Former members:   Ilaria Iuliani (PhD student), Lexuan Liu (PhD student), Gladys Mbemba (AI), Malik Yousuf (postdoc), Qing Zhang (postodc), Joachim Rambeau (postdoc), Debayan Saha (M2), Marine Baudin (PhD student), Elisa Brambilla (PhD student), Ipek Altinoglu (M2), Biljana Maricic (M2), Avelino Javier (M2), Damel Mektepbayeva (M2), Anne Olliver (Postdoc), Chiara Saggioro (PhD student).

Research

Our research focuses on the mechanisms that allow a population of bacterial cells to adapt to environnemental changes and to the presence of growth inhibitors. In order to rapidly change the expression of a large number of genes, to switch from a growth mode to a maintenance or stress response mode for example, these mechanisms often rely on a level of regulation that is independent of specific transcription factors. These global regulation mechanisms include changes in DNA topology, in the activity of abundant nucleoid proteins, or in the activity of RNA polymerase itself. 

We have shown for example that the physico-chemical properties of a DNA-binding protein can rapidly change the level of gene expression in response to a temperature change (Saggioro et al. 2013). Furthermore we have shown that the activity of highly abundant nucleoid proteins, such as H-NS, can change in magnitude as a function of genome position, which has important implications for genome plasticity and evolution (Brambilla and Sclavi, 2015). In some cases, this level of regulation can lead to the emergence of heterogeneity in an otherwise genetically identical population (Leh et al, 2017). More recent results explore in more detail the role of the genomic neighborhood in determining the growth rate dependence of gene expression (Yousuf et al 2020) and noise and the coordination of basic cellular processes, such as transcription and translation, in the adaptation to sublethal levels of antibiotics (Zhang et al 2020).

The role of global regulators

The regulation of expression of several genes involved in DNA replication and DNA repair also depends on the activity of global regulators, such as DNA topology and abundant DNA-binding nucleoid proteins. Our latest publication addresses the question of whether genome position could influence the activity of one of these global regulators, the H-NS protein. This work was the project of Elisa Brambilla's PhD thesis, that she carried out in part in Georgi Muskhelishvili's laboratory at Jacobs University in Bremen. In her work Elisa was able to show that, if one studies the growth rate and growth phase dependence of H-NS dependent gene regulation, it is possible to uncover genome-position specific effects that are correlated with the AT-content of the genomic region containing the promoter that is a target of H-NS-dependent repression.  These results suggest that the position of certain genes may be conserved throughout evolution in order to maintain a specific level of growth condition dependent regulation. Therefore the activity of the abundant DNA- binding protein H-NS is not always uniform along the genomic coordinate. In addition to the AT-content, its competition with other nucleoid proteins, such as Fis, may also play an important role in this level of regulation. We are currently testing this hypothesis in a set of promoters co-regulated by these two proteins. (Brambilla and Sclavi, 2015)

Mechanisms of regulation of DNA replication and the adaptation to changes in growth rate and in response to stress

During cellular growth, specific regulatory mechanisms ensure that each daughter cell inherits at least one integral copy of the genome and that division occurs in time with DNA replication. In addition, this control mechanism needs to respond to changes in the growth rate and to possible changes in the rate of genome duplication due to replication fork stalling because of DNA damage or of the presence of inhibitors of replication factory components. Even though most of the genes and protein factors in the DNA replication network, or hyperstructure, have been identified, the specific mechanisms involved in these control processes remain to be described. Our research aims to define the dynamics of the regulatory networks of these fundamental cellular mechanisms ensuring genome integrity for the next generations. More specifically we are interested in the role of the transcription network within this regulatory system.

(See Herrick and Sclavi, Ribonucleotide reductase and the regulation of DNA replication: an old story and an ancient heritage. Mol Microbiol. 2007 Jan;63(1):22-34).(PubMed link)

The DnaA story

In our previous publication in Biochemical Journal we describe the results obtained during Chiara Saggioro's PhD thesis where she studied how the temperature dependence of the interaction between the DnaA protein and the DNA influences its auto-regulation of gene expression. By careful quantitative footprinting experiments Chiara was able to show that the temperature dependence of DnaA binding is not the same depending on whether it is binding to its high affinity sites or it is binding via oligomerisation in its ATP-bound state to the lower affinity sites. These results could nicely explain the observed changes in the dnaA promoters' activities as a function of temperature in vivo. Via mutations of DnaA's specific sites Chiara showed that as the temperature is lowered, it is the loss of negative auto-regulation at one of its promoters, P1, that is most likely responsible for induction of DnaA expression. The other promoter, P2, on the other hand remains both positively and negatively auto-regulated. Was the temperature-dependence of DnaA-DNA interactions selected for in order to result in an induction of gene expression at lower temperatures? Or was the mechanism of auto-regulation selected for in order to compensate for the changes in protein activity? Is this part of a specific adaptation process to frequent changes in growth condition? How does it affect the expression of all the genes regulated by DnaA? These are the questions that we are currently addressing in our research.(PubMedLink)

We had previously described the mechanism of regulation of ribonucleoide reductase (RNR) expression by the DnaA protein. DnaA is a key factor in the initiation of DNA replication, while RNR is the enzyme necessary for the synthesis of the building blocks of DNA, the dNTPs. James Fuchs and coworkers had done extensive work on the study of the regulation of the expression of RNR and had established that this enzyme is expressed at the time of replication initiation.

In a bacterial cell the cell cycle is much less defined than in an eukaryotic cell, it is thus not easy to imagine how a gene's expression may be timed with DNA replication. The DnaA protein exists under two different forms in the cell, ATP or ADP-bound, and the ratio of DnaA-ATP to DnaA-ADP oscillates during the cell cycle. Regulation by DnaA was therefore a logical candidate for regulating the timing of RNR expression.

We used a combination of quantitative in vitro and in vivo measurements of protein-DNA affinities and gene expression activities to study the role of DnaA in RNR expression. We have shown that DnaA-ATP can act both as a repressor and an activator of transcription, depending on its concentration, thus resolving apparent contrasting results from the Fuchs and Beckwith laboratories on the role of DnaA at this promoter. In addition we determined that RNA polymerase itself can bind to the upstream region necessary for cell cycle control, suggesting that modulation of this interaction may be responsible for the timing of RNR expression. Finally by flow cytometry analysis of DNA and GFP content we have shown that the dual role of DnaA as an activator and a repressor is not the cause for RNR's timing of expression. Activation by DnaA however results in a cooperative induction of expression, probably contributing to synchronizing the various copies of the gene in the cell (in a similar fashion to the synchronization of the origins) while repression limits the amount of RNR expression to remain proportional to the number of active replication forks.

Anne Olliver, Chiara Saggioro, John Herrick, Bianca Sclavi (2010) DnaA-ATP acts as a molecular switch to control levels of ribonucleotide reductase expression in Escherichia coli. Mol Microbiol. 2010 Jun;76(6):1555-71 (PubMed link).

*****

"Theories have four stages of acceptance. i) this is worthless nonsense; ii) this is an interesting, but perverse, point of view, iii) this is true, but quite unimportant; iv) I always said so." 

JBS Haldane.

Less recent results...

The RNAP-promoter interactions story

Our paper on promoter recognition by RNA polymerase in Nucleic Acids Research : (pdf file)

DNA melting by RNA polymerase at the T7A1 promoter precedes the rate limiting step at 37°C and results in the accumulation of an off-pathway intermediate.

Anastasia Rogozina¥, Evgeny Zaychikov¥, Malcolm Buckle‡, Hermann Heumann¥, Bianca Sclavi‡*

‡ LBPA, UMR 8113 CNRS, ENS Cachan, 94235 Cachan, France

¥ Max Planck Institute of Biochemistry, Am Klopferspitz 18A, D82152 Martinsried bei Munchen, Germany

Abstract

The formation of a transcriptionally active complex by RNA polymerase involves a series of short-lived structural intermediates where protein conformational changes are coupled to DNA wrapping and melting. We have used time-resolved KMnO4 and hydroxyl-radical x-ray footprinting to directly probe conformational signatures of these complexes at the T7A1 promoter. Here we demonstrate that DNA melting from m12 to m4 precedes the rate-limiting step in the pathway and takes place prior to the formation of full downstream contacts. In addition, on the wild type promoter, we can detect the accumulation of a stable off-pathway intermediate that results from the absence of sequence-specific contacts with the melted non-consensus -10 region. Finally, the comparison of the results obtained at 37°C with those at 20°C reveals significant differences in the structure of the intermediates resulting in a different pathway for the formation of a transcriptionally active complex.

Figure 7. Proposed structures for the key intermediates in the pathway of formation of a transcriptionally active complex on the wild type T7A1 promoter at 37°C.

The model of the open complex proposed by Darst and coworkers from the crystal structure of the Thermus aquaticus RNAP was used as a starting point to create these images(17). α-CTDs and NTDs are shown in light and dark gray respectively. β and β’ are in blue and green respectively, while sigma is in red. The template strand is in orange and the nontemplate strand in yellow. The shaded parts of the DNA are those that have entered the active site channel and the jaws and are therefore placed behind the β subunit. These intermediates correspond to those shown in Figure 5a, C through G. Following the formation of early complexes stabilized by the interaction of the α-CTDs with the UP-element and s region 4 with the -35 region of the promoter (A, B and B’ not shown) the DNA is bent towards s regions 3 and 2 where contacts are made with the spacer and the upstream end of the -10 region, C. The E’ intermediate corresponds to the off-pathway complex. While the pattern of protection in complexes E, F and G does not change, the extent of protection increases as the complex isomerizes into a transcriptionally active structure where the template strand is placed at the active site (G). Protection of the DNA down to p20 is likely due to an interaction with the β DR1 and β’ NCD and their subsequent folding stabilizing the transcriptionally active complex (3,54).

From our publication in PNAS in 2005: We used time-resolved X-ray footprinting to determine the structure of the intermediates in the pathway of promoter recognition by E. coli RNA polymerase. This figure is a model of proposed conformational changes occurring during T7A1 promoter recognition and open complex formation by RNA polymerase. The structure of the polymerase is that determined by the laboratory of S. Darst. RNA polymerase subunits beta and beta' are in two different shades of green, whereas the alpha N-terminal domains are in light and dark blue. The alpha-CTD, absent in the crystal structure, have been added as blue spheres. The sigma subunit is in red. A simplified cartoon of the DNA is drawn to scale. The TS is in dark purple and the NTS is light purple. The contacts shown between the enzyme and the DNA reflect the decreased solvent accessibility of nucleotides in specific positions on the promoter during the formation of the complex as determined in this work by time-resolved hydroxyl radical footprinting. One of the important results of this study was the identification of several intermediates in the kinetic pathway that are in equilibrium with each other. This is possible because the rates of the isomerization steps are slower than the interconversion rates between the intermediates within a given group (A or B). This model for promoter recognition is specific to the T7A1 promoter studied here. This promoter possesses an AT rich sequence upstream, also known as an UP element, and a -10 sequence that is not optimal. The first group of intermediates, A, are stabilized by the interaction of the alpha-CTDs with the UP element and sigma region 4 with the -35 sequence. The nonconsensus -10 sequence probably slows down one or both of the subsequent isomerization steps because of the non optimal interactions between the protein and the DNA in this region that are formed in the B intermediates. T7A1 is one of the first promoters transcribed in the T7 phage genome. This is one of the strongest promoters known from which RNA polymerase can transcribe in the absence of activators. Because of its strength it can successfully compete for the limited amount of free RNA polymerases in the cellular pool.

Time-resolved X-ray footprinting

The radiolysis of water by X-rays results in the production of hydroxyl radicals. These small highly reactive molecules can abstract an hydrogen from the backbone sugars of either RNA or DNA resulting in the cleavage of the polynucleotide. Due to their small size the cleavage pattern of hydroxyl radicals can be directly correlated to the solvent accessibility of the minor groove.

By using a synchrotron white beam as the source of the X-rays, enough radicals for a footprinting reaction can be created in a few milliseconds or less, after the beam is turned off the radicals decay in a few microseconds. By using a stopped-flow to quickly mix the protein and the DNA and then expose the sample to the beam one can carry out time-resolved x-ray footprinting experiments with a time resolution of the order of a few tens of milliseconds (Ralston, et al. (2000) Methods in Enzymology, 317, 353-368. Sclavi, et al. (1998). Methods in Enzymology, 295, 379-402).

This technique was developed during my PhD in the laboratories of Michael Brenowitz and Mark Chance at Albert Einstein College of Medicine in the Bronx. There is now a beamline dedicated to x-ray footprinting, the Center for Synchrotron Biosciences, at the National Synchrotron Light Source in Brookhaven, NY.

We have now set up this technique at the ESRF synchrotron in Grenoble, France.

We are continuing the X-ray footprinting experiments at the new synchrotron near Paris, Soleil, in collaboration with three other French research groups interested in DNA-protein interactions, protein footprinting of large multi-protein complexes and protein footprinting of membrane proteins.