A novel carbohydrate-binding surface layer protein from the hyperthermophilic archaeon Pyrococcus horikoshii.

In Archaea and Bacteria, surface layer (S-layer) proteins form the cell envelope and are involved in cell protection. In the present study, a putative S-layer protein was purified from the crude extract of Pyrococcus horikoshii using affinity chromatography. The S-layer gene was cloned and expressed in Escherichia coli. Isothermal titration calorimetry analyses showed that the S-layer protein bound N-acetylglucosamine and induced agglutination of the gram-positive bacterium Micrococcus lysodeikticus. The protein comprised a 21-mer structure, with a molecular mass of 1,340 kDa, as determined using small-angle X-ray scattering. This protein showed high thermal stability, with a midpoint of thermal denaturation of 79 °C in dynamic light scattering experiments. This is the first description of the carbohydrate-binding archaeal S-layer protein and its characteristics.

A three-dimensional model of RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Ribonuclease P (RNase P) is an endoribonuclease involved in maturation of the 5'-end of tRNA. We found previously that RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of a catalytic RNase P RNA (PhopRNA) and five protein cofactors designated PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38. The crystal structures of the five proteins have been determined, a three-dimensional (3-D) model of PhopRNA has been constructed, and biochemical data, including protein-RNA interaction sites, have become available. Here, this information was combined to orient the crystallographic structures of the proteins relative to their RNA binding sites in the PhopRNA model. Some alterations were made to the PhopRNA model to improve the fit. In the resulting structure, a heterotetramer composed of PhoPop5 and PhoRpp30 bridges helices P3 and P16 in the PhopRNA C-domain, thereby probably stabilizing a double-stranded RNA structure (helix P4) containing catalytic Mg2+ ions, while a heterodimer of PhoRpp21 and PhoRpp29 locates on a single-stranded loop connecting helices P11 and P12 in the specificity domain (S-domain) in PhopRNA, probably forming an appropriate conformation of the precursor tRNA (pre-tRNA) binding site. The fifth protein PhoRpp38 binds each kink-turn (K-turn) motif in helices P12.1, P12.2, and P16 in PhopRNA. Comparison of the structure of the resulting 3-D model with that of bacterial RNase P suggests transition from RNA-RNA interactions in bacterial RNase P to protein-RNA interactions in archaeal RNase P. The proposed 3-D model of P. horikoshii RNase P will serve as a framework for further structural and functional studies on archaeal, as well as eukaryotic, RNase Ps.

Functional characterization of archaeal homologs of human nuclear RNase P proteins Rpp21 and Rpp29 provides insights into the molecular basis of their cooperativity in catalysis.

Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the processing of 5' leader sequences of precursor tRNAs (pre-tRNA). RNase P proteins PhoRpp21 and PhoRpp29 in the hyperthermophilic archaeon Pyrococcus horikoshii, homologs of human nuclear RNase P proteins Rpp21 and Rpp29 respectively, fold into a heterodimeric structure and synergistically function in the activation of the specificity domain (S-domain) in RNase P RNA (PhopRNA). To elucidate the molecular basis for their cooperativity, we first analyzed binding ability to PhopRNA using a pull-down assay. The result showed that PhoRpp21 is able to bind to PhopRNA in the absence of PhoRpp29, whereas PhoRpp29 alone has reduced affinity to PhopRNA, suggesting that PhoRpp21 primarily functions as a binding element for PhopRNA in the PhoRpp21-PhoRpp29 complex. Mutational analyses suggested that although individual positively charged clusters contribute little to the PhopRNA binding, Lys53, Lys54, and Lys56 at the N-terminal helix (α2) in PhoRpp21 and 10 C-terminal residues in PhoRpp29 are essential for PhopRNA activation. Moreover, deletion of a single stranded loop linking P11 and P12 helices in the PhopRNA S-domain impaired the PhoRpp21-PhoRpp29 complex binding to PhopRNA. Collectively, the present results suggest that PhoRpp21 binds the loop between P11 and P12 helices through overall positively charged clusters on the surface of the complex and serves as a scaffold for PhoRpp29 to optimize structural conformation of its N-terminal helix (α2) in PhoRpp21, as well as C-terminal residues in PhoRpp29, for RNase P activity.

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

PhoRpp38 in the hyperthermophilic archaeon Pyrococcus horikoshii, a homologue of human ribonuclease P (RNase P) protein Rpp38, belongs to the ribosomal protein L7Ae family that specifically recognizes a kink-turn (K-turn) motif. A previous biochemical study showed that PhoRpp38 specifically binds to two stem-loops, SL12 and SL16, containing helices P12.1/12.2 and P15/16 respectively, in P. horikoshii RNase P RNA (PhopRNA). In order to gain insight into the PhoRpp38 binding mode to PhopRNA, we determined the crystal structure of PhoRpp38 in complex with the SL12 mutant (SL12M) at a resolution of 3.4 Å. The structure revealed that Lys35 on the ß-strand (ß1) and Asn38, Glu39, and Lys42 on the α-helix (α2) in PhoRpp38 interact with characteristic G•A and A•G pairs in SL12M, where Ile93, Glu94, and Val95, on a loop between α4 and ß4 in PhoRpp38, interact with the 3-nucleotide bulge (G-G-U) in the SL12M. The structure demonstrates the previously proposed secondary structure of SL12, including helix P12.2. Structure-based mutational analysis indicated that amino acid residues involved in the binding to SL12 are also responsible for the binding to SL16. This result suggested that each PhoRpp38 binds to the K-turns in SL12 and SL16 in PhopRNA. A pull-down assay further suggested the presence of a second K-turn in SL12. Based on the present results, together with available data, we discuss a structural basis for recognition of K-turn motifs in PhopRNA by PhoRpp38.

Mutation of the gene encoding the ribonuclease P RNA in the hyperthermophilic archaeon Thermococcus kodakarensis causes decreased growth rate and impaired processing of tRNA precursors.

Ribonuclease P (RNase P) catalyzes the processing of 5' leader sequences of tRNA precursors in all three phylogenetic domains. RNase P also plays an essential role in non-tRNA biogenesis in bacterial and eukaryotic cells. For archaeal RNase Ps, additional functions, however, remain poorly understood. To gain insight into the biological function of archaeal RNase Ps in vivo, we prepared archaeal mutants KUWΔP3, KUWΔP8, and KUWΔP16, in which the gene segments encoding stem-loops containing helices, respectively, P3, P8 and P16 in RNase P RNA (TkopRNA) of the hyperthermophilic archaeon Thermococcus kodakarensis were deleted. Phenotypic analysis showed that KUWΔP3 and KUWΔP16 grew slowly compared with wild-type T. kodakarensis KUW1, while KUWΔP8 displayed no difference from T. kodakarensis KUW1. RNase P isolated using an affinity-tag from KUWΔP3 had reduced pre-tRNA cleavage activity compared with that from T. kodakarensis KUW1. Moreover, quantitative RT-PCR (qRT-PCR) and Northern blots analyses of KUWΔP3 showed greater accumulation of unprocessed transcripts for pre-tRNAs than that of T. kodakarensis KUW1. The current study represents the first attempt to prepare mutant T. kodakarensis with impaired RNase P for functional investigation. Comparative whole-transcriptome analysis of T. kodakarensis KUW1 and KUWΔP3 should allow for the comprehensive identification of RNA substrates for archaeal RNase Ps.

On archaeal homologs of the human RNase P proteins Pop5 and Rpp30 in the hyperthermophilic archaeon Thermococcus kodakarensis.

The ribonuclease P (RNase P) proteins TkoPop5 and TkoRpp30, homologs of human Pop5 and Rpp30, respectively, in the hyperthermophilic archaeon Thermococcus kodakarensis were prepared and characterized with respect to pre-tRNA cleavage activity using the reconstitution system of the well-studied Pyrococcus horikoshii RNase P. The reconstituted particle containing TkoPop5 in place of the P. horikoshii counterpart PhoPop5 retained pre-tRNA cleavage activity comparable to that of the reconstituted P. horikoshii RNase P, while that containing TkoRpp30 instead of its corresponding protein PhoRpp30 had slightly lower activity than the P. horikoshii RNase P. Moreover, we determined crystal structures of TkoRpp30 alone and in complex with TkoPop5. Like their P. horikoshii counterparts, whose structures were solved previously, TkoRpp30 and TkoPop5 fold into TIM barrel and RRM-like fold, respectively. This finding demonstrates that RNase P proteins in T. kodakarensis and P. horikoshii are interchangeable and that their three-dimensional structures are highly conserved.

Enhancement of RNA annealing and strand displacement found in archaeal ribonuclease P proteins is conserved in Escherichia coli protein C5 and yeast protein Rpr2.

We analyzed modes of action of ribonuclease P (RNase P) proteins, C5 in Escherichia coli and Rpr2 in Saccharomyces cerevisiae, using a pair of complementary fluorescence-labeled oligoribonucleotides. Fluorescence resonance energy transfer-based assays revealed that RNA annealing and strand displacement activities found in archaeal RNase P proteins are prevalent in eubacterial (C5) and eukaryotic (Rpr2) RNase P proteins.

Extra-structural elements in the RNA recognition motif in archaeal Pop5 play a crucial role in the activation of RNase P RNA from Pyrococcus horikoshii OT3.

Ribonuclease P (RNase P) is a ribonucleoprotein complex essential for the processing of 5' leader sequences of precursor tRNAs (pre-tRNA). PhoPop5 is an archaeal homolog of human RNase P protein hPop5 involved in the activation of RNase P RNA (PhopRNA) in the hyperthermophilic archaeon Pyrococcus horikoshii, probably by promoting RNA annealing (AN) and RNA strand displacement (SD). Although PhoPop5 folds into the RNA recognition motif (RRM), it is distinct from the typical RRM in that it has an insertion of α-helix (α2) between α1 and ß2. Biochemical and structural data have shown that the dimerization of PhoPop5 through the loop between α1 and α2 is required for the activation of PhopRNA. In addition, PhoPop5 has additional helices (α4 and α5) at the C-terminus, which pack against one face of the ß-sheet. In this study, we examined the contribution of the C-terminal helices to the activation of PhopRNA using mutation analyses. Reconstitution experiments and fluorescence resonance energy transfer (FRET)-based assays indicated that deletion of the C-terminal helices α4 and α5 significantly influenced on the pre-tRNA cleavage activity and abolished AN and SD activities, while that of α5 had little effect on these activities. Moreover, the FRET assay showed that deletion of the loop between α1 and α2 had no influence on the AN and SD activity. Further mutational analyses suggested that basic residues at α4 are involved in interaction with PhopRNA, while hydrophobic residues at α4 participate in interaction with hydrophobic residues at the ß-sheet, thereby stabilizing an appropriate orientation of the helix α4. Together, these results indicate that extra-structural elements in the RRM in PhoPop5 play a crucial role in the activation of PhopRNA.

Crystal structures of the archaeal RNase P protein Rpp38 in complex with RNA fragments containing a K-turn motif.

A characteristic feature of archaeal ribonuclease P (RNase P) RNAs is that they have extended helices P12.1 and P12.2 containing kink-turn (K-turn) motifs to which the archaeal RNase P protein Rpp38, a homologue of the human RNase P protein Rpp38, specifically binds. PhoRpp38 from the hyperthermophilic archaeon Pyrococcus horikoshii is involved in the elevation of the optimum temperature of the reconstituted RNase P by binding the K-turns in P12.1 and P12.2. Previously, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was determined at 3.4 Šresolution. In this study, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was improved to 2.1 Šresolution and the structure of PhoRpp38 in complex with the K-turn in P12.1 was also determined at a resolution of 3.1 Å. Both structures revealed that Lys35, Asn38 and Glu39 in PhoRpp38 interact with characteristic G·A and A·G pairs in the K-turn, while Thr37, Asp59, Lys84, Glu94, Ala96 and Ala98 in PhoRpp38 interact with the three-nucleotide bulge in the K-turn. Moreover, an extended stem-loop containing P10-P12.2 in complex with PhoRpp38, as well as PhoRpp21 and PhoRpp29, which are the archaeal homologues of the human proteins Rpp21 and Rpp29, respectively, was affinity-purified and crystallized. The crystals thus grown diffracted to a resolution of 6.35 Å. Structure determination of the crystals will demonstrate the previously proposed secondary structure of stem-loops including helices P12.1 and P12.2 and will also provide insight into the structural organization of the specificity domain in P. horikoshii RNase P RNA.

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

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 [PhoRpp30-(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 (α4) 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 C-terminal helices (α4) of PhoPop5 in the tetramer whose formation is assisted by PhoRpp30 act as binding elements and bridge SL3 and SL16 in PhopRNA.

Characterization of the peripheral structures of archaeal RNase P RNA from Pyrococcus horikoshii OT3.

Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5' leader sequence of precursor tRNA (pre-tRNA). The secondary structure of RNase P RNA (PhopRNA) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 has several peripheral stem-loops. In order to investigate their functional role, we prepared six mutants, ΔP1, ΔP3, ΔP8, ΔP9, ΔP12 and ΔP15, in which the stem-loops including helices P1, P3, P8, P9, P12/12.1/12.2 and P15/16 in PhopRNA were individually deleted, respectively, and characterized them with respect to pre-tRNA cleavage activity in the presence of five proteins and also to the ability to form a complex with the proteins. The reconstituted particles containing ΔP3, ΔP8 or ΔP9 retained considerable levels of activity (35-65%), while those containing ΔP1, ΔP12 or ΔP15 had markedly reduced activity (13%). It was further found that the reconstituted particles comprising ΔP3 or ΔP15 lacked PhoPop5 and PhoRpp30, whereas those containing ΔP1, ΔP8, ΔP9 or ΔP12 bound to all five proteins. Since it is known that PhoPop5 functions in a complex with PhoRpp30, the present result suggests that the peripheral stem-loops containing P3 or P15/16 are involved in the structural formation of a catalytic site by interacting with the protein complex PhoPop5-PhoRpp30.