State-of-the-art and preparatory work

The ribosome, a key component of the cellular gene expression apparatus, is a molecular machine responsible for translation of a messenger RNA (mRNA) into a protein. We have previously developed a strategy for characterizing rRNA regions that change their conformation in response to the alterations of the ribosome structure and which are spatially separated from the functional site under investigation. This strategy is based on combining various chemical approaches with sitedirected mutagenesis.

References

1. Sergiev, P.V., Serebryakova, M.V., Bogdanov, A.A. & Dontsova, O.A. (2008) The ybiN gene of Escherichia coli encodes adenine‐N6 methyltransferase specific for modification of A1618 of 23 S ribosomal RNA, a methylated residue located close to the ribosomal exit tunnel. J Mol Biol 375, 291‐300.

2. Golovina, A.Y., Sergiev, P.V., Golovin, A.V., Serebryakova, M.V., Demina, I., Govorun, V.M. & Dontsova, O.A. (2009) The yfiC gene of E. coli encodes an adenine‐N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC). RNA 15, 1134‐1141.

Aims

The overall aims of this project are

I. in vitro investigation of mRNA translation. The main attention will be devoted to the identification of proteins, biosynthesis of which is influenced by ribosomal RNA or protein modification;

II. modifications of the hairpin 31 in the 16S rRNA and ribosome protein S6.

Work programme and methods

The main attention will be devoted to the identification of proteins, biosynthesis of which is influenced by ribosomal RNA or protein modification. For the identification of proteins, biosynthesis of which is influenced by ribosomal RNA or protein modification, system biology methods will be exploited, such as 2D gel electrophoresis and real‐time PCR. A set of E. coli strains will be used where every strain carries one inactivated gene of an rRNA modification or a ribosomal protein modification. Comparison of the protein composition between the two strains which differ only in a ribosome modification will lead to the identification of the proteins, synthesis of which can be influenced by the transcription apparatus modification. This influence can be direct (when the translation efficiency of the given mRNA is changed) or indirect (when the ribosome modification influences translation of other mRNAs which are connected with the given mRNA through a regulatory network). These two categories can be discriminated by additional methods. The real‐time PCR will allow comparing mRNA amounts. When the amount of a certain mRNA in a cell remains constant while the amount of the corresponding protein changes, it is likely that the ribosome modification influences exactly the translation stage.

The final task of the study will be in vitro investigation of mRNA translation. We have in our laboratory a system of mRNA step‐by‐step translation where all the translation apparatus components are recombinant. The efficiency of every translation stage will be checked by toe printing. This method is based on the reverse transcription of the mRNA being translated and allows determining exactly the position of the ribosome moving along the mRNA. Among the rRNA modifications, we plan to study at first is the methylation of the nucleotide G1835 in the 23S rRNA.

This modified base is situated on the surface of the large subunit, between several molecular bridges with the small subunit. We plan to examine the influence of this modification onto the efficiency of the basic ribosome functions. The parameters of the subunit association, the efficiency of tRNA binding and the efficiency of the interaction of translation factors with the ribosome will be studied. Then, proteome analysis methods will be used to study the influence of the modification on the protein composition of a bacterial cell. Those genes will be identified whose expression is influenced by G1835 methylation. The expression stage influenced by G1835 methylation will be revealed using a set of reporter constructs. The regulatory networks which are subjected to such an influence will be identified.

The other object of our investigation will be modification of the hairpin 31 in the 16S rRNA. This hairpin contains two consecutive methylated bases, m2G966 and m5C967. According to data obtained by chemical footprinting and, later, by X‐ray crystallography, these bases form a part of the tRNA binding pocket in the P‐site. Since the direct tRNA binding with the P‐site takes place on the translation initiation stage, we will measure the efficiency of the individual steps of the initiationstage and the efficiency of the function of the initiation factors IF1, IF2 and IF3. Proteome analysis methods will be exploited to reveal the genes whose translation efficiency is influenced by the modification of the bases in the hairpin 31. The genes coding for the proteins detected by the proteome analysis will be investigated using the reporter constructs.

The third object of the study will be the modification of the ribosome protein S6. This protein is modified in a very uncommon way – four glutamic acid residues are enzymatically connected with its C‐terminus. Surprisingly, a bacterial cell has a special enzyme for this purpose, RimK, while these four residues could have been encoded in the gene of the S6 protein. The functional role of this modification will be studied by proteome analysis. A set of bacterial strains will also be constructed which lack at all these residues or have them encoded in the S6 gene. The protein composition of these strains will be studied by the proteome analysis and by the analysis of the gene expression regulation using the reporter constructions.

Titles for dissertations (prospective)

• Translational control by rRNA modification: role of RlmG methyltransferase

• Interaction of ribosome with rRNA‐specific methyltransferases: protein‐RNA recognition and application for site‐specific labeling of ribosomes

• Role of m2G966 and m5C967 16S rRNA modifications in translation initiation and translational control

Relationships/connections within the research training group

Hartmann: RNase P Enzymes

Kubareva/Оretskaya: Investigation of nucleic acids‐protein and protein‐protein interactions taking part in gene expression regulation in bacterial cells using modified oligonucleotides

Benefits of the scientific exchange

Hartmann: RNA‐protein interactions

Kubareva/Оretskaya: Application of azobenzene derivatives and modified oligoribonucleotides to study ribosome functioning