RNase P of the Cyanophora paradoxa cyanelle: a plastid ribozyme

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that generates the mature 5' ends of tRNAs. Ubiquitous across all three kingdoms of life, the composition and functional contributions of the RNA and protein components of RNase P differ between the kingdoms. RNA-alone catalytic activity has been reported throughout bacteria, but only for some archaea, and only as trace activity for eukarya. Available information for RNase P from photosynthetic organelles points to large differences to bacterial as well as to eukaryotic RNase P: for spinach chloroplasts, protein-alone activity has been discussed; for RNase P from the cyanelle of the glaucophyte Cyanophora paradoxa, a type of organelle sharing properties of both cyanobacteria and chloroplasts, the proportion of protein was found to be around 80% rather than the usual 10% in bacteria. Furthermore, the latter RNase P was previously found catalytically inactive in the absence of protein under a variety of conditions; however, the RNA could be activated by a cyanobacterial protein, but not by the bacterial RNase P protein from Escherichia coli. Here we demonstrate that, under very high enzyme concentrations, the RNase P RNA from the cyanelle of C. paradoxa displays RNA-alone activity well above the detection level. Moreover, the RNA can be complemented to a functional holoenzyme by the E. coli RNase P protein, further supporting its overall bacterial-like architecture. Mutational analysis and domain swaps revealed that this A,U-rich cyanelle RNase P RNA is globally optimized but conformationally unstable, since changes as little as a single point mutation or a base pair identity switch at positions that are not part of the universally conserved catalytic core led to a complete loss of RNA-alone activity. Likely related to this low robustness, extensive structural changes towards an E. coli-type P5-7/P15-17 subdomain as a canonical interaction site for tRNA 3'-CCA termini could not be coaxed into increased ribozyme activity.     

Plant mitochondrial RNase P

Molecular investigations in mitochondria of higher plants have to take in account the complicated genomic structure of these organelles and their complex mode of gene expression. Recently tRNA processing activities and particulary RNase P-like activities have been described for mitochondria of mono- and dicot plants. The determined biochemical characteristics of these plant mitochondrial tRNA processing enzymes now allow a comparison to the bacterial prototype from which they evolved. The substrate specifity of the plant mitochondrial RNase P in particular has unique selection parameters distinct from theE. coli RNase P.     

RNase P RNA mediated cleavage: substrate recognition and catalysis

The universally conserved endoribonuclease P consists of one RNA subunit and, depending on its origin, a variable number of protein subunits. RNase P is involved in the processing of a large variety of substrates in the cell, the preferred substrate being tRNA precursors. Cleavage activity does not require the presence of the protein subunit(s) in vitro. This is true for both prokaryotic and eukaryotic RNase P RNA suggesting that the RNA based catalytic activity has been preserved during evolution. Progress has been made in our understanding of the contribution of residues and chemical groups both in the substrate as well as in RNase P RNA to substrate binding and catalysis. Moreover, we have access to two crystal structures of bacterial RNase P RNA but we still lack the structure of RNase P RNA in complex with its substrate and/or the protein subunit. Nevertheless, these recent advancements put us in a new position to study the way and nature of interactions between in particular RNase P RNA and its substrate. In this review I will discuss various aspects of the RNA component of RNase P with an emphasis on our current understanding of the interaction between RNase P RNA and its substrate.     

Relationship between the structure and function of Escherichia coli initiator tRNA

Through functional studies of mutant tRNAs, we have identified sequence and/or structural features important for specifying the many distinctive properties of E coli initiator tRNA. Many of the mutant tRNAs contain an anticodon sequence change from CAU→CUA and are now substrates for E coli glutaminyl-tRNA synthetase (GlnRS). We describe here the effect of further mutating the discriminator base 73 and nucleotide 72 at the end of the acceptor stem on: i) recognition of the mutant tRNAs by E coli GlnRS; ii) recognition by E coli methionyl-tRNA transformylase; and iii) activity of the mutant tRNAs in initiation in E coli. For GlnRS recognition, our results are, in general, consistent with interactions found in the crystal structure of the E coli GlnRS-glutamine tRNA complex. The results also support our previous conclusion that formylation of initiator tRNA is important for its function in initiation.     

Analysis of the tertiary structure of bacterial RNase P RNA

The ubiquitous occurrence of ribonuclease P (RNase P) as a ribonucleoprotein and the catalytic properties of bacterial RNase P RNAs indicate that RNA fulfills an ancient and important role in the function of this enzyme. This review focuses on efforts to determine the structure of the bacterial RNase P RNA ribozyme. Phylogenetic comparative analysis of a library of bacterial RNase P RNA sequences has resulted in a well-developed secondary structure model and allowed identification of some elements of tertiary structure. The native structure has been redesigned by circular permutation to facilitate intra- and inter-molecular crosslinking experiments in order to gain further structural information. The crosslinking constraints, together with the constraints provided by comparative analyses, have been incorporated into a first-order model of the structure of the ribozyme-substrate complex. The developing structural perspective allows the design of self-cleaving pre-tRNA-RNase P RNA conjugates which are useful tools for additional structure-probing experiments.Abbreviations cpRNA circularly permuted RNA     

Alternative Substrate Kinetics of Escherichia coli Ribonuclease P: DETERMINATION OF RELATIVE RATE CONSTANTS BY INTERNAL COMPETITION*

A single enzyme, ribonuclease P (RNase P), processes the 5′ ends of tRNA precursors (ptRNA) in cells and organelles that carry out tRNA biosynthesis. This substrate population includes over 80 different competing ptRNAs in Escherichia coli. Although the reaction kinetics and molecular recognition of a few individual model substrates of bacterial RNase P have been well described, the competitive substrate kinetics of the enzyme are comparatively unexplored. To understand the factors that determine how different ptRNA substrates compete for processing by E. coli RNase P, we compared the steady state reaction kinetics of two ptRNAs that differ at sequences that are contacted by the enzyme. For both ptRNAs, substrate cleavage is fast relative to dissociation. As a consequence, V/K, the rate constant for the reaction at limiting substrate concentrations, reflects the substrate association step for both ptRNAs. Reactions containing two or more ptRNAs follow simple competitive alternative substrate kinetics in which the relative rates of processing are determined by ptRNA concentration and their V/K. The relative V/K values for eight different ptRNAs, which were selected to represent the range of structure variation at sites contacted by RNase P, were determined by internal competition in reactions in which all eight substrates were present simultaneously. The results reveal a relatively narrow range of V/K values, suggesting that rates of ptRNA processing by RNase P are tuned for uniform specificity and consequently optimal coupling to precursor biosynthesis.     

RNase P from bacteria. Substrate recognition and function of the protein subunit

RNase P recognizes many different precursor tRNAs as well as other substrates and cleaves all of them accurately at the expected position. RNase P recognizes the tRNA structure of the precursor tRNA by a set of interactions between the catalytic RNA subunit and the T- and acceptor-stems mainly, although residues in the 5-leader sequence as well as the 3-terminal CCA are important. These conclusions have been reached by several studies on mutant precursor tRNAs as well as cross-linking studies between RNase P RNA and precursor tRNAs. The protein subunit of RNase P seems also to affect the way that the substrate is recognized as well as the range of substrates that can be used by RNase P, although the protein does not seem to interact directly with the substrates. The interaction between the protein and RNA subunits of RNase P has been extensively studiedin vitro. The protein subunit sequence is not highly conserved among bacteria, however different proteins are functionally equivalent as heterologous reconstitution of the RNase P holoenzyme can be achieved in many cases.Abbreviations C5 protein protein subunit fromE. coli RNase P - EGS external guide sequence - M1 RNA RNA subunit formE. coli RNase P - ptRNA precursor tRNA - RNase P ribonuclease P     

Binding and cleavage of unstructured RNA by nuclear RNase P

Ribonuclease P (RNase P) is an essential endoribonuclease for which the best-characterized function is processing the 5' leader of pre-tRNAs. Compared to bacterial RNase P, which contains a single small protein subunit and a large catalytic RNA subunit, eukaryotic nuclear RNase P is more complex, containing nine proteins and an RNA subunit in Saccharomyces cerevisiae. Consistent with this, nuclear RNase P has been shown to possess unique RNA binding capabilities. To understand the unique molecular recognition of nuclear RNase P, the interaction of S. cerevisiae RNase P with single-stranded RNA was characterized. Unstructured, single-stranded RNA inhibits RNase P in a size-dependent manner, suggesting that multiple interactions are required for high affinity binding. Mixed-sequence RNAs from protein-coding regions also bind strongly to the RNase P holoenzyme. However, in contrast to poly(U) homopolymer RNA that is not cleaved, a variety of mixed-sequence RNAs have multiple preferential cleavage sites that do not correspond to identifiable consensus structures or sequences. In addition, pre-tRNA(Tyr), poly(U)(50) RNA, and mixed-sequence RNA cross-link with purified RNase P in the RNA subunit Rpr1 near the active site in "Conserved Region I," although the exact positions vary. Additional contacts between poly(U)(50) and the RNase P proteins Rpr2p and Pop4p were identified. We conclude that unstructured RNAs interact with multiple protein and RNA contacts near the RNase P RNA active site, but that cleavage depends on the nature of interaction with the active site.     

Genetic studies on the beta subunit of Escherichia coli RNA polymerase. IX. The role of the carboxy-terminus in enzyme assembly

Summary The assembly of RNA polymerase was studied in Escherichia coli mutants encoding large N-terminal amber fragments of the subunit. Whereas the removal of up to 20% of the carboxy-terminus does not prevent the formation of premature core enzyme, the amber fragments seem to interfere with holoenzyme production. These studies permit, therefore, the localization of a region on the polypeptide involved in sigma binding.Paper VIII is Glass et al. (1986a)     

Trials, Travails and Triumphs: An Account of RNA Catalysis in RNase P

Last December marked the 20th anniversary of the Nobel Prize in Chemistry to Sidney Altman and Thomas Cech for their discovery of RNA catalysts in bacterial ribonuclease P (an enzyme catalyzing 5′ maturation of tRNAs) and a self-splicing rRNA of Tetrahymena, respectively. Coinciding with the publication of a treatise on RNase P,¹ this review provides a historical narrative, a brief report on our current knowledge, and a discussion of some research prospects on RNase P.

“…the great thing about science is that you can actually solve a problem. You can take something which is confused, a mess, and not only find a solution, but prove it's the right one.”²

-Sydney Brenner

“In research the front line is almost always in a fog.”³

-Francis Crick

     

Genetic analysis of polynucleotide phosphorylase structure and functions

Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.     

Genetic and physical analysis of plasmid genes expressing inducible resistance of tellurite in Escherichia coli

Summary A large (>250 kb) conjugative plasmid, pMER610, specifying resistance to tellurium and mercury was isolated from an Alcaligenes strain and transferred by conjugation to Escherichia coli AB1157. The acquisition of pMER610 by AB1157 increased the resistance to both tellurite and tellurate by 100-fold. Expression of tellurite resistance by pMER610 and the cloned Te^r determinant was inducible by prior exposure to tellurite at levels sub-toxic to the sesitive AB1157. Physical analysis of the cloned Te^r fragment located the resistance determinant to a 3.55 kb region. Insertion of Tn 1000 () into this region produced two classes of sensitive mutations, fully sensitive and intermediate or hyposensitive, which map in adjacent regions and form two complementation groups. Maxicell analysis identified four polypeptides (15.5, 22, 23 and 41 kDa) expressed by the Te^r clone. The 23 kDa polypeptide may not be required for resistance since tellurium-sensitive insertion mutations were not detected in the 23 kDa coding region.     

Role of precursor sequences in the ordered maturation of E. coli 23S ribosomal RNA

The maturation of ribosomal RNAs (rRNAs) is an important but incompletely understood process required for rRNAs to become functional. In order to determine the enzymes responsible for initiating 3' end maturation of 23S rRNA in Escherichia coli, we analyzed a number of strains lacking different combinations of 3' to 5' exo-RNases. Through these analyses, we identified RNase PH as a key effector of 3' end maturation. Further analysis of the processing reaction revealed that the 23S rRNA precursor contains a CC dinucleotide sequence that prevents maturation from being performed by RNase T instead. Mutation of this dinucleotide resulted in a growth defect, suggesting a strategic significance for this RNase T stalling sequence to prevent premature processing by RNase T. To further explore the roles of RNase PH and RNase T in RNA processing, we identified a subset of transfer RNAs (tRNAs) that contain an RNase T stall sequence, and showed that RNase PH activity is particularly important to process these tRNAs. Overall, the results obtained point to a key role of RNase PH in 23S rRNA processing and to an interplay between this enzyme and RNase T in the processing of different species of RNA molecules in the cell.     

Structural modeling of RNase P RNA of the hyperthermophilic archaeon 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 RNase P RNA (PhopRNA) of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is central to the catalytic process and binds five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) which contribute to the enzymatic activity of the holoenzyme. Despite significant progress in determining the crystal structure of the proteins, the structure of PhopRNA remains elusive. Comparative analysis of the RNase P RNA sequences and existing crystallographic structural information of the bacterial RNase P RNAs were combined to generate a phylogenetically supported three-dimensional (3-D) model of the PhopRNA. The model structure shows an essentially flat disk with 16 tightly packed helices and a conserved face suitable for the binding of pre-tRNA. Moreover, the structure in solution was investigated by enzymatic probing and small-angle X-ray scattering (SAXS) analysis. The low resolution model derived from SAXS and the comparative 3-D model have similar overall shapes. The 3-D model provides a framework for a better understanding of structure–function relationships of this multifaceted primordial ribozyme.     

Ribonuclease P: the diversity of a ubiquitous RNA processing enzyme

Ribonuclease P is the endonuclease required for generating the mature tRNA 5'-end. The ribonucleoprotein character of this enzyme has now been proven in most organisms and organelles. Exceptions, however, are still the chloroplasts, plant nuclei and animal mitochondria where no associated RNAs have been detected to date. In contrast to the known RNA subunits, which are fairly well-conserved in size and structure among diverse phylogenetic groups, the protein contribution to the holoenzyme is highly variable in size and number of the individual components. The structure of the bacterial protein component has recently been solved. In contrast, the spatial arrangement of the multiple subunits in eukaryotic enzymes is still enigmatic. Substrate requirements of the enzymes or their catalytic RNA subunits are equally diverse, ranging from simple single domain mimics to an almost intact three-dimensional structure of the pre-tRNA substrate. As an example for an intermediate in the enzyme evolution, ribonuclease P from the Cyanophora paradoxa cyanelle will be discussed in more detail. This enzyme is unique, as it combines cyanobacterial and eukaryotic features in its function, subunit composition and holoenzyme topology.