Roles moleculaires des proteines de choc thermique dans le fonctionnement des organismes a des temperatures normales et suite a des chocs thermiques; differents genes impliques

The heat-shock proteins

The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence.

/pdf/compilation-and-analysis-of-escherichia-coli-promoter-dna-3laukojglx.pdf

Compilation and analysis of Escherichia coli promoter DNA sequences.

When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins. The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the "N-end rule"). The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule. The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule. Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.

https://science.sciencemag.org/content/sci/234/4773/179.full.pdf

In Vivo Half-Life of a Protein is a Function of its Amino-Terminal Residue

https://mmbr.asm.org/content/mmbr/48/1/60.full.pdf

Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli.

Increases in ultraviolet radiation at the Earth's surface due to the depletion of the stratospheric ozone layer have recently fuelled interest in the mechanisms of various effects it might have on organisms. DNA is certainly one of the key targets for UV-induced damage in a variety of organisms ranging from bacteria to humans. UV radiation induces two of the most abundant mutagenic and cytotoxic DNA lesions such as cyclobutane–pyrimidine dimers (CPDs) and 6–4 photoproducts (6–4PPs) and their Dewar valence isomers. However, cells have developed a number of repair or tolerance mechanisms to counteract the DNA damage caused by UV or any other stressors. Photoreactivation with the help of the enzyme photolyase is one of the most important and frequently occurring repair mechanisms in a variety of organisms. Excision repair, which can be distinguished into base excision repair (BER) and nucleotide excision repair (NER), also plays an important role in DNA repair
in several organisms with the help of a number of glycosylases and polymerases, respectively. In addition, mechanisms such as mutagenic repair or dimer bypass, recombinational repair, cell-cycle checkpoints, apoptosis and certain alternative repair pathways are also operative in various organisms. This review deals with UV-induced DNA damage and the associated repair mechanisms as well as methods of detecting DNA damage and its future perspectives.

/pdf/uv-induced-dna-damage-and-repair-a-review-28aup0s77v.pdf

UV-induced DNA damage and repair: a review

BASE INSERTION SELECTIVITY BY DNA POL YMERASES . The Base 1''lSertion Pathway . Structural Aspects to Base Selection by DNA Polymerases . Polymerase Insertion Errors . Fidelity Assays and Major Conclusions . Error Avoidance in Base Insertion----General Conclusions ..... .

Fidelity mechanisms in DNA replication.

The mutation rate of Escherichia coli increases approximately 100-fold after treatment with replication-inhibiting agents such as UV light. This enhanced mutation rate requires the action of the UmuD and UmuC proteins, which are induced as part of the SOS response to DNA damage. To initiate a biochemical characterization of the role of these proteins, we have developed a plasmid system that gives efficient expression of the umuD and umuC genes. The umuD and umuC genes were placed under the control of a regulated phage lambda PL promoter and a synthetic ribosome-binding site, and the distance to the UmuD start was adjusted to maximize gene expression. Starting from this overproduction system, we have purified the UmuD protein and studied its interaction with RecA. The SOS response is turned on by the capacity of RecA protein to mediate cleavage of the LexA repressor for SOS-controlled operons. Others have shown that UmuD exhibits sequence homology to LexA around the cleavage site, suggesting a possible cleavage reaction for UmuD. We show that RecA mediates cleavage of UmuD, probably at this site. As with LexA, UmuD also undergoes a self-cleavage reaction. We infer that RecA-mediated cleavage of UmuD is another role for RecA in SOS mutagenesis, probably activating UmuD for its mutagenic function.

https://www.pnas.org/content/pnas/85/6/1811.full.pdf

UmuD mutagenesis protein of Escherichia coli: Overproduction, purification, and cleavage by RecA

The NtrC protein activates transcription of the glnA operon of enteric bacteria by stimulating the formation of stable "open" complexes by RNA polymerase (sigma 54-holoenzyme form). To regulate the glnA promoter, NtrC binds to sites that have the properties of transcriptional enhancers: the sites will function far from the promoter and in an orientation-independent fashion. To investigate the mechanism of enhancer function, we have used electron microscopy to visualize the interactions of purified NtrC and RNA polymerase with their DNA binding sites and with each other. Under conditions that allow the formation of open complexes, about 30% of DNA molecules carry both RNA polymerase and NtrC bound to their specific sites. Of these, about 15% form looped structures in which NtrC and the RNA polymerase-promoter complex are in contact. The length of the looped DNA is that predicted from the spacing that was engineered between the enhancer and the glnA promoter (390 base pairs). As expected for activation intermediates, the looped structures disappear when RNA polymerase is allowed to transcribe the DNA. We conclude that the NtrC enhancer functions by means of a direct association between DNA-bound NtrC and RNA polymerase (DNA-looping model). Association of DNA-bound proteins appears to be the major mechanism by which different types of site-specific DNA transactions are localized and controlled.

https://www.pnas.org/content/pnas/87/14/5504.full.pdf

DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter.

The precise association of DNA-binding proteins with localized regions of DNA is crucial for regulated replication and expression of the genome. For certain DNA transactions, the requirement for precision in localization and control is extremely high. High-precision events amenable to detailed biochemical analysis are the initiation of DNA replication and site-specific recombination by bacteriophage lambda and Escherichia coli. Recent experiments indicate that site-localization and control in these reactions involves the association of DNA-bound proteins to generate organized nucleoprotein structures in which the DNA is folded or wound. These specialized nucleoprotein structures are likely to provide the requisite accuracy for site localization and the necessary regulated reactivity to direct the DNA transaction. Multiple DNA-protein interactions are also required for controlled transcription of the eukaryotic genome. Distant upstream regulator and enhancer sequences may define protein-binding sites that form part of a reactive nucleoprotein structure capable of initiating transcription.

https://science.sciencemag.org/content/sci/233/4768/1050.full.pdf

Multiple DNA-protein interactions governing high-precision DNA transactions.

DNA polymerase III (polIII) holoenzyme of Escherichia coli has 3'----5' exonuclease ("editing") activity in addition to its polymerase activity, a property shared by other prokaryotic DNA polymerases. The polymerization activity is carried by the large alpha subunit, the product of the dnaE gene. Mutations affecting the fidelity of DNA replication in vivo and the activity of 3'----5' exonuclease assayed in vitro are found in the dnaQ gene, which specifies the epsilon subunit. To determine whether epsilon carries the 3'----5' exonuclease activity, we have used an overproduction protocol to purify epsilon separately from the other subunits of polIII holoenzyme. We find that epsilon has 3'----5' exonuclease activity indistinguishable from that of polIII core, the subassembly of polIII holoenzyme consisting of the alpha, epsilon, and theta subunits. We conclude that the editing and polymerization activities of polIII holoenzyme reside on distinct subunits, in contrast to DNA polymerase I of E. coli and DNA polymerase of phage T4. This functional separation may provide for regulation of exonucleolytic editing independently of polymerization, allowing cellular control of replication fidelity.

https://www.pnas.org/content/pnas/81/24/7747.full.pdf

Harrison Echols

Papers

Fidelity mechanisms in DNA replication.

UmuD mutagenesis protein of Escherichia coli: Overproduction, purification, and cleavage by RecA

DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial glnA promoter.

Multiple DNA-protein interactions governing high-precision DNA transactions.

A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme