Investigation of RNase P active site residues and catalytic domain interaction
- Date: 22 February, 09:30
- Location: B/C4:305, Biomedical Center, Husargatan 3, Uppsala
- Doctoral student: Mao, Guanzhong
- About the dissertation
- Organiser: Institutionen för cell- och molekylärbiologi
- Contact person: Mao, Guanzhong
RNase P is an essential endoribonuclease responsible for the maturation of the tRNA 5’end. The RNase P family encompasses the ribozyme based, RNase P RNP, and proteinaceous RNase P (PRORP). The ribozyme based RNase P is widely distributed in most species while PRORP has so far mainly been found in some eukaryotic cells.
The RNase P RNP contains one RNA subunit (RPR), which is the catalytic moiety, and one or more protein subunits. The structural topology of the RPR is crucial for RNase P RNP to correctly and efficiently maintain its function. The RPR is composed of domains such as the specificity (S) and catalytic (C) domains, and structural elements that connect these.
The objectives of my thesis were to study the importance of structural elements in the C-domain of the RPR with respect to substrate interaction and catalysis. Another objective was to study substrate interaction in PRORP-mediated catalysis, and to compare RNase P RNP- and PRORP-mediated cleavage. To achieve this I have studied cleavage of both pre-tRNA and model hairpin loop substrates with RPR variants carrying deletions and base substitutions, and PRORP1 from Arabidopsis thaliana.
My data provide evidence for an intra domain interaction, referred to as the P6-mimic, in the RPR C-domain. The P6-mimic forms when the S-domain of the RPR is deleted and it contributes to catalysis. The inter domain P8/P18 interaction, which connects the S- and the C-domains, plays an important role for catalysis. My data suggest that, in the absence of the S-domain, P18 does not contribute to catalysis raising the possibility that the P8/ P18-interaction acts as a structural mediator between the TSL/ TBS-interaction site in the S-domain and the active center that ensures correct and efficient cleavage. This is consistent with that RNase P RNP operates through an induced fit mechanism.
Furthermore, on the basis of biochemical and genetic data the well-conserved A248 in the RPR has been proposed to form a cis Watson-Crick/Watson-Crick (cis WC/WC) pair with the residue immediately 5' of the cleavage site, N-1, in the substrate. My data does not support this cis WC/WC pairing. Rather, the data are consistent with a model where the structural topology of the active site varies and depends on the identity of the nucleobases at, and in proximity to, the cleavage site and their potential to interact. As a consequence, this affects the positioning of Mg2+ that activates the water that acts as the nucleophile resulting in efficient and correct cleavage. In this scenario it is suggested that the role of A248 is to exclude bulk water from accessing the amino acid acceptor stem and thereby prevent non-specific hydrolysis of the pre-tRNA. In a broader perspective, base stacking might be a way to prevent access of water to functionally important base pairing interactions, and thereby ensuring high fidelity during RNA processing and decoding of mRNA.
As for RNase P RNP, my studies on PRORP1 indicate the importance of the identity of N-1 and the N-1: N+73 base pair in the substrate for efficient and correct cleavage. Although, the data indicate similarities they also provide key differences in substrate recognition by RNase P RNP and PRORP1 where the RNP form appears to require more recognition determinants for cleavage site selection.