msc final project - alvaro diaz mendoza

Master’s Degree in Condensed Matter Physics and Biological Systems Universidad Autonoma de Madrid (UAM)-Facultad de Ciencias

MSc Final Project – 2016

Interaction of the Hfq-protein from E.coli bacteria with DNA

Alvaro Diaz Mendoza Veronique Arluison1, Jean François Berret2 & Marisela Velez Tirado3

Abstract— The interaction between the Hfq protein from the Escherichia coli bacteria and DNA has been investigated by isothermal

titration calorimetry and electro-mobility shift assay. The proteins used for this analysis consist of two forms, which are some specific parts

of the wild type protein produced as synthetic peptides and some point mutations of the full-length protein. Besides, the project allows to

characterize the binding of Hfq carboxyl terminal region on DNA by using two approaches: (i) binding of carboxyl terminal region directly

and (ii) binding of carboxyl terminal region when interaction with the amino terminal region is abolished. This analysis has been conducted

within a previous study of the peptides, making experiments with an atomic force microscopy in order to characterize the kinetics of

self-assembly. This has been done in order to interpret isothermal titration calorimetry experiments.

Experimentally, we observed an interaction between the protein and DNA with both characterization techniques. Isothermal tiatration

calorymetry results, show an interaction between the protein and DNA. The titration experiments present an exothermic interaction with

given results such as the binding constant, stoichiometry ratio or enthalpy of the process. Concerning the electrophoretic technique,

it proves the absence of interaction for two of the mutated proteins as it has been observed in past publications. Results will be discussed

herein.

Definitions— Atomic Force Microscopy (AFM), Amino Terminal Region (NTR), Carboxyl Terminal Region (CTR), Electrophoretic Mobility

Shift Assay (EMSA), Hfq, Isothermal Titration Calorimetry (ITC), kilo base pair (kbp), PCR (Polymerase Chain Reaction).

Annotation— The figures named with a letter appear at the appendix as supplementary material, while the ones with numbers in the

present report.

_________________________________________________________________________________________________________________

1. INTRODUCTION

HE Hfq protein (standing for Host factor for bacterio-phage Qβ replication) is a bacterial protein from

Escherichia coli, which main function is to regulate bacterial gene expression. Hfq mainly acts as a RNA-chaperone, making possible the interaction between small RNA (sRNA) and messenger RNA (mRNA) and thus silencing the transla-tion [1,2]. Nevertheless, it has been demonstrated that this protein is also capable to interact with DNA and is involved in processes such as DNA replication, transposition, and possibly also in transcription [1] (Figure A). The interest of studying this protein stands with its general role in RNA and DNA metabolism, with important consequences for bacterial fitness, stress response in bacteria and in virulence [3]. This is particularly important given that the protein is present in at least half of bacterial species [4].

From the structural point of view, the protein has a toroidal hexameric ring shape. Each monomer consists of 102 residues, distributed in an amino and carboxyl terminal regions (NTR - CTR).

The NTR, comprising approximately the 65 first amino acids domains, folds into a 5-stranded β-sheet. The β-sheets from six monomers interact with each other to assemble in a toroidal structure with two non-equivalent faces, i.e. the proximal and distal surfaces. The CTR appears intrinsically unstructured and comprises about 35 terminal amino acid residues [5]. The function in RNA binding mainly lies in the NTR, while the CTR has been proved to self-assemble into fibrillary amyloid structures in vitro [5] (Figure 1.B.).

T

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Alvaro Diaz Mendoza is currently pursuing his Masters studies in Biophysics at the Universidad Autonoma de Madrid, Spain. E-mail: [email protected]

1Laboratoire Léon Brillouin, CEA – Centre de Saclay, France. 2Matière et Systèmes Complexes, Université Denis Diderot , France. 3Instituto de Catálisis y Petroleoquímica, CSIC – Cantoblanco, España.

Figure 1.A. Tridimensional structure of Hfq NTR obtained by

radiocrystallography indicate a ring diameter of about 7 nm. 1.B. Electronic microscope image shows the self-assembly of the CTR

into amyloid fibers with a section of aound 6 nm.

A

B

mailto:[email protected]

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Indeed, the two regions of the protein interact with DNA. Specific amino acids of the NTR are involved in nucleation on high affinity sites; while the edge of the NTR and the CTR are involved in the consolidation of binding. Besides, the self-assembly region of the CTR, is dedicated to propagation on DNA neighbouring regions. Note that inside the CTR some regions do not interact with DNA, such as the 11 amino acids sequence just responsible of the self-assembly. In this project we intend to characterize the interaction between different regions of Hfq with DNA. More precisely, the regions we will use are the CTR of Hfq (38 residues), one part of the CTR responsible of the protein self-assembly (11 residues) and some mutants in the NTR region. In the latter case, the mutations are introduced in order to switch a specific amino acid of the original sequences into another one. The aim of this mutagenesis is to observe the binding of the CTR when interaction with the NTR is abolished. The muta-tions are located at the distal face and the edge of Hfq protein; and have been chosen following Updegrove et al publication [6]. The mutations were introduced by directed mutagenesis into plasmidic DNA encoding the Hfq protein. After this molecular biology step, a subsequent purification of the protein is made. On the other hand, while the NTR mutants were produced in the laboratory, CTR synthetic peptides were produced by a company by chemical synthesis. The reason why the full-length protein was not produced with a chemical process is because a wrong folding of the NTR torus occurs (unpublished result). Then, two main techniques are used to analyze and characterize the interaction of Hfq with DNA. The first one is called Electro Mobility Shift Assay (EMSA) and consists in observing and quantifying the formation of the complex on a native gel. The second technique used, Isothermal Titration Calorimetry (ITC), consists in analyzing the thermodynamics of the interaction between the two components. Before using ITC, a previous study of the peptides was made with an atomic force microscopy (AFM), in order to observe the self-assembly kinetics. With this characterization, we can better interpret the isothermal titration calorimetry behavior of the isolated protein. Note that EMSA has been previously used to characterize the interaction of both full-length proteins and CTR peptides. Thus, this project will focus on studying with this technique, the interaction of the mutated proteins localized on the NTR. In parallel, it was also characterize the interaction of CTR from the thermodynamically point of view. For this goal it was used ITC, obtaining values like enthalpy (ΔH) or stoichiometry (n). However, with both techniques is possible to obtain the binding constant (KB) of the process.

The work was performed in three different laboratories: Instituto de Catálisis y Petroleoquímica (CSIC-Cantoblanco), Matière et Systèmes Complexes (MSC-Université Paris Diderot) and Laboratoire Léon Brillouin, (CEA - Centre de Saclay).

2. METHODS AND MATERIALS

2.1. Materials

Three different types of materials were used: the DNA, the

synthetic Hfq-peptide and the Hfq mutated protein. For DNA,

random sequences of 1 kbp and 2 kbp were used (NOLIMIT,

ThermoFisher Scientific Inc). As for the Hfq-peptide, two syn-

thetic lyophilized peptides (Proteogenix Inc.) have been used,

with their corresponding amino acid sequence presented in

Table 1. Finally, three mutated forms of full-length protein

have been produced, which means that at the end they will

have 102 residues. In our project we will focus on the

mutantions: R16A (on the edge), Y25A (distal face) and K31A

(distal face) like it is shown in Figure 2. These ones were based

on Updegrove et al. publication, which mainly consists in

changing one specific amino acid into another from the

original sequence; for instance in the mutant R16A the amino

acid R (Arg) of the position 16 will be changed into an A (Ala), maintaining the remain structure of the protein intact.

Figure 2. Three mutations: R16A, Y25A and K31A and their

location (distal face and edge) are shown, selected from

Updegrove et al. publication.

TABLE 1. SEQUENCE OF THE AMINO ACIDS

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2.2. Characterization techniques

Three different techniques were used to perform the experiments: the AFM, the ITC and the EMSA. Following, it is explained their principles and how they were implemented. The AFM samples were measured with an Agilent 5000 microscope, dissolving the lyophilized powder of the two peptides with Mili-Q water at 20 mg/mL and later diluting to 1:100 with the same solvent. The images were obtained with tapping mode at air condition with a resonance frequency of 280-310 kHz applied to silicon stiff tips (ScienceTec Inc.) of 25-75 N/m. One drop of 1 μL was placed over cleaved mica, used as substrate, allowing to air dry at room temperature. For the AFM, they were used for the characterization only two synthetic peptides to understand their behavior and regulate the time for the self-assembly, an important fact for later design the ITC experiments. Concerning ITC, we used an instrument that was capable to measure the power generated by the interaction between two elements which are a ligand and a receptor, injected one over the other. The system is formed by two chambers, as it is shown in Figure B, one that will conserve a set temperature (reference cell) and another that will measure the interaction of the elements (reaction cell) [7]. The results will show how the system produces heat along the titration to maintain constant the temperature difference between the two cells. To accomplish the experiment a VP-ITC calorimeter (MicroCal Inc.) was used, compound of a syringe volume of 283 μL and a reaction cell volume equal to 1.4643 mL (Figure B). In the syringe, DNA was loaded ten times more concentrated than the peptide, located in the reaction cell. The experiment begins with the injection of DNA, first with just one addition of 2 μL and subsequently 28 injections of 10 μL on the peptide. The injection duration was equal to 20 s with a delay of 300 s in between two successives and with a stirring speed of 307 rpm. In addition, before starting the experiments all the solutions were degasified in order to avoid bubbles while the experiment was running. Note that to measure both DNA and peptide at different concentrations, a buffer solution of pH 7.0 (10mM Tris-HCl and 5.0mM NaCl) was used to create the respectively dilutions. For this technique the two synthetic peptides (38 and 11 residues) and 2kbp DNA were used. Regarding EMSA, it is a classical biochemical technique used to observe the formation of the complex on a native gel. Each gel is loaded keeping constant the concentration of DNA, while the protein concentration varies. Then DNA is stained with a blue dye in order to observe it. The products that suffer interaction will migrate slowly due to their higher size, in comparison with those that does not interact and thus migrate faster [8]. The EMSA was made with acrylamide gels (GE Healthcare Inc.) which concentration was 4-12%, varying along the gel for a better resolution (conditions have been optimize previously). Experimentally, the gel does not have buffer inside, in order to allow any other one to be used; i.e. a gel for protein or nucleic acid migration. In order to place the gel in the correct buffer, the gel was pre-run at 100 V with a running buffer (Tris-Acetate and EDTA, TAE) during 30 minutes before loading the products.

Before migration, the protein and the DNA are mixed in buffer solution at pH 7.5 (50mM Tris-HCl pH 7.5, 50mM NH4Cl, 0.5mM EDTA with 10% of glycerol). The final volume was 10 μL and the mixture was left at room temperature for 20 minutes in order to allow the interaction of both biomole-cules to reach equilibrium. Then, the gel wells are launched with the 10 μL solution and the gel is run for 75 minutes at 100V. Once the gel is run, the gel was stained with a dye called GelRed, a safer alternative to Ethidium bromide for staining DNA. The last step was the analysis of the gel under UV light to take a final picture with the desired contrast and luminosity. For this technique we use the three mutated proteins (R16A, Y25A, and K31A) and the 1kbp DNA. This was chosen instead of 2 kbp in order to have a better resolu-tion in the gel.

2.3. Mutagenesis and purification

The protocol used to produce the mutated protein consist of

molecular biology and biochemistry procedures. Below are

explained the steps that were: mutagenesis, bacteria

transformation, bacteria culturing and protein purification.

The first step of the mutagenesis is the PCR (Polymerase

Chain Reaction), in order to create a high number of copies of

the mutated DNA. The template used for mutagenesis was a

plasmid encoding the full-length protein. Two primers, the

sense and anti-sense primers, are used to amplify the whole

plasmid (Figure C). One of the primer introduce the desired

point mutation and the primers are different for each mutants

(R16A, Y25A and K31A). Note that in order to replicate the

whole plasmid, a specific polymerase has to be used

(included in the mutagenesis kit and called Q5). In order to

perform the PCR it is also necessary to add deoxyribonucleo-

tides triphosphate (dNTPs) and a buffer containing

Magnesium ions. The PCR will consist on applying controlled

heat cycles of discrete temperatures; this will allow the

denaturation of the original DNA, annealing of the primer and

synthesis of the new one. After producing mutated DNA

fragments, an extra-step is necessary to remove the original

not mutated DNA. This is achieved by the digestion of the

original DNA, which is methylated while the new PCR

generated fragment is not. For this goal, an enzyme called

DpnI is used. Note that at this step the plasmids are still linear

and not re-circularized. This will be achieved directly by the

bacteria after introducing the DNA inside the cell. This step is

called the transformation of the bacteria and consists on

putting in plasmid into competent E. coli cells. For this

objective, cells are incubated in CaCl2 followed by a thermal

shock. Then the bacteria need to recover for one hour in rich

media, before being plated on LB-agar plates with ampicillin

(the resistance given by the plasmid). Only bacteria containing

the plasmid will grow overnight at 37⁰ C as the plasmid allows

the resistance to the antibiotic. Nevertheless, the DpnI

digestion is not 100% efficient and it is necessary to ensure

that the mutation occurs by sequencing the plasmid.

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Peptide of 11 amino acids after 2 hours

To achieve this, DNA of each colony is purified with a process

called mini-Prep.

The second main step of the purification process consists first

in the culture of the bacteria to produce the protein. Note that

for this step a specific bacteria called BL21(DE3) must be used.

Thus, a second transformation of the plasmid purified and

sequenced has to be done. After the second transformation, a

colony was grown overnight in a 5 mL preculture. Then the

5 mL are added to a larger culture of 1L. The process takes

about few hours, including not only the culturing but also the

induction of the protein that consists in the addition of a

compound called IPTG to induce the protein production.

The protein encoded in the plasmid is under control of an

inducible promoter and adding IPTG will allow to express the

protein at a high level. In between of the culturing, the

turbidity of the cell suspension is measured to know when it is

necessary to induce the culture (at 0.6 OD which means

exponential phase). Once it is produced all, the purification

begins. This one consist first on disrupting the cells with a

sonication process. The following step of the purification is the

heating of the lysate, as Hfq is thermostable. The heating step

will denature other proteins, but Hfq will remain in solution.

Before going to the purification, the solution is mixed with

DNase and RNase in order to remove nucleic acids that would

co-purify with Hfq. Afterwards comes the chromatographic

separation with a Ni+2 column which is the principal step of

the purification process. The Hfq protein binds to the column

as it contains a natural track of Histidines, which is not the

case of other proteins still present in the lysate that are eluted.

Then, Hfq is eluted from the column with a buffer containing

imidazole, an analog of Histidine. Finally, this process ends

with a dialysis of the resulting protein, in order to remove all

the excess of Ni+2 and salts that would abolish DNA binding.

3. RESULTS AND DISCUSSION

The project comprises the research of interaction between two different parts of the Hfq protein with DNA strands, focusing on the synthetic peptides of the CTR and certain amino acids from the NTR. The interesting point is to understand the interaction of both regions and appreciate the link between them. Results from diverse techniques are presented in this section with their corresponding conclusions. It is important to say that for all the experiments the DNA concentration was calculated in terms of base pair and not according to the chains length.

3.1. Synthetic peptide interaction – 38 and 11 amino acid

3.1.1. AFM

The AFM characterization was the starting point of these experiments, which main objective was to have a first information of how long it takes the peptide to create fibers.

Indeed, this is an important fact to consider because if the ITC experiment was performed with the peptide already assem-bled, the DNA would probably not bind to it. The design of the experiment consists in diluting the peptide in Mili-Q water, putting over cleaved mica and leaving to air dry at room temperature; for then observe it with the microscope and obtain the presented images. The reason of working at air and not liquid conditions was to examine the fibers, without removing them with the tip, while the image in tapping mode was taken. This procedure was performed through several days, starting with the stock solution of 20 mg/mL. Then, aliquots were removed and diluted before observation, in order to examine how the peptide evolves with the time. The reason to proceed in this way was to achieve an enough concentration to create fibers by self-assembly. These results are exposed on the images from Figure 3, showing that the peptide of 38 amino acid takes around 4 days to create fibers, which means that the self-assembly occurs in a long period time, not being the same for the 11 amino acids which just require 2 hours. Therefore, it is revealed the operational time needed to run the ITC after diluting the peptide. So in the case of the 38 amino acids the degasification before starting the experiment took longer, in comparison with the 11 amino acids that only was 10 minutes.

Moreover, the shape of the fibers from both peptides was characterized, with an average length of 400 - 500 nm and an average thickness of 25 - 40 nm for the 38 amino acids.

Peptide of 38 amino acids through several days

Figure 3. AFM micrographs presented to study the polymerization

time of both synthetic peptides before been diluted. 3.A. At the

upper side appear the 38 amino acids peptides, taking around

5 days to get into the fiber conformation. 3.B. Below, are shown

the 11 amino acids creating fibers in 2 hours.

B

A

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In the case of the 11 amino acids, the created fibers were few times smaller with an average length and thickness of 250 - 300 nm and 30 - 50 nm respectively. Figure 4 present one image at high resolution with further zooms to explore the conformation that the fibers of the 38 amino acids acquire at smaller resolutions. The same study was made with the 11 amino acids obtaining similar results. One interesting aspect of these images is the pattern created by the fibers; at the top the longest ones and underneath the smallest. It is thought that the longest fibers are created due to the agglomeration of the smallest by the drying effect, being the last size the real one. However, the aim of imaging the peptide with AFM was to know the polymerization timing and not to characterize the conformation. Future experiments are proposed for a further characterization in order to know the real shape of the fibers; making the measurements first at dry conditions and then at liquid, rewetting the sample with few microliters.

3.1.2. ITC

As soon as the peptide polymerization time was known, the ITC experiments were accomplished. The principle objective of ITC was to determine the interaction of the two synthetic peptides from the Hfq protein (38 and 11 amino acids) and 2kbp DNA. The results are presented in two types of curves, the thermograms and the binding curves. In the case of the thermograms, it is represented the heat that the instrument needs to produce in order to recover the set reference temperature (coming from the reference cell). In other words, this means how much energy the system needs to produce in each injection due to the heat produced by the interaction occurring along the titration. Hence, this will give the differential power for each of the 28 injections that corresponds to each thermogram’s peak. It is important to comment that the first peak is small in comparison with the 27 following ones, injecting 2 µL instead of 10 µL in order to equilibrate the system. A thermogram is shown at the upper side of Figure 5.A. in which is plotted the differential power versus the time while the titration runs.

Besides, on the bottom of Figure 5.A. is plotted the binding curve that corresponds to the thermogram discussed before. This curve represents the integration of the thermograms in terms of the injectant mole concentration, which is the DNA. Afterwards, the integrated data is fitted with the model known as “one set of sites” applying 100 iterations (red line), in order to determine parameters such as the binding constant KB or the reaction stoichiometry n. Specify that the enthalpy value is obtained from the inflection point of the fitted curve, the stoichiometry from the intercept with the vertical axis and the binding constant from the slope created at the inflection point of the fitted curve. Owing to this, the rest of the data from Figure 5 is presented with the binding curve rather than thermograms. The experiments were performed at different concentrations for the two biomacromolecules, with 0.2 - 0.5 mM and 1.6 - 2.5 mM for the peptide and DNA respectively, making an interaction ratio of 5 and 8. This bring us interesting results for the 38 amino acids, where an exothermic interaction of around 2.4 - 2.6 kcal mol-1 takes place (Figure 5.A.). The interaction can be classified as a stronger one, for instance if we compare our results with F. Loosli et al. [7], in where it is studied the mechanisms involved in the nanoparticles interaction. The differential power of their experiment is 2.1 kcal mol-1, which is relatively close to ours, keeping in mind that both experiments come from different scenarios. In the same figure (Figure 5.B.), they are also presented several binding curves of the 38 amino acids interacting with the DNA 2kbp at different concentrations. Additionally to his results, the thermodynamic values from the curves of Figure 5.B. are set out in Table 2, bringing us to conclude that in average the interaction has a stoichiometry of 0.54, a binding constant of 2.76 x 104 M-1 and an enthalpy of 5.48 kcal.mol-1. Therefore, this supports the fact mentioned previously that exists a significant interaction. Finally, Figure 5.C. shows how different is the interaction behavior of the 38 and 11 amino acids with the DNA. From one side the blue curve, that represents the 11 amino acids, shows values of -0.05 0.11 kcal.mol-1, meanwhile the green that corresponds to the 38 amino acids, values of -4.12 0.10 kcal.mol-1. These results clearly explain the absence of interaction of the 11 amino acids and DNA, in comparison with the 38 amino acids; this is in accordance to what we expected from previous EMSA experiments, as mentioned in the introduction. Moreover, two thermograms of this last amino acid are presented in the appendix in Figure D. To conclude, it would be interesting to design certain experi-ments, like measuring different interaction ratios keeping constant the peptide concentration until appreciating a notable change in the thermogram. This could show us the optimal interaction ratio so that the model curve can fit in a better way, and therefore obtain results with less variation in between the experiments. Also, the accomplishment of this experiment could prove the existence of a needed threshold concentration in order to have interaction.

Figure 4. AFM micrographs of the synthetic peptide of 38 amino

acids at big and small scale. The left image has the bigger scale

while the left one has the smaller, making a progressive zoom from

the left to the right side.

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3.2. Mutated protein interaction – R16A, Y25A and K31A

3.2.1. EMSA

This second part of the experiments begins with the production of the mutants from the Hfq protein, which protocol is presented in the methods and materials section. Once the mutations were done, it was studied how they inter-act with 1kbp DNA using the electrophoretic shift mobility technique. The results obtained with the EMSA experiments are shown in Figure 6. In this figure each well has the same concentration of mutated protein, in order to compare the migration at the same conditions. The free DNA was also included as a marker, in this case in the first lane. From these results, we conclude that the mutations R16A and K31A almost did not interact with the DNA while the Y25A interacts even more than the wild type protein.

Although, the obtained results are not the same than the presented in Updegrove et al. paper, whom expose that the interaction might not occur for Y25A mutation, major conclusion of our analysis are the same. Additionally, on Figure E which is attached to the appendix, it is presented a second set of results with one gel per mutated protein. In this case, the protein has different concentration in each well, with an increase from the right to the left side of the gel. Note that as soon as the concentration increases, the compound of protein and DNA migrate less, meaning that the interaction took place. It is also possible to observe that, similarly to the wild type protein, we have a cooperative binding on DNA and supershift occurs in the Y25 mutation, in agreement with the binding of more than Hfq on DNA. Then a further characteri-zation will need to be accomplished in order to observe how these mutants polymerize on DNA. Nevertheless, this is planned to do as future experiments.

Figure 5.A. The interaction of 38 amino acids and 2kbp DNA is presented in a thermogram with its corresponding binding

curve. At the same time, in the binding curve is presented a fit curve (red) in order to obtain the thermodynamic values.

5.B. Binding curves of five experiments with 38 amino acids and 2kbp DNA at different concentrations are plotted.

The values set out in Table 2 were obtained from these curves, these are the stoichiometry (n), the binding constant (KB),

enthalpy (ΔH) and the entropy (ΔS). 5.C. Two binding curves are presented for the two synthetic peptides. The green

curve belongs to the peptide of 11 amino acids and the blue one to the peptide of 38 amino acids.

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4. SUMMARY AND CONCLUSION

The project intend to characterize the interaction among the different regions of the Hfq protein with double stranded DNA. The results obtained are the ones that were set in the objectives, measuring an interaction with two characterization techniques. In past researching works, it was measured in an extensively way how this protein interacted with RNA, but in this case we want to go further in how it interacts with another nucleoid acid, DNA. Two main conclusions can be drawn. Thermodynamics of DNA assembly can be characterized by ITC and consists in an exothermic interaction with higher differential power values. EMSA demonstrates that NTR mutants are affected in DNA binding, but do not completely abolish it. This needs to be investigated in more details in order to see how the coopera-tivity of binding is modified when the NTR is mutated.

ACKNOWLEDGMENT

The author wish to thank the support of Marisela Velez Tirado (ICP-CSIC, Cantoblanco) who made possible the internship in Paris and showed him how to use the AFM. Also be really grateful with Veronique Arluison (LLB-CEA, Saclay) and Jean-François Berret (MSC-Diderot, Paris), the two mentors that guide all the work in Paris having fruitful discussions with them. Do not forget Frédéric Loosli, Javier Morgado, Antonie Malabirade and David Partouche that helped the author to accomplish several experiments showing the techniques and giving their support.

APPENDIXES

All the figures cited with a letter along the document are at-tached at the appendix. These are from Figure A to Figure E.

REFERENCES

[1] G. M. Cech, A. Szalewska-Pałasz, K. Kubiak, A. Malabirade, W.

Grange, V. Arluison, G. Węgrzyn. “The Escherichia coli Hfq protein:

an unattended DNA-transactions regulator” (2016).

[2] J. Vogel and Ben F. Luisi. “Hfq and its constellation of RNA”.

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[3] Tsui, Leung, H. C. & Winkler, M. E. “Characterization of broadly

pleiotropic phenotypes caused by an Hfq insertion mutation in

Escherichia coli”. Mol. Microbiol. 13, 35–49 (1994).

[4] Valentin-Hansen, P. Eriksen, M. & Udesen. “The bacterial Sm-like

protein Hfq: a key player in RNA transactions”. Mol. Microbiol. 51,

1525–1533 (2004).

[5] E.Fortas, F. Piccirilli, A. Malabirade, V. Militello, S, Trépout,

S. Marco, A. Taghbalout & V. Arluison. “New insight into the struc-

ture and function of Hfq C-terminus” Biosci. Rep. 35 art: 190 (2015).

[6] T. B. Updegrove, J. J. Correia, R. Galletto, W. Bujalowski & R. M.

Wartell. “E. coli DNA associated with isolated Hfq interacts with

Hfq's distal surface and C-terminal domain”. Biochimica et

Biophysica Acta 1799, 588-596 (2010).

[7] F. Loosli, L. Vitorazi, J-F. Berret & S. Stoll. “Isothermal titration

calorimetry as a powerful tool to quantify and better understand

agglomeration mechanisms during interaction processes between

TiO2 nanoparticles and humic acids”. Environ. Sci.: Nano, 541 (2016).

[8] M.Garner & A. Revzin. “A gel electrophoresis method for

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tion to components of the Escherichia coli lactose operon regulatory

system”. IRL Press Limited, volume 9, Number 13 (1981).

Figure 6. EMSA made with mutants (R16A, Y25A and K31A)

and WT protein. The interaction was performed with 1kbp DNA at

15.5nM concentration and 1.5 µM for the proteins.

msc final project - alvaro diaz mendoza

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