Ribosomal RNA methylation studied by NMR spectroscopy

Enzymes are the biochemical workhorses of living organisms, facilitating essential chemical reactions and controlling their speed. To fulfil this task, enzymes fold into a specific three-dimensional structure. However, for catalyzing chemical reactions a certain level of enzyme structural flexibility is required. For example, structural flexibility can be necessary to facilitate effective substrate recognition and binding, and to enable re- arrangements at the catalytic site of the enzyme that are required for optimal efficiency. In addition, it has bee recognized in the last decade that even low-populated conformers that are only transiently formed can be of relevance and must be taken into account. With few exceptions, however, standard X-ray diffraction and nuclear magnetic resonance (NMR) spectroscopic studies of enzyme structures focus on the most stable conformer and can therefore not explain all experimental observations. The aim of this stand-alone project of the FWF is to provide a comprehensive de- scription of the conformational flexibility of the enzyme RlmJ. This protein catalyzes the methylation of ribosomal ribonucleic acid (ribosomal RNA) at a single position and with high specificity. Ribosomal RNA is a component of the ribosome, a complex molecular machine that serves to translate genetic information into proteins in all living cells. Methylation of ribosomal RNA is one way of regulating the efficacy of transcription. For RlmJ, three-dimensional structural data are available, but the exact structural determinants of enzymatic catalysis are unclear. For example, it is not known how binding of substrate RNA and release of product RNA are regulated, how the catalytic sites of this enzyme is arranged, and what the functional role of structural flexibility could be. In our project we will use dynamic NMR spectroscopy to study the structural flexibility of RlmJ. This technique enables us to directly monitor the transitions between different conformers at atomic resolution, including low-populated conformers that are only transiently formed. Using chemical synthesis of isotope labeled and/or chemically modified substrate RNA, these data will be complemented by structure determination, NMR- observed enzymatic catalysis and other biophysical techniques. The integration of diverse sets of experimental data will enable us to observe both components of the system, protein and RNA, and to provide a quantitative description of the interplay between structure, flexibility and enzymatic catalysis, and to create a basis for understanding how methyltransferases work.

In this project of the Austrian Science Fund FWF we characterized the molecular function of an enzyme at atomic resolution. Enzymes are the biochemical workhorses of living organisms, facilitating essential chemical reactions and controlling their speed. To fulfil this task, enzymes fold into a specific three-dimensional structure. However, for catalyzing chemical reactions a certain level of enzyme structural flexibility is required. Structural flexibility can be necessary to facilitate effective substrate recognition and binding, and to enable re-arrangements at the catalytic site of the enzyme that are required for optimal efficiency. In addition, it has been recognized in the last decade that even low-populated conformers that are only transiently formed can be of relevance and must be taken into account. In our project we established a comprehensive de-scription of the conformational flexibility of the enzyme RlmJ. This particular enzyme catalyzes the methylation of ribosomal ribonucleic acid (ribosomal RNA) at a single position and with high specificity. Ribosomal RNA is a component of the ribosome, a complex molecular machine that serves to translate genetic information into proteins in all living cells. Methylation of ribosomal RNA is one way of regulating the efficacy of transcription. For RlmJ, the exact structural determinants of enzymatic catalysis are unclear. In our work we used dynamic nuclear magnetic resonance (NMR) spectroscopy to study the structural flexibility of RlmJ in detail. This technique enables us to directly monitor the transitions between different conformers at atomic resolution, including low-populated conformers that are only transiently formed. Chemical synthesis of isotope labeled and/or chemically modified substrate RNA, was used to complement this information, as well as NMR-observed enzymatic catalysis and other biophysical techniques. The integration of diverse sets of experimental data enabled us to observe both components of the system, protein and RNA, and to provide a detailed description of the interplay between structure, flexibility and enzymatic catalysis, and to create a basis for understanding how methyltransferases work. Our results show that structural flexibility indeed plays a decisive role for RNA methylation.