The Conformational Space of RNase P RNA in Solution

Academic Background

The conformational diversity of RNA plays a crucial role in biology, particularly in processes such as RNA splicing, packaging, cellular transcriptional activation, and responses to environmental stimuli. However, traditional biophysical techniques have been unable to directly visualize the complete conformational space of RNA in solution. RNase P RNA is an RNA enzyme found in all forms of life, responsible for editing the 5’ end of precursor tRNA (pre-tRNA). Due to its broad substrate specificity, RNase P RNA is believed to exhibit significant conformational flexibility, which exists in a complex balance with the structural rigidity required for its enzymatic activity. Understanding this relationship between conformational flexibility and structural rigidity is essential for elucidating the function and biological significance of RNA.

Source of the Paper

This paper was co-authored by Yun-Tzai Lee, Maximilia F. S. Degenhardt, Ilias Skeparnias, Hermann F. Degenhardt, Yuba R. Bhandari, Ping Yu, Jason R. Stagno, Lixin Fan, Jinwei Zhang, and Yun-Xing Wang. The research team is affiliated with the National Cancer Institute (NCI), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and Leidos Biomedical Research, Inc. The paper was published in Nature in 2024.

Research Process

1. Preparation of the Research Object

The study used full-length RNase P RNA from Geobacillus stearothermophilus (Gst). The RNA was prepared through in vitro transcription (IVT) and purified using fast protein liquid chromatography (FPLC). The purified RNA was imaged using atomic force microscopy (AFM) at a physiologically relevant Mg²⁺ concentration of 1 mM.

2. Solution Atomic Force Microscopy (AFM) Imaging

AFM imaging was conducted at 4°C using a Cypher VRS AFM instrument. RNA molecules were immobilized on a mica surface treated with 1-(3-aminopropyl)silatrane (APS). The resolution of the AFM images was 10-15 Å, allowing for clear identification of the RNA’s major structural features and overall folding.

3. Determination of Conformational Structures

A total of 158 individual RNA molecules were selected from the AFM images, and the three-dimensional topological structures of each molecule were determined using the HORNET software package. These conformational structures were validated using small-angle X-ray scattering (SAXS) data and classified into three categories based on their radius of gyration (Rg): C1 (compact), C2 (intermediate), and C3 (extended).

4. Analysis of the Conformational Space

The conformational space of the RNA molecules was analyzed using principal component analysis (PCA) and atomic motion similarity matrix (AMSM). The results showed that the S-domain of RNase P RNA (including P7-P9, P10.1, and P12) exhibited the largest spatial motions, with amplitudes reaching up to 55 Å. Additionally, the study revealed correlated motions between different structural domains of the RNA.

5. Effect of Mg²⁺ on the Conformational Space

The effect of Mg²⁺ concentration on the conformational space of RNase P RNA was investigated using SAXS and enzymatic activity assays. The results showed that as the Mg²⁺ concentration increased, the RNA molecules became more compact, and their enzymatic activity increased. Notably, at Mg²⁺ concentrations above 1 mM, the conformational space of the RNA significantly narrowed, and its enzymatic activity markedly improved.

6. Correlation Between Sequence and Conformational Dynamics

The relationship between sequence conservation and conformational dynamics in RNase P RNA was studied through phylogenetic analysis and conformational dynamics analysis. The results indicated that certain regions of the RNA, despite having low sequence conservation, exhibited high conformational conservation, likely driven by structural complementarity. Conversely, other regions with high sequence conservation displayed high conformational dynamics, which may be functionally relevant.

Main Results

1. Conformational Diversity: RNase P RNA exhibited extensive conformational diversity in solution, with the S-domain (including P7-P9, P10.1, and P12) showing the largest spatial motions, with amplitudes up to 55 Å.
2. Effect of Mg²⁺: As the Mg²⁺ concentration increased, the RNA molecules became more compact, and their enzymatic activity increased. Notably, at Mg²⁺ concentrations above 1 mM, the conformational space of the RNA significantly narrowed, and its enzymatic activity markedly improved.
3. Correlation Between Sequence and Conformational Dynamics: Certain regions of the RNA, despite having low sequence conservation, exhibited high conformational conservation, likely driven by structural complementarity. Conversely, other regions with high sequence conservation displayed high conformational dynamics, which may be functionally relevant.

Conclusion

This study, through solution AFM, deep neural networks, and statistical analysis, directly visualized the complete conformational space of RNase P RNA in solution for the first time. The research revealed the conformational diversity of RNase P RNA and its relationship with enzymatic activity and substrate specificity. Additionally, the study demonstrated a complex correlation between sequence conservation and conformational dynamics, providing new insights into the function and evolution of RNA.

Research Highlights

1. Direct Visualization of RNA Conformational Space: This study is the first to directly visualize the complete conformational space of RNA in solution using solution AFM, providing a new method for studying RNA structure and dynamics.
2. Revealing RNA Conformational Diversity: The research revealed the extensive conformational diversity of RNase P RNA in solution, particularly the large spatial motions in the S-domain.
3. Effect of Mg²⁺ on RNA Conformation and Enzymatic Activity: The study showed that increasing Mg²⁺ concentration significantly narrowed the conformational space of RNA and enhanced its enzymatic activity.
4. Correlation Between Sequence and Conformational Dynamics: The research revealed a complex correlation between RNA sequence conservation and conformational dynamics, providing new perspectives for understanding RNA function and evolution.

Research Significance

This study not only provides new insights into the structure and function of RNase P RNA but also offers a new method for studying the conformational dynamics of other RNA molecules. Additionally, the research revealed a complex correlation between RNA sequence conservation and conformational dynamics, providing new perspectives for understanding RNA function and evolution.