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Substrate Recognition by Human Mitochondrial RNase P: Moonlighting of a tRNA Methyltransferase
Author(s) -
Liu Xin,
Howard Michael,
Klemm Bradley,
Fierke Carol
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.573.14
Subject(s) - transfer rna , rnase p , ribonucleoprotein , ribonuclease , rna , biochemistry , rnase mrp , biology , protein subunit , microbiology and biotechnology , chemistry , gene
Ribonuclease P (RNase P) catalyzes removal of 5′ end leader sequences from precursor tRNA (pre‐tRNA). In most organisms, RNase P is a ribonucleoprotein complex with a catalytic RNA subunit. However, a recently discovered human mitochondrial RNase P (mtRNase P) is composed solely of three protein subunits: MRPP1, 2 and 3. The MRPP1•MRPP2 subcomplex contains m 1 N9 tRNA methyltransferase (MRPP1), while MRPP3 belongs to a new class of metallonucleases. We reconstituted human mtRNase P proteins in vitro and measured reactivity with different types of pre‐tRNA substrate to study substrate recognition. For a canonical pre‐tRNA, transient kinetic measurements show that the MRPP1•MRPP2 subcomplex activates the endonuclease activity of MRPP3 by about 2000‐fold and enhances substrate affinity by 50‐fold. For a non‐canonical human mt‐pre‐tRNA, significant miscleavage was observed with MRPP3 alone but the MRPP1•MRPP2 subcomplex rescued miscleavage and enhanced the cleavage rate constant by 1000‐fold. In addition, in vitro pull‐down results indicate that MRPP3 mainly interacts with the (MRPP1•MRPP2)•pre‐tRNA complex. These data suggest that MRPP3 recognizes the MRPP1•MRPP2‐bound pre‐tRNA as substrate. The unusual structural features of human mitochondrial tRNA may have been part of the evolutionary driving force for this unique strategy for substrate recognition. We are carrying out further investigations into the molecular details of this mechanism using transient and steady‐state kinetics, structure probing, and mass spectrometry. [(This work is supported by funding from NIH (GM 55387)]