins will be different.

Colicin import and pore formation: a system for studying protein transport across membranes?

Pore‐forming colicins are a family of protein toxins (Mr40–70kDa) produced by Escherichia coli and related bacteria. They are bactericidal by virtue of their ability to form ion channels in the inner membrane of target cells. They provide a useful means of studying questions such as toxin action, polypeptide translocation across and into membranes, voltage‐gated channels and receptor function. These colicins bind to a receptor in the outer membrane before being translocated across the cell envelope with the aid of helper proteins that belong to nutrient‐uptake systems and the so‐called‘Tol’proteins, the function of which has not yet been properly defined. A distinct domain appears to be associated with each of three steps (receptor binding, translocation and formation of voltage‐gated channels). The Tol‐dependent uptake pathway is described here. The structures and interactions of TolA, B, Q and R have by now been quite clearly defined. Transmembrane α‐helix interactions are required for the functional assembly of the E. coli Tol complex, which is preferentially located at contact sites between the inner and outer membranes. The number of colicin translocation sites is about 1000 per cell. The role and the involvement of the OmpF porin (with colicins A and N) have been described in a recent study on the structural and functional interactions of a colicin‐resistant mutant of OmpF. The X‐ray crystal structure of the channel‐forming fragment of colicin A and that of the entire colicin la have provided the basis for biophysical and site‐directed muta‐genesis studies. Thanks to this powerful combination, it has been established that the interaction with the receptor in the outer membrane leads to a very substantial conformational change, as a result of which the N‐terminal domains of colicins interact with the lumen of the OmpF pore and then with the C‐terminal domain of TolA. A molten globular conformation of colicins probably constitutes the intermediate translocation/insertion competent state. Once the pore has formed, the polypeptide chain spans the whole cell envelope. Three distinct steps occur in the last stage of the process: (i) fast binding of the C‐terminal domain to the outer face of the cytoplasmic membrane; (ii) a slow insertion of the polypeptide chain into the outer face of the inner membrane in the absence of Δψ and (iii) a profound reorganization of the helix association, triggered by the transmembrane potential and resulting in the formation of the colicin channel.