State-of-the-art and preparatory work

The project is dedicated to studying new mechanisms of gene expression regulation in bacterial cells and to characterising the interactions between the biomacromolecules recruited in this process. The main goal of the project is the analysis of the topology of supramolecular complexes which are formed during transcription of prokaryotic genes by bacterial RNA polymerase with participation of 6S RNA and the (cytosine‐5)‐DNA methyltransferase SsoII. M.SsoII was shown to inhibit transcription of its own gene by binding to a regulatory sequence near its promoter and to activate transcription of the second gene from this restriction‐modification system – R.SsoII (1, 2).

6S RNAs are new types of regulators – small nontranslated RNAs which are able to control transcription initiation by direct binding to bacterial RNA polymerase without interaction with the promotor (3). Similar mechanisms of regulation are studied not only for prokaryotic but also for eukaryotic nontranslated RNAs (4). Although the mechanism of this regulation seems to be known for the 6S RNA from E. coli, detection of two different 6S RNAs from Bacillus subtilis (6S‐1 and 6S‐2) (5) and different sensitivities of promotors to NTP concentration (6) suggest a more complex character of transcription inhibition in the Bacillus system and call for further investigations.

Another project in our laboratory is dedicated to the study of DNA containing widespread genetically harmful modifications (e.g. thymidine glycol, apurinic sites, non‐canonic pairs) and the development of methods which allow testing and analysis of their influence on their interactions with the proteins of a cell.

References

1. Karyagina, A., Shilov, I., Tashlitskii, V., Khodoun, M., Vasil'ev, S., Lau, P.C. & Nikolskaya, I. (1997) Specific binding of SsoII DNA methyltransferase to its promoter region provides the regulation of SsoII restriction‐modification gene expression. Nucleic Acids Res. 25, 2114‐2120.

2. Shilov, I., Tashlitsky, V., Khodoun, M., Vasil'ev, S., Alekseev, Y., Kuzubov, A., Kubareva, E. & Karyagina, A. (1998) DNAmethyltransferase SsoII interaction with own promoter region binding site. Nucleic Acids Res.26, 2659‐2664.

3. Willkomm, D. K. & Hartmann, R. K. (2005) 6S RNA – an ancient regulator of bacterial RNA‐polymerase rediscovered. Biol Chem 86, 1273–1277.

4. Goodrich, J. A. & Kugel, J. F. (2006) Non‐coding‐RNA regulators of RNA polymerase II transcription. Nature Reviews Molecular Cell Biology 7, 612‐616.

5. Trotochaud, A. E. & Wassarman, K. M. (2005) A highly conserved 6S RNA structure is required for regulation of transcription. Nat Struct Mol Biol 12, 313‐319.

6. Krasny, L., Tiserova, H., Jonak, J., Rejman, D. & Sanderova, H. (2008). The identity of the transcription +1 position is crucial for changes in gene expression in response to aminoacid starvation in Bacillus subtilis. Molecular Microbiology. 69, 42‐54.

Aims

The overall aims of these projects are

I. to study the interaction of different 6S RNA from B. subtilis with RNA polymerase from B. subtilis in order to elucidate their functions and to identify RNA‐protein contacts.

II. to develop reactive analogues of nucleic acids for identification of sites of interaction between RNA polymerase and a regulatory 6S RNA.

III. to synthesize azobenzene containing oligonucleotides and to characterize them with respect to their influence on the thermal stability of duplexes under illumination.

Work programme and methods

The main focus of our project is on the role of a new type of regulator – small nontranslated RNAs (6S RNA) which are able to control transcription initiation by direct binding to bacterial RNA polymerase without interacting with the promoter. We will study the promoter organization to find out which features determine the sensitivity to 6S RNA‐mediated regulation. A possibility will be to adapt the SsoII R‐M system for studying the regulation of gene transcription in the presence of not only M.SsoII, but also 6S RNA. Interactions of different 6S RNAs from B.subtilis with RNA polymerase from B. subtilis will be studied in order to elucidate their functions and to identify RNA‐protein contacts. Another aspect of this project concerns the regulation of transcription of the bsrA and bsrB genes (coding for 6S‐1 and 6S‐2 RNA, respectively) which is sensitive to 6S RNA‐mediated inhibition depending on the nucleoside triphosphate concentration. Chemically active modified ribo‐ and deoxyribooligonucleotides will be used as tools for studying DNA‐protein, RNA‐protein and proteinprotein complexes of different complexity. DNA and RNA fragments will be synthesized with 2’‐ deoxy‐2’‐aminouridine or 2’‐deoxy-2’‐O‐trifluoroacetamidopropyl containing nucleosides. These functional groups will be located at different positions of the oligonucleotide strand and will be converted further into reactive centres aimed at cysteine residues, e.g. iodacetamide, maleimide, disulfide groups. As reagents capable of interacting with arginine residues, oligonucleotides containing a β‐diketo group in the 2’‐position of the sugar fragment will be tested. Arginine plays a key role in protein‐nucleic acid interactions. There is no specific reagent for arginine residues in proteins up to date. Designing such reagents would help identifying specific arginine residues which take part in DNA‐protein interactions. We have obtained preliminary positive results while studying guanidine interactions with oligonucleotides containing β‐diketo groups. The methodology will be developed in a relatively simple system consisting of R.SsoII in complex with DNA. Then the affinity modification of protein with reactive RNA analogues will be used in order to study RNA‐protein interactions. The affinity modification of protein with reactive RNA fragments containing the functional groups in the 2’‐position of ribose will be developed for bacterial RNase P as a model system, and then for identification of interaction sites of RNA polymerase with a regulatory 6S RNA. This part of the work will be performed in collaboration with R. Hartmann.

Studying chemical and biological properties of DNA containing widespread genetically harmful modifications (e.g. residues of thymidine glycol, apurinic sites, non‐canonical pairs), will allow developping methods for testing DNA damages and analysis of their influence on DNA interactions with the proteins of a cell. The approach is based on the concept of molecular recognition of increased flexibility and/or special properties of the damaged or mismatched bases by chemical reagents or by enzymes acting on DNA. Specially constructed model systems based on modified oligonucleotides which contain single or multiple thymidine glycol residues or tetrahydrofuran residues (mimicking apurinic sites) will be used for studying the influence of structural defects on the thermal stability and structure of DNA. Fast methods for direct photometric detection of apurinic sites will be developed, as well as a method for testing oxidation damages in DNA using commercially available restriction endonucleases. The influence of thymidine glycol residues which are integrated into the transcription factor NF‐κB recognition site on the DNA binding capability of NF‐κB will be studied, as well as the influence of oxidation damages of thymidine in the non‐canonical G/T pair on mismatch repair (in cooperation with P. Friedhoff).

Titles for dissertations (prospective)

• Construction of photoregulated restriction endonucleases (ongoing project with A. Pingoud)

• Structural and functional studies of the DNA methyltransferase SsoII and its complexes with DNA

• DNA duplexes with chemically reactive groups as tools for the investigation of DNA‐protein interactions in repair and restriction/modification systems

• Molecular "anatomy" of nucleic acid/protein complexes involved in transcription regulation in bacterial cells

• Synthesis of modified DNAs containing thymidine glycol residues and investigation of the structure and biological properties of DNA fragments containing thymidine glycol residues

• Reactive 2'‐aldehyde oligonucleotides for the study of methyltransferase SsoII and ribonuclease P by cross‐linking

Relationships/connections within the research training group

Hartmann: RNase P Enzymes

Pingoud: Photocontrol of restriction enzyme activity

Wende: Chimeric nucleases for controlled specific gene targeting

Friedhoff: Free MutL ‐ the central coordinator in DNA mismatch repair

Alexeevski/Karyagina/Spirin: Comparative analysis of sequences and 3D structures of related biomolecules

Dontsova: Functional role of ribosome modification

Georgieva: The role of SAYP and ENY2 in transcription activation and formation of mRNP particle

Benefits of the scientific exchange

• Hartmann, Dontsova, Georgieva: RNA‐protein interactions, mapping of RNA‐protein binding interfaces

• Wende/Pingoud: Application of bifunctional azobenzene and dithienylethylene derivatives and modified oligodeoxyribonucleotides to control nuclease activity by light

• Friedhoff: Cross‐linking of MutS and MutL to DNA

• Alexeevski/Kariagina/Spirin: Molecular modelling of NA‐protein interactions, access to bioinformatic tools for students