Henri Buc

SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope.

Changes in the transcriptional state of genes have been correlated with their repositioning within the nuclear space. Tethering reporter genes to the nuclear envelope alone can impose repression and recent reports have shown that, after activation, certain genes can also be found closer to the nuclear periphery. The molecular mechanisms underlying these phenomena have remained elusive. Here, with the use of dynamic three-dimensional tracking of a single locus in live yeast (Saccharomyces cerevisiae) cells, we show that the activation of GAL genes (GAL7, GAL10 and GAL1) leads to a confinement in dynamic motility. We demonstrate that the GAL locus is subject to sub-diffusive movement, which after activation can become constrained to a two-dimensional sliding motion along the nuclear envelope. RNA-fluorescence in situ hybridization analysis after activation reveals a higher transcriptional activity for the peripherally constrained GAL genes than for loci remaining intranuclear. This confinement was mediated by Sus1 and Ada2, members of the SAGA histone acetyltransferase complex, and Sac3, a messenger RNA export factor, physically linking the activated GAL genes to the nuclear-pore-complex component Nup1. Deleting ADA2 or NUP1 abrogates perinuclear GAL confinement without affecting GAL1 transcription. Accordingly, transcriptional activation is necessary but not sufficient for the confinement of GAL genes at the nuclear periphery. The observed real-time dynamic mooring of active GAL genes to the inner side of the nuclear pore complex is in accordance with the ‘gene gating’ hypothesis.

Kinetics of the allosteric interactions of phosphofructokinase from Escherichia coli

Abstract Phosphofructokinase has been partially purified from Escherichia coli , and its kinetic properties investigated. It shows co-operative interactions with respect to one of its substrates, fructose-6-phosphate, but not towards the second, namely ATP. ADP and other diphosphonucleosides act as activators and phosphoenolpyruvate as an inhibitor. Both effectors decrease the homotropic interactions between fructose-6-phosphate molecules; but, whereas the activators increase the affinity of the enzyme for this substrate, the inhibitor decreases it. These ligands have no effect on the maximum velocity of the reaction, except in the case of ADP which is a competitive inhibitor of ATP. These homotropic and heterotropic interactions are qualitatively and quantitatively accounted for by the concerted transition theory proposed by Monod, Wyman & Changeux (1965), assuming the enzyme to be in equilibrium between two conformational states which differ in their dissociation constants for fructose-6-phosphate, activators and inhibitor. A convenient method of obtaining these intrinsic dissociation constants has been derived from the equations of the theory. From the kinetic data, it is also possible to obtain the value of the equilibrium constant between the two states, if it is assumed that the enzyme is a tetramer made up of four identical subunits and that the transition is perfectly concerted.

Synthetic curved DNA sequences can act as transcriptional activators in Escherichia coli.

Can a transcriptional activator known to bend DNA be functionally replaced by a sequence‐directed bend in Escherichia coli? To investigate this question, a partially truncated promoter was used, deleted of its ‐35 region and of its CRP binding site, leaving only two Pribnow boxes as functional elements. Synthetic and naturally occurring curved DNA sequences introduced upstream from these elements could restore transcription at either one of the two natural starts. Some of these hybrid promoters turned out to be more efficient than the CRP activated wild‐type gal promoter in vivo. Control experiments performed with very similar sequences devoid of any curvature produced weak promoters only. Minimal changes in the location of the centre of curvature or perturbation in the amount of curvature strongly affected the level of expression. No significant stimulation of transcription could be detected in vitro. Furthermore, both gal P1 and P2 starts could be activated in vivo but also in vitro via a properly positioned CRP binding site. This partial analogy suggests that bending induced by the cAMP‐CRP complex upon binding to its site may be biologically relevant to the mechanism of transcriptional activation.

Stringent spacing requirements for transcription activation by CRP.

The cyclic AMP receptor protein-cAMP complex (CRP-cAMP) binds at a variety of distances upstream of several E. coli promoters and activates transcription. We have constructed a model system in which a consensus CRP binding site is placed at different distances upstream of the melR promoter. CRP-cAMP activates transcription from melR when bound at a number of positions, all of which lie on the same face of the DNA helix. The two distances at which transcription is strongly activated correspond exactly to those at which CRP-cAMP binds upstream of the well-studied galP1 and lac promoters. Footprinting of the synthetic promoters reveals that RNA polymerase makes identical contacts with their -10 regions even though CRP-cAMP binds at a different distance in each case. Kinetic analysis in vitro indicates that CRP-cAMP activates transcription from these promoters in similar but distinct ways. A model is proposed to explain this two-position activation.

[11] Phosphofructokinases from Escherichia coli

Publisher Summary This chapter describes the enzymic assay and purification of two phosphofructokinases in Escherichia. coli K 12. The most studied one, PFK1, is coded by the pfk A gene, while the other enzyme, PFK2, is specified by the pfk B gene. The two enzymes differ in their biochemical and catalytic properties. Phosphofructokinase activity is routinely assayed by following the formation of fructose 1,6-bisphosphate, which is converted to a-glycerophosphate by aldolase, triosephosphate isomerase, and glycerol-3-phosphate dehydrogenase in the presence of β -NADH. Purification of PFK1 involves preparation of crude extract, affinity chromatography, concentration, and heat denaturation of protein contaminants. Unlike PFK1, PFK2 in crude extracts does not bind to Blue Dextran–Sepharose, while the heat treatment does not affect the biochemical properties of PFK1 or PFK2. ATP is the best phosphoryl donor for both PFK1 and PFK2, and fructose 6-phosphate is the best phosphoryl acceptor. Growth conditions have no influence on the biosynthesis of PFK2 but the relative amount of PFK1 depends on the nature of the carbon source and on the oxygen tension.

The bacteriophage T4 AsiA protein: a molecular switch for sigma 70-dependent promoters

The AsiA protein, encoded by bacteriophage T4, inhibits Eσ70‐dependent transcription at bacterial and early‐phage promoters. We demonstrate that the inhibitory action of AsiA involves interference with the recognition of the −35 consensus promoter sequence by host RNA polymerase. In vitro experiments were performed with a C‐terminally labelled sigma factor that is competent for functional holoenzyme reconstitution. By protease and hydroxyl radical protein footprinting, we show that AsiA binds region 4.2 of σ70, which recognizes the −35 sequence. Direct interference with the recognition of the promoter at this locus is supported by two parallel experiments. The stationary‐phase sigma factor containing holoenzyme, which can initiate transcription at promoters devoid of a −35 region, is insensitive to AsiA inhibition. The recognition of a galP1 promoter by Eσ70 is not affected by the presence of AsiA. Therefore, we conclude that AsiA inhibits transcription from Escherichia coli and T4 early promoters by counteracting the recognition of region 4.2 of σ70 with the −35 hexamer.

Modulated expression of promoters containing upstream curved DNA sequences by the Escherichia coli nucleoid protein H‐NS

Replacement of the CRP‐binding site of the gal control region by curved sequences can lead to the restoration of promoter strength in vivo. One curved sequence called 5A6A, however, failed to do so. The gene hns exerts a strong negative control on the resulting 5A6A gal promoter as well as on the distant bla promoter, specifically in a 5A6A gal context. The product of this gene, H‐NS, displays a better affinity for this particular insert compared to other curved sequences. Mechanisms by which H‐NS may repress promoters both at short and long distances from a favoured binding site are discussed.

On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions.

Using DNase footprinting and transcription assays in vitro we have probed the effect of the cAMP‐cAMP receptor protein complex (cAMP‐CRP) on the positioning of RNA polymerase and on the location of the transcription start point at the Escherichia coli gal and lac operon regulatory regions. In both cases, RNA polymerase can form two alternative complexes which promote transcription from two different start points, S1 and S2: pre‐incubation of promoter DNA with cAMP‐CRP results in a shift of the transcription start from S2 to S1 and in an increase in the rate of open complex formation. Moreover, the rate of formation of each heparin‐resistant complex parallels the establishment of the corresponding footprint, showing that the stable binding corresponds to open complex formation. We show that, in the case of gal, RNA polymerase, which is bound so as to transcribe from S2, cannot be diverted to S1 by subsequent addition of cAMP‐CRP. In contrast, in the case of lac, when cAMP‐CRP is added after RNA polymerase, complexes which initiate transcription at S2 are rapidly converted to complexes which initiate at S1. Finally, we present data which suggest that protein‐protein interactions are essential for CRP‐induced activation at both the lac and gal promoters.

Regulation of the amount and of the activity of phosphofructokinases and pyruvate kinases in Escherichia coli

Two isozymes of fructose-6-phosphate kinase and two isozymes of pyruvate kinase have been detected in Escherichia coli under a wide variety of growth conditions. Their kinetic behavior has been characteriized with respect to different effectors and substrates. The conclusions reached on one hand by Malcovati and Kornberg (Biochim. Biophys. Acta (1969) 178, 420-423), on the other hand by Fraenkel, Kotlarz and Buc (J. Biol. Chem. (1973) 248, 4865-4866) have been found to be true in aerobiosis as well as in anaerobiosis. The biosynthesis of the four proteins is sensitive to the nature of the carbon sources as well as to the shift from aerobic to anaerobic conditions. Kinetics of depression after a shift to anaerobiosis have been followed and found to be of the order of the doubling time.

Attenuation of Rabies Virulence: Takeover by the Cytoplasmic Domain of Its Envelope Protein

Survival of rabies virus–infected neurons depends on a single amino acid in the PDZ-binding site of a viral protein. Tipping the Balance Strains of rabies virus, which infects neurons, may be virulent, in which case the cells survive long enough for the virus to replicate and spread, or they may be attenuated, in which case the infected cells die by apoptosis. Préhaud et al. compared one attenuated and one virulent viral strain and found that a single amino acid change in a region of a viral envelope protein that binds to host cell proteins was sufficient to account for the death or survival of infected cells. The binding properties of the attenuated virus protein were expanded, thereby affecting the balance in the activities of host kinases and phosphatases sufficiently to trigger cell death. These findings may inform strategies to engineer attenuated viruses, which are often used in live vaccines. The capacity of a rabies virus to promote neuronal survival (a signature of virulence) or death (a marker of attenuation) depends on the cellular partners recruited by the PDZ-binding site (PDZ-BS) of its envelope glycoprotein (G). Neuronal survival requires the selective association of the PDZ-BS of G with the PDZ domains of two closely related serine-threonine kinases, MAST1 and MAST2. Here, we found that a single amino acid change in the PDZ-BS triggered the apoptotic death of infected neurons and enabled G to interact with additional PDZ partners, in particular the tyrosine phosphatase PTPN4. Knockdown of PTPN4 abrogated virus-mediated apoptosis. Thus, we propose that attenuation of rabies virus requires expansion of the set of host PDZ proteins with which G interacts, which interferes with the finely tuned homeostasis required for survival of the infected neuron.