Ribosomal RNA dynamics studied by NMR-spectroscopy

Abstract

The ribosome is a large macromolecular machine that consists of both ribosomal proteins and ribosomal RNA (rRNA). It is a complex consisting of two subunits held together by non-covalent interactions, intersubunit bridges and some of these bridging interactions are mediated by the rRNA. In this PhD-project the dynamics of such regions of rRNA, participating in intersubunit bridges and tertiary interaction within the rRNA have been investigated with solution state NMR-spectroscopy. These studies have been performed in the context of several miniaturized RNA systems, containing sequences of E. coli 16S rRNA. In particular, regions along helix 44 (h44), the penultimate stem of E. coli 16S rRNA have been studied. The stem-loop part of h44 has been studied in detail, this part of the rRNA contains a naturally occurring UUCG-loop and adenines participating in a tertiary interaction with helix (h8) in the 16S rRNA.

In order to characterize the dynamics of these RNA constructs with NMR-spectroscopy, purified RNA material in large amounts is a necessity. Because of this we have developed an RNA-sample production method (Paper I) as well an NMR-experiment method (Paper II) that we call SELOPE, a method that can reduce the need of using isotopically enriched RNA material for NMR-studies. The first chapter of this thesis introduces the underlying theory for RNA-sample preparation as well as alternative techniques compared to the ones used in Paper I. In a similar manner the underlying theory for the NMR-technique is introduced and with some emphasis on concepts crucial for understanding the SELOPE experiment, to contextualize Paper II. The usage of NMR-spectroscopy for the measurements of dynamics in RNA molecules is also introduced. The first chapter of the thesis also includes a description of the ribosome to help further understanding of Paper III. In addition, during chapter 2-5 of this thesis some work related to 1H-R1r characterization of chemical exchange and cross-relaxation among RNA imino protons is described and discussed.

In Paper I, the development of an RNA-sample preparation method is described. The method is based on in vitro transcription of the wanted RNA sequence followed by a HPLC-purification procedure that uses two different HPLC techniques for the purification, both Reverse Phase Ion Pairing (RP-IP) and Ion Exchange (IE) HPLC. The complete method offers a robust and versatile alternative to other RNA sample preparation methods such as preparative gel electrophoresis techniques. In Paper II, we describe the development of an NMR pulse sequence that utilize a homonuclear magnetization transfer block in unlabeled RNA molecules. The pulse sequence can then for instance be used to transfer NMR signal of unwanted signals to other spectral regions and can for instance be used to remove the signal of pyrimidine H6s from the region of H6/H8/H2 in RNAs. In Paper III, the work of characterizing the stem-loop part of E. coli 16S rRNA h44 is described. This work both shows a UUCG-loop with dynamics on a millisecond time-scale as well as the dynamical behavior of a group of unpaired adenine bases, the study of dynamics of these adenines could aid the understanding of tertiary interactions within rRNA.