Functional implication of archaeal homologues of human RNase P protein pair Pop5 and Rpp30

Abstract

PhoPop5 and PhoRpp30 in the hyperthermophilic archaeon Pyrococcus horikoshii, homologues of human ribonuclease P (RNase P) proteins hPop5 and Rpp30, respectively, fold into a heterotetramer [PhoRpp 30(PhoPop5) 2 PhoRpp30], which plays a crucial role in the activation of RNase P RNA (PhopRNA). Here, we examined the functional implication of PhoPop5 and PhoRpp30 in the tetramer. Surface plasmon resonance (SPR) analysis revealed that the tetramer strongly interacts with an oligonucleotide including the nucleotide sequence of a stem-loop SL3 in PhopRNA. In contrast, PhoPop5 had markedly reduced affinity to SL3, whereas PhoRpp30 had little affinity to SL3. SPR studies of PhoPop5 mutants further revealed that the C-terminal helix (a4) in PhoPop5 functions as a molecular recognition element for SL3. Moreover, gel filtration indicated that PhoRpp30 exists as a monomer, whereas PhoPop5 is an oligomer in solution, suggesting that PhoRpp30 assists PhoPop5 in attaining a functionally active conformation by shielding hydrophobic surfaces of PhoPop5. These results, together with available data, allow us to generate a structural and mechanistic model for the PhopRNA activation by PhoPop5 and PhoRpp30, in which the two Cterminal helices (a4) of PhoPop5 in the tetramer whose formation is assisted by PhoRpp30 act as binding elements and bridge SL3 and SL16 in PhopRNA.Keywords: archaea/proteinRNA interaction/ Pyrococcus horikoshii/ribonuclease P/surface plasmon resonance.Abbreviations: 3-D, three-dimensional; PhopRNA, ribonuclease P RNA from P. horikoshii; pre-tRNA, precursor tRNA; RNase P, ribonuclease P; RNP, ribonucleoprotein; RRM, RNA recognition motif; SL3, stem-loop containing P3 helix; SL16, stem-loop containing P16 helix; SPR, surface plasmon resonance.Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that catalyses the processing of 5 0 leader sequences from tRNA precursors (pre-tRNA) and other noncoding RNAs in all living cells (1, 2). In contrast to eubacterial RNase P RNAs, the RNA components in archaea and eukaryotes alone have little catalytic activity in vitro and function in cooperation with protein subunits in substrate recognition and catalysis (3). Hence, archaeal and eukaryotic RNase Ps may serve as a model ribonucleoprotein (RNP) for studying how a functional RNA can be activated by protein cofactors and how the RNP enzymes catalyse biological processes.We earlier found that RNase P RNA (PhopRNA) and five proteins in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 reconstituted RNase P activity that exhibits enzymatic properties like those of the authentic enzyme (4, 5). The P. horikoshii RNase P proteins were designated PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30 and PhoRpp38, according to their sequence homology with the human RNase P proteins hPop5, Rpp21, Rpp29, Rpp30 and Rpp38, respectively (6). Biochemical and structural studies revealed that PhoPop5 and PhoRpp21 form a complex with PhoRpp30 and PhoRpp29, and the resulting complexes, PhoPop5PhoRpp30 and Pho Rpp21PhoRpp29, are involved i...

Structural basis for recognition of a kink-turn motif by an archaeal homologue of human RNase P protein Rpp38

“…It is known that the binding of L7Ae family proteins to their cognate RNAs forms additional protein binding sites on RNAs, thereby enabling subsequent assembly of RNPs [23,24]. Since P16 in SL16, to which the PhoPop5-PhoRpp30 complex binds, is in close proximity to the K-turn in SL16 [25], it is likely that the PhoRpp38 binding to the K-turn in SL16 leads to a specific conformation of the 3-nucleotide bulge structure, the K-turn thus optimizing a double-stranded structure In this docking model, three PhoRpp38 were readily able to locate on K-turns in SL12 and SL16 without steric hindrance of the PhoPop5-PhoRpp30 complex which bridges P3 and P16 helices in PhopRNA.…”

Structural basis for activation of an archaeal ribonuclease P RNA by protein cofactors

“…SPR studies of PhoPop5 mutants further revealed that the C-terminal helix (α4) in PhoPop5 function as a molecular recognition element for SL3. 62) These results, together with available data, allow us to generate a mechanistic model for the PhopRNA activation by PhoPop5 and PhoRpp30, as shown in Fig. 4(C).…”

“…58) Moreover, surface plasmon resonance (SPR) analysis revealed that the tetramer strongly interacts with an oligonucleotide including the nucleotide sequence of SL3 in PhopRNA. 62) In contrast, PhoPop5 had markedly reduced affinity to SL3, and PhoRpp30 had little affinity to SL3. SPR studies of PhoPop5 mutants further revealed that the C-terminal helix (α4) in PhoPop5 function as a molecular recognition element for SL3.…”

“…61) A gel filtration study further indicated that PhoRpp30 exists as a monomer, whereas PhoPop5 is an oligomer in solution. 62) The X-ray structure shows that the N-terminal residues Leu5 to Pro9 in PhoPop5 interact with residues Pro110 to Ile112 in PhoRpp30 by hydrophobic interactions. 61) These findings suggest that PhoRpp30 serves as molecular chaperone which assists PhoPop5 in attaining a functionally active conformation by shielding hydrophobic surfaces of PhoPop5.…”

New insights into the role of ribonuclease P protein subunit p30 from tumor to internal reference

“…In archaea, bacteria, and yeast, RPP30/RPP30 paired with Pop5/Pop5, may be functionally reconstituted with the phylogenetically-conserved core catalytic domain (C domain) of the RNA subunit to promote the assembly of RNase P providing substrate RNA binding sites and activating the RNA subunit (probably by RNA annealing and strand displacement ( 41 ) and stabilize ionic interactions with the RNA subunit or the substrate pre-tRNA at a relatively lower salt concentration ( 1 , 22 , 42 – 44 ). In the hyperthermophilic archaeon Pyrococcushorikoshii , PhoRPP30 is homologous to human RPP30 and acts as a molecular chaperone of PhoPop5, which recognizes the stem-loop containing the P3 helix in PhopRNA ( 45 ). RPP30-Pop5 is a tight heterotetrameric complex that increases the affinity of the holoenzyme for Mg 2+ and protects the RNase P M1 RNA’s C domain from RNase T1 cleavage, especially near conserved nucleotides of RNase P in archaea whose RNase P protein is homologous to eukaryotic counterparts ( 36 , 46 – 48 ).…”