RNase P can cleave a variety of substrates1,10,38
, but pre-tRNA is the only one that is common among all organisms. To decipher its function, it is important to understand two different aspects of pre-tRNA processing by RNase P: substrate specificity and the chemical mechanism of cleavage.
tRNA recognition by RNase P involves the highly conserved tRNA TΨC and D loops and the CR-II and CR-III in the S-domain of P RNA. Thus, regions with high sequence and structure conservation are involved in specific tertiary interactions, suggesting a universal mode of recognition among all RNase P’s. The presence of unpaired nucleotides next to the cleavage site is also an important feature for pre-tRNA recognition, although it is unclear whether this is a universal feature of all natural substrates1
. Finally, pre-tRNA is usually processed to form a 7 base pair long acceptor stem. An additional role of the interactions between CR-II and CR-III and tRNA may be to serve as a ‘ruler’ that ensures that the correct lengths are processed, although there is some flexibility as tRNA’s with acceptor stems 8 base pairs long can be processed39
. The interaction with the 3′ CCA end is also a key recognition feature, but may not be necessarily an RNA-RNA interaction in higher organisms. The L15 loop of P RNA is not found in eukarya or some archea40
and its function may be replaced by additional protein(s), suggesting that 3′ CCA intermolecular base pairing is not a universal interaction.
The second important aspect of RNase P function is the chemical mechanism of cleavage. Hydrolysis of a phosphodiester bond generates the mature 5′ RNA product. While it is not possible to propose a complete mechanism from a structure at this resolution, the RNase P-tRNA structures, together with extensive biochemical information, help identify the major active site components. The structure suggests that at least two distinct metals play a direct role. It is possible to propose a transition state model () where the M1 metal directly positions the scissile phosphate oxygens of the substrate and enables a hydroxyl ion to perform an SN
2-type nucleophilic substitution. In this scenario, the M2 metal ion stabilizes the transition state and mediates proton transfer to the 3′ scissile oxygen during product release, as previously proposed7
. Other universally conserved nucleotides in the vicinity appear to play a structural role in forming the correct structure and are not directly involved in catalysis, consistent with proposals that sequence conservation is largely the result of strong structural constraints19
. Hence, the RNaseP/tRNA complex reveals how the P RNA structure can serve as a scaffold to bind and orient metals and substrate properly. It appears that RNase P employs a two-metal ion catalytic mechanism, similar to other mechanisms proposed based on other large ribozyme structures41,42
and originally put forth as a general mechanism for many ribozymes43
The structural studies of the holoenzyme/tRNA complex help to show that all RNase P ribozymes share a common, RNA-based mechanism of RNA cleavage and recognition that involves two universally conserved structural modules. Adaptation through the addition of protein increases RNase P functionality by accurately positioning the 5′ leader pre-tRNA substrate and by contacting conserved regions of the P RNA structure. The unique tertiary fold of the P RNA utilizes shape complementarity, specific RNA-RNA contacts, and intermolecular base pairing to recognize its substrate efficiently. Within this tertiary fold, the universally conserved regions are crucial to form the active site scaffold and to create regions involved in tRNA recognition. In addition, both P RNA and the pre-tRNA help to coordinate two catalytically important metal ions essential for the putative mechanism of pre-tRNA cleavage. The RNase P/tRNA complex offers a glimpse into the transition from an ancient, RNA-based world to the present, protein-catalyst dominated world and affirms that RNA molecules can display comparable versatility and complexity.