The L7Ae protein binds to two kink-turns in the Pyrococcus furiosus RNase P RNA

Tanimoto

et al. 2017

Nucleic Acids Research

Self Cite

RNase P is primarily responsible for the 5΄ maturation of transfer RNAs (tRNAs) in all domains of life. Archaeal RNase P is a ribonucleoprotein made up of one catalytic RNA and five protein cofactors including L7Ae, which is known to bind the kink-turn (K-turn), an RNA structural element that causes axial bending. However, the number and location of K-turns in archaeal RNase P RNAs (RPRs) are unclear. As part of an integrated approach, we used native mass spectrometry to assess the number of L7Ae copies that bound the RPR and site-specific hydroxyl radical-mediated footprinting to localize the K-turns. Mutagenesis of each of the putative K-turns singly or in combination decreased the number of bound L7Ae copies, and either eliminated or changed the L7Ae footprint on the mutant RPRs. In addition, our results support an unprecedented ‘double K-turn’ module in type A and type M archaeal RPR variants.

“…Moreover, to correlate binding of L7Ae to the multiple sites, the occupancy of L7Ae at each site in the wild-type and mutant PfuRPRs was mapped using OH • -mediated footprinting (32). …”

Section: Resultsmentioning

confidence: 99%

“…RPRs were refolded as described earlier for MS (35), footprinting (32) and pre-tRNA processing (11,16) experiments. See Supplementary Information for additional details.…”

Section: Methodsmentioning

confidence: 99%

A novel double kink-turn module in euryarchaeal RNase P RNAs

Tanimoto

et al. 2017

Nucleic Acids Research

Self Cite

Annals of the New York Academy of Sciences

“…K‐turn motifs are found in a wide group of RNAs, including rRNAs, C/D box s(no)RNAs, and mRNAs, and can act as binding sites for diverse RNA binding proteins (RBPs) . L7Ae can specifically recognize k‐turn motifs using two RNA‐binding interfaces . An RNA immunoprecipitation‐seq approach was used to identify RNA molecules that are copurified with genomically tagged L7Ae in the hypertermophilic crenarchaeon S. acidocaldarius .…”

Section: Rna‐binding Proteinsmentioning

confidence: 99%

“…38 L7Ae can specifically recognize k-turn motifs using two RNA-binding interfaces. 40 An RNA immunoprecipitation-seq approach was used to identify RNA molecules that are copurified with genomically tagged L7Ae in the hypertermophilic crenarchaeon S. acidocaldarius. These experiments identified more than hundred different RNA interactors of L7Ae, including 59 C/D box sRNAs, RNase P, and SRP RNA.…”

Section: Rna-binding Proteinsmentioning

confidence: 99%

RNA stabilization in hyperthermophilic archaea

Gomes-Filho

Randau

2019

Analyses of the RNA metabolism of hyperthermophilic archaea highlight the efficiency of regulatory RNAs and RNA‐guided processes at extreme temperatures. These organisms must overcome the intrinsic thermolability of RNAs. Elevated levels of RNA modifications and structured GC‐rich regions are observed for many universal noncoding RNA families. Guide RNAs are often protected from degradation by their presence within ribonucleoprotein complexes. Modification and ligation of RNA termini can be employed to impair exonucleolytic degradation. Finally, antisense strand transcription promotes the formation of RNA duplexes and can be used to stabilize RNA regions. In our review, we provide examples of these RNA stabilization mechanisms that have been observed in hyperthermophilic archaeal model organisms.

Biochemical and Biophysical Research Communications

“…Based on the consensus sequence, we found possible K-turn motifs at positions 141-148 and 170-174 in SL12 and at positons 241-249 and 259-264 in SL16, where they contain asymmetric internal loops with G-C rich pairs [9,10]. Recently, hydroxyl radical-mediated footprinting carried out by Lai et al predicted the presence of two K-turn motifs in stem-loop structures containing helices P12 and P16 in P. furiosus RNase P RNA (PfupRNA), which correspond to SL12 and SL16 in PhopRNA [11]. However, nucleotides predicted for the K-turn in PfupRNA P12 are slightly distinct from those predicted to be folded into the K-turn in SL12, although nucleotides in PfupRNA P16 are in perfect agreement with those in SL16 in PhopRNA [11].…”

Section: Introductionmentioning

confidence: 99%

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

Oshima

Kakiuchi

Tanaka

et al. 2016

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 β-strand (β1) 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 β4 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.