Membrane Protein Production in Escherichia coli: Protocols and Rules.

Despite recent progresses in the use of eukaryotic expression system, production of membrane proteins for structural studies still relies on microbial expression systems. In this review, we provide protocols to achieve high level expression of membrane proteins in Escherichia coli, especially using the T7 RNA polymerase based expression system. From the design of the construct, the choice of the appropriate vector-host combination, the assessment of the bacterial fitness, to the selection of bacterial mutant adapted to the production of the target membrane protein, the chapter covers all necessary methods for a rapid optimization of a specific target membrane protein. In addition, we provide a protocol for membrane protein solubilization based on our recent analysis of the Protein Data Bank.

[Glomerulopathy associated with lecithin-cholesterol-acyltransferase deficiency: A case report and literature review]. / Glomérulopathie associée à un déficit en lécithine-cholestérol-acyltransférase : rapport de cas et revue de la littérature.

Glomerulopathy associated with lecithin-cholesterol-acyltransferase deficiency (LCAT) is a rare automosal recessive disease. Acquired LCAT deficiency due to inhibitory autoantibodies against LCAT are also described. This disease is induced by systemic deposits related to a lipid metabolism disorder and lead to multi-organ involvement including renal involvement. Lipid profile usually shows variable cholesterol levels but very low HDL levels. Here we describe the case of a 33-year-old man presenting a nephrotic syndrome associated with moderate renal insufficiency for which the pathological analysis allowed to guide towards the diagnosis of LCAT deficiency. Laboratory and genetic data confirmed this diagnosis. Familial history and lipid profile abnormalities are important in the identification of this disease.

A novel regulation mechanism of the T7 RNA polymerase based expression system improves overproduction and folding of membrane proteins.

Membrane protein (MP) overproduction is one of the major bottlenecks in structural genomics and biotechnology. Despite the emergence of eukaryotic expression systems, bacteria remain a cost effective and powerful tool for protein production. The T7 RNA polymerase (T7RNAP)-based expression system is a successful and efficient expression system, which achieves high-level production of proteins. However some foreign MPs require a fine-tuning of their expression to minimize the toxicity associated with their production. Here we report a novel regulation mechanism for the T7 expression system. We have isolated two bacterial hosts, namely C44(DE3) and C45(DE3), harboring a stop codon in the T7RNAP gene, whose translation is under the control of the basal nonsense suppressive activity of the BL21(DE3) host. Evaluation of hosts with superfolder green fluorescent protein (sfGFP) revealed an unprecedented tighter control of transgene expression with a marked accumulation of the recombinant protein during stationary phase. Analysis of a collection of twenty MP fused to GFP showed an improved production yield and quality of several bacterial MPs and of one human monotopic MP. These mutant hosts are complementary to the other existing T7 hosts and will increase the versatility of the T7 expression system.

Membrane Protein Production in Escherichia coli: Protocols and Rules.

Functional and structural studies on membrane proteins are limited by the difficulty to produce them in large amount and in a functional state. In this review, we provide protocols to achieve high-level expression of membrane proteins in Escherichia coli. The T7 RNA polymerase-based expression system is presented in detail and protocols to assess and improve its efficiency are discussed. Protocols to isolate either membrane or inclusion bodies and to perform an initial qualitative test to assess the solubility of the recombinant protein are also included.

Resonance assignment of the ribosome binding domain of E. coli ribosomal protein S1.

Ribosomal protein S1 is an essential actor for protein synthesis in Escherichia coli. It is involved in mRNA recruitment by the 30S ribosomal subunit and recognition of the correct start codon during translation initiation. E. coli S1 is a modular protein that contains six repeats of an S1 motif, which have distinct functions despite structural homology. Whereas the three central repeats have been shown to be involved in mRNA recognition, the two first repeats that constitute the N-terminal domain of S1 are responsible for binding to the 30S subunit. Here we report the almost complete (1)H, (13)C and (15)N resonance assignment of two fragments of the 30S binding region of S1. The first fragment comprises only the first repeat. The second corresponds to the entire ribosome binding domain. Since S1 is absent from all high resolution X-ray structures of prokaryotic ribosomes, these data provide a first step towards atomic level structural characterization of this domain by NMR. Chemical shift analysis of the first repeat provides evidence for structural divergence from the canonical OB-fold of an S1 motif. In contrast the second domain displays the expected topology for an S1 motif, which rationalizes the functional specialization of the two subdomains.

Bacteriophage T4 polynucleotide kinase triggers degradation of mRNAs.

The bacteriophage T4-encoded RegB endoribonuclease is produced during the early stage of phage development and targets mostly (but not exclusively) the Shine-Dalgarno sequences of early genes. In this work, we show that the degradation of RegB-cleaved mRNAs depends on a functional T4 polynucleotide kinase/phosphatase (PNK). The 5'-OH produced by RegB cleavage is phosphorylated by the kinase activity of PNK. This modification allows host RNases G and E, with activity that is strongly stimulated by 5'-monophosphate termini, to attack mRNAs from the 5'-end, causing their destabilization. The PNK-dependent pathway of degradation becomes effective 5 min postinfection, consistent with our finding that several minutes are required for PNK to accumulate after infection. Our work emphasizes the importance of the nature of the 5' terminus for mRNA stability and depicts a pathway of mRNA degradation with 5'- to 3'-polarity in cells devoid of 5'-3' exonucleases. It also ascribes a role for T4 PNK during normal phage development.

Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation.

Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10-15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC) and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit); and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes) of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context.

Probing the relationship between Gram-negative and Gram-positive S1 proteins by sequence analysis.

Escherichia coli ribosomal protein S1 is required for the translation initiation of messenger RNAs, in particular when their Shine-Dalgarno sequence is degenerated. Closely related forms of the protein, composed of the same number of domains (six), are found in all Gram-negative bacteria. More distant proteins, generally formed of fewer domains, have been identified, by sequence similarities, in Gram-positive bacteria and are also termed 'S1 proteins'. However in the absence of functional information, it is generally difficult to ascertain their relationship with Gram-negative S1. In this article, we report the solution structure of the fourth and sixth domains of the E. coli protein S1 and show that it is possible to characterize their beta-barrel by a consensus sequence that allows a precise identification of all domains in Gram-negative and Gram-positive S1 proteins. In addition, we show that it is possible to discriminate between five domain types corresponding to the domains 1, 2, 3, 4-5 and 6 of E. coli S1 on the basis of their sequence. This enabled us to identify the nature of the domains present in Gram-positive proteins and, subsequently, to probe the filiations between all forms of S1.

RNA processing and decay in bacteriophage T4.

Bacteriophage T4 is the archetype of virulent phage. It has evolved very efficient strategies to subvert host functions to its benefit and to impose the expression of its genome. T4 utilizes a combination of host and phage-encoded RNases and factors to degrade its mRNAs in a stage-dependent manner. The host endonuclease RNase E is used throughout the phage development. The sequence-specific, T4-encoded RegB endoribonuclease functions in association with the ribosomal protein S1 to functionally inactivate early transcripts and expedite their degradation. T4 polynucleotide kinase plays a role in this process. Later, the viral factor Dmd protects middle and late mRNAs from degradation by the host RNase LS. T4 codes for a set of eight tRNAs and two small, stable RNA of unknown function that may contribute to phage virulence. Their maturation is assured by host enzymes, but one phage factor, Cef, is required for the biogenesis of some of them. The tRNA gene cluster also codes for a homing DNA endonuclease, SegB, responsible for spreading the tRNA genes to other T4-related phage.

[Dialin: infection surveillance network for haemodialysis patients. First results]. / Présentation de Dialin : réseau de surveillance des infections chez les patients hémodialysés en centre. Premiers résultats.

AIM AND BACKGROUND: To show results of the first year of an infection surveillance network for haemodialysis patients (Dialin). In order to improve the security and quality of care, six haemodialysis centers have organized an infection watching network. The purpose of the network is to compare of the watching results between centers. This comparison includes vascular access infection (VAI), bacteraemia and C viral hepatitis. The heterogeneous pattern has been also taken into account. SURVEY TYPE: Multicenter prospective permanent survey. POPULATION: Six hundred and sixty-four haemodialyzed chronic patients, followed during one year (2005), in six voluntary haemodialysis centers. This survey has based on 71,688 treatment sessions corresponding to 6257.5 months of haemodialysis (HM). METHODS: As with the heterogeneity among centers, the acquired infection standardized ratios (observed/expected) (AISR) and 95% confidence interval are computed with Cox model which includes confounding factors found in literature or in the preliminary stage of the survey. RESULTS: VAI crude rate was 0.47 per 100HM, 0.10 per 1000 native fistulae utilisation days, 0.45 per 1000 days of prosthetic graft utilisation and 0.44 per 1000 days of catheter utilisation. Bacteraemia crude incidence rate was 0.69 per 100HM, 0.02 per 1000 days of native fistulae utilisation, 0.00 per 1000 days of prosthetic graft utilisation and 0.39 per 1000 days of catheter utilisation. No new case of C viral hepatitis was found. Prevalence rate at the beginning of the survey was 5.3% (35 over 664). Two centers had a significantly high AISR for VAI and two centers had a significantly low AISR for VAI. One center had a significantly high AISR for bacteraemia and one center had a significantly low AISR for bacteraemia. CONCLUSIONS: The first year of Dialin running demonstrates the importance of standardised surveillance method in VAI and bacteraemia surveillance but not for viral hepatitis.

S1 ribosomal protein functions in translation initiation and ribonuclease RegB activation are mediated by similar RNA-protein interactions: an NMR and SAXS analysis.

The ribosomal protein S1, in Escherichia coli, is necessary for the recognition by the ribosome of the translation initiation codon of most messenger RNAs. It also participates in other functions. In particular, it stimulates the T4 endoribonuclease RegB, which inactivates some of the phage mRNAs, when their translation is no longer required, by cleaving them in the middle of their Shine-Dalgarno sequence. In each function, S1 seems to target very different RNAs, which led to the hypothesis that it possesses different RNA-binding sites. We previously demonstrated that the ability of S1 to activate RegB is carried by a fragment of the protein formed of three consecutive domains (domains D3, D4, and D5). The same fragment plays a central role in all other functions. We analyzed its structural organization and its interactions with three RNAs: two RegB substrates and a translation initiation region. We show that these three RNAs bind the same area of the protein through a set of systematic (common to the three RNAs) and specific (RNA-dependent) interactions. We also show that, in the absence of RNA, the D4 and D5 domains are associated, whereas the D3 and D4 domains are in equilibrium between open (noninteracting) and closed (weakly interacting) forms and that RNA binding induces a structural reorganization of the fragment. All of these results suggest that the ability of S1 to recognize different RNAs results from a high adaptability of both its structure and its binding surface.

Structural and functional studies of RegB, a new member of a family of sequence-specific ribonucleases involved in mRNA inactivation on the ribosome.

The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome.

Activation of RegB endoribonuclease by S1 ribosomal protein requires an 11 nt conserved sequence.

The T4 RegB endoribonuclease cleaves specifically in the middle of the -GGAG- sequence, leading to inactivation and degradation of early phage mRNAs. In vitro, RegB activity is very weak but can be enhanced 10- to 100-fold by the Escherichia coli ribosomal protein S1. Not all RNAs carrying the GGAG motif are cleaved by RegB, suggesting that additional information is required to obtain a complete RegB target site. In this work, we find that in the presence of S1, the RegB target site is an 11 nt long single-stranded RNA carrying the 100% conserved GGA triplet at the 5' end and a degenerate, A-rich, consensus sequence immediately downstream. Our data support the notion that RegB alone recognizes only the trinucleotide GGA, which it cleaves very inefficiently, and that stimulation of RegB activity by S1 depends on the nucleotide immediately 3' to -GGA-.

Phage T4 early promoters are resistant to inhibition by the anti-sigma factor AsiA.

Phage T4 early promoters are transcribed in vivo and in vitro by the Escherichia coli RNA polymerase holoenzyme Esigma(70). We studied in vitro the effects of the T4 anti-sigma(70) factor AsiA on the activity of several T4 early promoters. In single-round transcription, promoters motB, denV, mrh.2, motA wild type and UP element-deleted motA are strongly resistant to inhibition by AsiA. The alpha-C-terminal domain of Esigma(70) is crucial to this resistance. DNase I footprinting of Esigma(70) and Esigma(70)AsiA on motA and mrh.2 shows extended contacts between the holoenzyme with or without AsiA and upstream regions of these promoters. A TG --> TC mutation of the extended -10 motif in the motA UP element-deleted promoter strongly increases susceptibility to inhibition by AsiA, but has no effect on the motA wild-type promoter: either the UP element or the extended -10 site confers resistance to AsiA. Potassium permanganate reactivity shows that the two structure elements are not equivalent: with AsiA, the motA UP element-deleted promoter opens more slowly whereas the motA TC promoter opens like the wild type. Changes in UV laser photoreactivity at position +4 on variants of motA reveal an analogous distinction in the roles of the extended -10 and UP promoter elements.

First structural investigation of the restriction ribonuclease RegB: NMR spectroscopic conditions, 13C/15N double-isotopic labelling and two-dimensional heteronuclear spectra.

The bacteriophage T4 genome-encoded ribonuclease RegB is the unique well-defined restriction endoribonuclease. This protein cleaves with an almost absolute specificity its RNA substrate in the middle of the GGAG tetranucleotide mainly found in the Shine-Dalgarno sequence (required for the prokaryotic initiation of the translation). This protein has no significant homology to any known ribonuclease and its structure has never been investigated. The extreme toxicity of this ribonuclease prevents the expression of large quantities for structural studies. Here, we show that the toxicity of RegB can be bypassed by using the RegB H48A point mutant and explain why resolving the structure of this mutant is relevant. For nuclear magnetic resonance (NMR) purposes, we report the preparation of highly pure (13)C/(15)N double-labelled 1.2mM samples of RegB H48A using a high yield expression procedure in minimal medium (30 mg/L). We also present a set of solution conditions that maintain the concentrated samples of this protein stable for long periods at the NMR-required temperature. Finally, we present the first (1)H/(15)N and (1)H/(13)C two-dimensional NMR spectra of RegB H48A. These spectra show that the protein is folded and that the full structural analysis of RegB by NMR is feasible.

New system for positive selection of recombinant plasmids and dual expression in yeast and bacteria based on the restriction ribonuclease RegB.

By coupling the toxic restriction endoribonuclease RegB, from the bacteriophage T4, to the prokaryotic T7 and the eukaryotic GAL1 promoters, we constructed a two-function plasmid called pTOXR-1. This plasmid is a zero-background cloning vector. It allows an efficient positive selection of recombinant plasmids without the need to completely digest, dephosphorylate, or purify the vector prior to the ligation step. The pTOXR-1 positive selection system requires no special Escherichia coli strains, no special culture media, and no addition of inducer to the selective plates. In addition, since this vector carries all signals required for both prokaryotic and eukaryotic expression, it allows the overproduction of heterologous proteins, fused to a polyhistidine tag, in the bacterium E. coli and in the yeast Saccharomyces cerevisiae from a single plasmid. Hence, this vector may be a useful time-saving tool for one-step cloning and versatile protein expression in bacteria and yeast.

The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active.

The regB gene, from the bacteriophage T4, codes for an endoribonuclease that controls the expression of a number of phage early genes. The RegB protein cleaves its mRNA substrates with an almost absolute specificity in the middle of the tertranucleotide GGAG, making it a unique well-defined restriction endoribonuclease. This striking protein has no homology to any known RNase and its catalytic mechanism has never been investigated. Here, we show, using 31P nuclear magnetic resonance (NMR), that RegB produces a cyclic 2',3'-phosphodiester product. In order to determine the residues crucial for its activity, we prepared all the histidine-to- alanine point mutants of RegB. The activity of these mutants was characterized both in vivo and in vitro. In addition, their binding capability was quantified by surface plasmon resonance and their structural integrity was probed by 1H/15N NMR correlation spectroscopy. The results obtained show that only the H48A and the H68A substitutions significantly reduce RegB activity without changing its ability to bind the substrate or affecting its overall structure. Altogether, our results define RegB as a new cyclizing RNase and present His48 and His68 as potent catalytic residues. The effect of the in vivo selected R52L mutation is also described and discussed.

Activation of the RegB endoribonuclease by the S1 ribosomal protein is due to cooperation between the S1 four C-terminal modules in a substrate-dependant manner.

The RegB protein, encoded by the T4 bacteriophage genome, is a ribonuclease involved in the inactivation of the phage early messenger RNAs. Its in vitro activity is very low but can be enhanced up to 100-fold in the presence of the ribosomal protein S1. The latter is made of six repeats of a conserved module found in many other proteins of RNA metabolism. Considering the difference between its size (556 amino acids) and that of several RegB substrates (10 nucleotides), we wondered whether all six modules are necessary for RegB activation. We studied the influence of twelve S1 fragments on the cleavage efficiency of three short substrates. RegB activation requires the cooperation of different sets of modules depending on the substrates. Two RNAs are quite well cleaved in the presence of the fragment formed by the fourth and fifth modules, whereas the third requires the presence of the four C-terminal domains. However, NMR interaction experiments showed that, despite these differences, the interactions of the substrates with either the bi- or tetra-modules are similar, suggesting a common interaction surface. In the case of the tetra-module the interactions involve all four domains, raising the question of the spatial organization of this region.

Microarray analysis of gene expression during bacteriophage T4 infection.

Genomic microarrays were used to examine the complex temporal program of gene expression exhibited by bacteriophage T4 during the course of development. The microarray data confirm the existence of distinct early, middle, and late transcriptional classes during the bacteriophage replicative cycle. This approach allows assignment of previously uncharacterized genes to specific temporal classes. The genomic expression data verify many promoter assignments and predict the existence of previously unidentified promoters.