Recognition of the 5′ leader of pre-tRNA substrates by the active site of ribonuclease P

Zahler, Nathan H.; Christian, Eric L.; Harris, Michael E.

doi:10.1261/rna.5220703

Cited by 78 publications

(167 citation statements)

References 44 publications

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“…Our assignment of the chemically active site is further corroborated by the spatial proximity of structural elements implicated in the recognition of the substrate precursors of tRNA. The base of the highly conserved A256 is thought to pair with nucleotide N -1 of the 5Ј-precursor sequence (49,50) and is positioned next to the cleft. Loop L15, shown to be involved in binding the substrate 3Ј-CCA (51), although partially disordered is also within consistent distance to the proposed active site (Fig.…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of a bacterial ribonuclease P RNA

Kazantsev

Krivenko

Harrington

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

202

230

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The x-ray crystal structure of a 417-nt ribonuclease P RNA from Bacillus stearothermophilus was solved to 3.3-Å resolution. This RNA enzyme is constructed from a number of coaxially stacked helical domains joined together by local and long-range interactions. These helical domains are arranged to form a remarkably flat surface, which is implicated by a wealth of biochemical data in the binding and cleavage of the precursors of transfer RNA substrate. Previous photoaffinity crosslinking data are used to position the substrate on the crystal structure and to identify the chemically active site of the ribozyme. This site is located in a highly conserved core structure formed by intricately interlaced long-range interactions between interhelical sequences.ribozyme ͉ RNA crystallography ͉ tRNA processing R Nase P catalyzes hydrolysis of a phosphodiester bond in precursors of transfer RNA (tRNA) to form the 5Ј-phosphorylated mature tRNA with the release of a 5Ј-precursor fragment (1, 2). RNase P homologs occur in all organisms, and the cellular RNase P always is a ribonucleoprotein that consists of one large RNA and one or more protein component. In bacteria, RNase P is typically comprised of a 350-to 400-nt RNA and one Ϸ120-aa basic protein. Although both RNA and protein components are necessary for cell viability, in vitro at high salt concentrations, bacterial RNase P RNA can act as a catalyst independently of protein (3). Bacterial RNase P is a ribozyme, an RNA-based enzyme.Knowledge of the structure of RNase P RNA is essential for understanding its function, and structure has been the focus of numerous studies of the RNA. Phylogenetic comparative analyses of RNase P RNA sequences have established the secondary and some tertiary structure of the RNA in a broad diversity of organisms (4-8). Photochemical crosslinking studies provided structural information to orient the helical elements and identified nucleotides associated with the active site of the RNA (9, 10). There are two major structural types of bacterial RNase P RNA, A (ancestral) and B (Bacillus), which differ in a number of structural elements attached to a homologous conserved structure. About two-thirds of any bacterial RNase P RNA is shown by sequence covariations to be involved in Watson-Crick base-pairing interactions, but the interactions that form the global structure have been speculative.To gain a better understanding of bacterial RNase P, we crystallized and solved the structure of a 417-nt B-type RNase P RNA from Bacillus stearothermophilus, a moderately thermophilic, low GϩC Gram-positive bacterium. Although the structure does not yet explain the chemical mechanism of catalysis, it is in agreement with a wealth of available biochemical and comparative data, and it provides a structural context for the chemically active site of this ribozyme. Materials and MethodsRNA Purification, Crystallization, and Data Collection. As detailed in supporting information, which is published on the PNAS web site, RNA was transcribed in vitro with T7 phage RNA ...

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Section: Discussionmentioning

confidence: 99%

Crystal structure of a bacterial ribonuclease P RNA

Kazantsev

Krivenko

Harrington

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

202

230

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“…Previous work has focused on the characterization of the interactions between substrate and PRNA in the ribozyme reaction (Christian et al 1998;Loria and Pan 1998;Christian and Harris 1999;Siew et al 1999;Zahler et al 2003) and demonstrated that the substrate directly contacts the P protein in RNase P (Crary et al 1998;Niranjanakumari et al 1998b). However, less is known about the interactions between the PRNA and the P protein within the holoenzyme complex.…”

Section: Introductionmentioning

confidence: 99%

Ionic interactions between PRNA and P protein in Bacillus subtilis RNase P characterized using a magnetocapture-based assay

Day-Storms¹,

Niranjanakumari²,

Fierke³

2004

RNA

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Ribonuclease P (RNase P) is a ribonucleoprotein complex that catalyzes the cleavage of the 5 end of precursor tRNA. To characterize the interface between the Bacillus subtilis RNA (PRNA) and protein (P protein) components, the intraholoenzyme K D is determined as a function of ionic strength using a magnetocapture-based assay. Three distinct phases are evident. At low ionic strength, the affinity of PRNA for P protein is enhanced as the ionic strength increases mainly due to stabilization of the PRNA structure by cations. Lithium substitution in lieu of potassium enhances the affinity at low ionic strength, whereas the addition of ATP, known to stabilize the structure of P protein, does not affect the affinity. At high ionic strength, the observed affinity decreases as the ionic strength increases, consistent with disruption of ionic interactions. These data indicate that three to four ions are released on formation of holoenzyme, reflecting the number of ion pairs that occur between the P protein and PRNA. At moderate ionic strength, the two effects balance so that the apparent K D is not dependent on the ionic strength. The K D between the catalytic domain (C domain) and P protein has a similar triphasic dependence on ionic strength. Furthermore, the intraholoenzyme K D is identical to or tighter than that of full-length PRNA, demonstrating that the P protein binds solely to the C domain. Finally, pre-tRNA asp (but not tRNA asp ) stabilizes the PRNA·P protein complex, as predicted by the direct interaction between the P protein and pre-tRNA leader.

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“…This nucleotide change occurs in a conserved site considered to be important for function (Zahler et al, 2003). An ARU mutation at this position (E. coli position 248) has been shown to be of particular significance to the binding affinity and catalysis relevant to pre-tRNA processing (Zahler et al, 2003). The change in the base at this site, which is otherwise conserved throughout the Bacteria, is consistent with the divergent position of the planctomycetes.…”

Section: Resultsmentioning

confidence: 95%

“…strain 1, has revealed the mutation of a base universally conserved in Archaea and Bacteria (Zahler et al, 2003). Thought to be invariant, A248 has been replaced by a U in these sequences.…”

Section: Resultsmentioning

confidence: 99%

“…Thought to be invariant, A248 has been replaced by a U in these sequences. This nucleotide change occurs in a conserved site considered to be important for function (Zahler et al, 2003). An ARU mutation at this position (E. coli position 248) has been shown to be of particular significance to the binding affinity and catalysis relevant to pre-tRNA processing (Zahler et al, 2003).…”

Section: Resultsmentioning

confidence: 99%

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Comparative analysis of ribonuclease P RNA of the planctomycetes

Butler

Fuerst

2004

International Journal of Systematic and Evolutionary Microbiology

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The planctomycetes, order Planctomycetales, are a distinct phylum of domain Bacteria. Genes encoding the RNA portion of ribonuclease P (RNase P) of some planctomycete members were sequenced and compared with existing database planctomycete sequences. rnpB gene sequences encoding RNase P RNA were generated by a conserved primer PCR strategy for Planctomyces brasiliensis, Planctomyces limnophilus, Pirellula marina, Pirellula staleyi strain ATCC 35122, Isosphaera pallida, one other Isosphaera strain, Gemmata obscuriglobus and three other strains of the Gemmata group. These sequences were aligned against reference bacterial sequences and secondary structures of corresponding RNase P RNAs deduced by a comparative approach. P12 helices were found to be highly variable in length, as were helices P16.1 and P19, when present. RNase P RNA secondary structures of Gemmata isolates were found to have unusual features relative to other planctomycetes, including a long P9 helix and an insert in the P13 helix not found in any other member of domain Bacteria. These unique features are consistent with other unusual properties of this genus, distinguishing it from other bacteria. Phylogenetic analyses indicate that relationships between planctomycetes derived from RNase P RNA are consistent with 16S rRNA-based analyses. INTRODUCTIONRibonuclease P (RNase P) is the most widespread ribonuclease (Condon & Putzer, 2002), found in all organisms so far examined (Altman et al., 1989) except the bacterial species 'Aquifex aeolicus' (Condon & Putzer, 2002), and also found within the subcellular compartments of eukaryotes known to synthesize tRNA (Frank & Pace, 1998). It is one of the key enzymes involved in the processing of tRNA, responsible for the generation of the 59 termini of mature tRNA (Altman, 1975), via a single endonucleolytic cleavage (Deutscher, 1984). Due to its universal existence, it is most likely an ancient enzyme, possibly descended from the postulated 'RNA world', prior to the development of protein-directed catalysis (Brown & Pace, 1991;Jeffares et al., 1998). In bacteria, the RNase P molecule is composed of a small protein and a large RNA moiety (Pace & Brown, 1995), the latter of which is capable of in vitro catalysis (Guerrier-Takada et al., 1983). RNase P RNA molecules in bacteria can generally be divided into two structurally separate classes: type A, the most common form, and type B, which is found in the low-G+C Gram-positive bacteria . Type A is distinguished by the presence of helix P6 and the absence of helix P5.1. A third intermediate type, type C, has been observed in the green nonsulfur bacteria (Haas & Brown, 1998). RNase P RNA has proven useful for phylogenetic analysis of the Chlamydiales (Herrmann et al., 2000), Prochlorococcus (Schön et al., 2002), the LL-2,6-diaminopimelic acid-containing actinomycetes (Yoon & Park, 2000), Bartonella (Pitulle et al., 2002) and Vibrio core species (Maeda et al., 2001), among others.The phylum Planctomycetes (Garrity et al., 2001) comprises a distinct group of the do...

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Recognition of the 5′ leader of pre-tRNA substrates by the active site of ribonuclease P

Cited by 78 publications

References 44 publications

Crystal structure of a bacterial ribonuclease P RNA

Crystal structure of a bacterial ribonuclease P RNA

Ionic interactions between PRNA and P protein in Bacillus subtilis RNase P characterized using a magnetocapture-based assay

Comparative analysis of ribonuclease P RNA of the planctomycetes

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