Ribosomal protein L7Ae is a subunit of archaeal RNase P

Abstract

To the mounting evidence of nonribosomal functions for ribosomal proteins, we now add L7Ae as a subunit of archaeal RNase P, a ribonucleoprotein (RNP) that catalyzes 5′-maturation of precursor tRNAs (pre-tRNAs). We first demonstrate that L7Ae coelutes with partially purified Methanococcus maripaludis (Mma) RNase P activity. After establishing in vitro reconstitution of the single RNA with four previously known protein subunits (POP5, RPP21, RPP29, and RPP30), we show that addition of L7Ae to this RNase P complex increases the optimal reaction temperature and k cat ∕K m (by ∼360-fold) for pre-tRNA cleavage to those observed with partially purified native Mma RNase P. We identify in the Mma RNase P RNA a putative kink-turn (K-turn), the structural motif recognized by L7Ae. The large stimulatory effect of Mma L7Ae on RNase P activity decreases to ≤4% of wild type upon mutating either the conserved nucleotides in this K-turn or amino acids in L7Ae shown to be essential for K-turn binding. The critical, multifunctional role of archaeal L7Ae in RNPs acting in tRNA processing (RNase P), RNA modification (H/ACA, C/D snoRNPs), and translation (ribosomes), especially by employing the same RNA-recognition surface, suggests coevolution of various translation-related functions, presumably to facilitate their coordinate regulation.pre-tRNA processing | RPP38 | protein-aided RNA catalysis R Nase P is a Mg 2þ -dependent endoribonuclease that is primarily responsible for catalyzing the removal of the 5′ leaders of precursor-tRNAs (pre-tRNAs) (1-3). Except for some unique organellar variants, RNase P functions in all three domains of life as a ribonucleoprotein (RNP) (1, 2). Although catalysis rests with the essential RNase P RNA (RPR) in all three domains of life (4-6), the RNase P protein (RPP) cofactors play essential roles. In the simple one RPR-one RPP bacterial RNase P, the RPP aids RPR catalysis by enhancing cleavage efficiency and affinity for substrate and Mg 2þ (7-9). The bacterial RPP has not been found in any archaeal or eukaryal genome (10). Eukaryal (nuclear) RNase P, which comprises an RPR and 9 or 10 RPPs (11, 12), has not been reconstituted from recombinant subunits, thus thwarting efforts to uncover the individual functions of eukaryal RPPs. Archaeal RNase P, with an RPR and four RPPs (all homologous to eukaryal RPPs), has therefore been explored as an experimental surrogate for its so-far-intractable eukaryal cousin (13-16). Although native archaeal RNase P has not been characterized, Western analysis and immunoprecipitation validated these four RPPs (POP5, RPP21, RPP29, and RPP30) as being associated with partially purified Methanothermobacter thermautotrophicus (Mth) RNase P activity (14). Subsequent structural and biochemical reconstitution studies using recombinant subunits have proven the utility of archaeal RNase P as a model system to dissect the role of multiple protein cofactors in facilitating RNA catalysis (16).Besides POP5, RPP21, RPP29, and RPP30, weak homologies are evident in the archaeal genomes...

tRNA biology charges to the front

“…The accepted dogma of tRNA processing has always involved synthesis of a precursor tRNA containing a 59 leader sequence, which was subsequently removed by the endonuclease RNase P (Walker and Engelke 2006). It has also been accepted dogma since the early 1980s that RNase P is a ribonucleoprotein (RNP), and that RNA is the catalytic component in bacteria (Guerrier-Takada et al 1983;Torres-Larios et al 2005), which have one protein component, and in archaea (Pannucci et al 1999), which have five protein components (Cho et al 2010). However, the source of RNase P catalysis was unknown in eukaryotes such as yeast or humans, which have nine (Table 1) or 10 protein subunits, respectively, in addition to their RNA components (Walker and Engelke 2006), particularly since their RNA components have substantial differences in regions important for stability and catalysis (Marquez et al 2005(Marquez et al , 2006.…”

Functional Coupling between a Distal Interaction and the Cleavage Site in Bacterial RNase-P-RNA-Mediated Cleavage

“…[1][2][3][4][5] Regardless of origin, RPR alone, without protein, is capable of cleaving various substrates. [6][7][8][9][10] Recent reports, however, demonstrate protein-based RNase-P-like activity.…”

The molecular recognition of kink-turn structure by the L7Ae class of proteins

“…In mammals, the 15.5-kDa protein binds to a k-turn in the U4 snRNA during the spliceosome cycle (Nottrott et al 1999;Vidovic et al 2000). L7Ae has recently been shown to be a subunit of archaeal RNaseP (Cho et al 2010) required for tRNA maturation. Thus, L7Ae-family proteins are rather general k-turn binding proteins that are important in ribosome structure, spliceosome assembly, and the site-specific modification of RNA.…”