Protein-only RNase P function in Escherichia coli: viability, processing defects and differences between PRORP...

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• The RNase P enzyme family includes diverse ribonucleoproteins and single polypeptides.
• RNase P is essential for tRNA maturation in all domains of life.

Purpose of the Study:

• To investigate the functional conservation and substrate specificity of Arabidopsis thaliana single-polypeptide RNase P enzymes (AtPRORP1, 2, 3) in a bacterial system.
• To compare the substrate spectrum of plant RNase P enzymes with bacterial RNase P.

Main Methods:

• Complementation assay in Escherichia coli with lethal knockdown of endogenous RNase P.
• RNA sequencing (RNA-Seq) to analyze precursor tRNA processing.
• Assessment of non-tRNA substrate processing (4.5S RNA).

Main Results:

• AtPRORP1, 2, and 3 conferred viability to E. coli lacking endogenous RNase P.
• AtPRORP1 exhibited aberrant cleavage of specific precursor tRNAs in E. coli, particularly those with G:C-rich acceptor stem extensions.
• AtPRORP1 and 3 were defective in processing E. coli pre-tRNASec, leading to its degradation.
• AtPRORP1, 2, and 3 failed to process the primary transcript of 4.5S RNA, unlike bacterial RNase P.

Conclusions:

• Plant single-polypeptide RNase P enzymes (PRORPs) can functionally substitute for bacterial RNase P in vivo.
• PRORPs possess a narrower, more tRNA-centric substrate spectrum compared to bacterial RNase P.
• The essential function of the signal recognition particle can be maintained with a 5'-extended 4.5S RNA in E. coli.