People involved:
Ryszard Stolarski
Anna Niedzwiecka
Anna Modrak-Wójcik
Katarzyna Ruszczyńska
Remigiusz Worch

Collaboration with (in Department of Biophysics):
Edward Darżynkiewicz
Jan Antosiewicz

Further collaboration:
Robert E. Rhoads, Department of Biochemistry and Molecular Biology, Louisiana State University Health Center (LSUHSC), Shreveport, Louisiana, U.S.A.;
Stephen Cusack, European Molecular Biology Laboratory (EMBL), Grenoble, France;
Richard E. Davis, Department of Biochemistry and Molecular Genetics, University of Colorado, Aurora CO U.S.A.;
Jacek Wójcik, Nuclear Magnetic Resonance Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland (Head: Dr. hab. Andrzej Ejchart);
Michał Dadlez, Department of Biophysics and Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland (Head: Prof. Andrzej Bierzyński);
Andrzej Jaworski, Faculty of Physics, Warsaw University of Technology;
 

Research interests:
A general aim of our research is to establish the molecular mechanisms of specific, mutual recognition of macromolecules and thermodynamically based stability of the protein - RNA complexes in the gene expression and regulation, i. e. translation initiation, RNA splicing, and intracellular transport. We are interested in interactions between various protein factors and RNA 5' termini (cap) composed of 7-methylguanosine linked by a triphosphate chain with the first transcribed nucleoside. For this purpose we have used synthetic cap analogues provided within collaboration with the research group of Prof. E. Darżynkiewicz (Department of Biophysics) and recombinant proteins, overexpressed in our Laboratory of Gene Expression or provided by the collaborating groups. The proteins include the translation initiation factors: various isoforms of eukaryotic initiation factor 4E (eIF4E), regulatory 4E-binding protein (4E-BP1 or PHAS-I, phosphorylated heat- and acid-stable protein regulated by insulin), and peptide fragments of the eIF4G scaffolding factor. Further on, we study the multifunctional nuclear cap - binding complex CBC composed of two subunits CBP80 and CBP20 engaged e. g. in the nucleus - cytoplasm transport of RNA, and the RNA splicing, and enzymes taking part in RNA degradation, like decapping scavenger DcpS. We have exploited various experimental and theoretical methods, including emission spectroscopy (steady state and stopped-flow fluorescence), isothermal microcalorimetry, nuclear magnetic resonance NMR, mass spectrometry, X-ray crystallography, molecular dynamics computer simulations and quantum calculations of the stacking energy of some model complexes composed of aromatic amino acids and nucleic acid bases by means of the second order perturbation approach MP2 at SCF-HF level. The intrinsic fluoresence quenching data, gathered by a our new time synchronized titration method, provided very precise values of the protein - ligand equilibrium association constants Kas at various temperatures and solution conditions. The method also allowed for determination of an amount of the active protein in the sample, and of the stoichiometry of enzymatic complexes with the ligands. Molecular spectroscopy, calorimetry and crystallography were employed to study interactions of various analogues of the eukaryotic mRNA 5'-terminus (cap) with the recombinant, murine and human 25 kDa cap-binding proteins eIF4E, as well as with the newly-isolated isoforms of eIF4E (IFE) from the nematode C. elegans. The results enabled to parse the Gibbs free energy of the binding into contributions originating from the individual intermolecular atomic contacts. The studies were extended on ternary complexes, composed of eIF4E, the cap, and synthetic peptide fragments of the eIF4G factors (I and II) or regulatory 4E-BP1 proteins. The steady-state fluorescence data correspond to those obtained by the dynamic fluorescence measurements using stopped-flow technique, regarding electrostatic steering of the cap-analogues toward the eIF4E active centre. Contributions to the Gibbs free energy of single structural cap modifications showed that the 5'-phosphate chain is a primary anchor to eIF4E. The electrostaticly driven encounter of the cap with the protein is followed by cooperative cation-pi stacking and the Watson-Crick type hydrogen bonding in the eIF4E cap-binding slot. Dependence of Kas on ionic strength and pH gave further information concerning ion and water exchange upon formation of the eIF4E-cap complexes as well as conformational rearrangement of the protein. A van't Hoff dependence of Kas gave thermodynamic parameters of the complexes formation, i. e. standard enthalpy DH°, standard entropy DS°, and standard heat capacity change under constant pressure DC°P. The unusual, positive DC°P values upon formation of some eIF4E-cap complexes were confirmed by a modified isothermal titration calorymetry method (ITC). Intreresting results were also obtained on the way to find biological role of eIF4E phosphorylation in regulation of the translation process. Similar studies of the CBC - cap complexes showed a crucial role of the second cap nucleoside (first transcribed) as well as of conformational changes of the CBP20 subunit in recognition and stabilization of the RNA 5' terminus by CBC. Values of the interaction energy of the aromatic amino acids stacked with the RNA bases for a set of the protein - RNA complexes (structures known from crystallography) pointed to a specific role of entropy effects in mutual recognition of both types of macromolecules. Distribution of the electric charge in the 7-methylguanine aromatic ring were linked to the coupling constants and chemical shifts, derived both from the NMR measurements for N-15/C-13 double labelled cap-analogues and from theoretical calculations. This allowed for localization of the net positive charge at N(7) of the five member ring, contrary to the previously accepted model of its delocalization between N(7) and N(9).

Supported by:

Ministry of Science and Higher Education: grant 3 P04A 021 25 (2003-2006):
"Specific interactions of RNA oligomers containing 5'-cap with proteins; Biophysical studies of molecular mechanisms and application to drug design"


Ministry of Science and Higher Education: grant N301 007 31/0154 (2006-2007):
"Analysis of interaction between nuclear protein complex and the structure of RNA 5' terminus by molecular biophysics"

Ministry of Science and Higher Education: grant 2 P04A 006 28 (2005-2008):
"Structure and function of cap 4 in early eukaryotes - trypanosomatides; the chemical analogues as potential drugs against Leishmania."