RNA Subunit Of RNase Research Articles

Characterization of the mitogenome of Gongronella sp. w5 reveals substantial variation in Mucoromycota.

Gongronella is a genus of fungi in Mucorales (Mucoromycota). Some of its members have important biotechnological applications, but until now, not a single mitogenome has been characterized in Gongronella. Here, we present the complete mitogenome assembly of Gongronella sp. w5, a soil isolate known to interact with plants and several fungi. Its 36,593-bp circular mitogenome encodes the large and small subunit rRNAs, 14 standard mitochondrial proteins, 24 tRNAs, three free-standing ORF proteins, and the RNA subunit of RNase P (rnpB). These genes arrange in an order novel to known fungal mitogenomes. Three group I introns are present in the cob, cox1, and nad5 genes, respectively, and they are probably acquired by horizontal gene transfer. Phylogenetic analysis based on mitochondrion-encoded proteins supports the grouping of Gongronella sp. w5 with Absidia glauca, forming the Cunninghamellaceae clade within Mucoromycota. Gongronella and most other Mucoromycota species are predicted to use the standard genetic code in mitochondrial translation, rather than code 4 assigned by GenBank. A comparison among seven publicly available mitogenomes in Mucoromycota reveals the presence of the same 14 typical protein-coding genes plus rnpB, yet substantial variation in mitogenome size, intron number, gene order, and orientation. In this comparison, the uniqueness of Gongronella is evident from similarly large differences to its closest phylogenetic neighbor, A. glauca. This study promotes our understanding of fungal evolution in Mucoromycota. KEY POINTS: • This study reports the first mitogenome in Gongronella, which presents a novel gene order. • Different Mucoromycota mitogenomes show substantial variation of gene organizations. • Most Mucoromycota species use the standard genetic code to translate mitochondrial genes.

Rid7C, a Member of the YjgF/YER057c/UK114 (Rid) Protein Family, Is a Novel Endoribonuclease That Regulates the Expression of a Specialist RNA Polymerase Involved in Differentiation in Nonomuraea gerenzanensis.

The YjgF/YER057c/UK114 (Rid) is a protein family breadth conserved in all domains of life and includes the widely distributed archetypal RidA (YjgF) subfamily and seven other subfamilies (Rid1 to Rid7). Among these subfamilies, RidA is the only family to have been biochemically well characterized and is involved in the deamination of the reactive enamine/imine intermediates. In this study, we have characterized a protein of the Rid7 subfamily, named Rid7C, in Nonomuraea gerenzanensis, an actinomycete that is characterized by the presence of two types of RNA polymerases. This is due to the coexistence in its genome of two RNA polymerase (RNAP) β chain-encoding genes, rpoB(S) (the wild-type rpoB gene) and rpoB(R) (a specialist, mutant-type rpoB gene) that controls A40926 antibiotic production and a wide range of metabolic adaptive behaviors. Here, we found that expression of rpoB(R) is regulated posttranscriptionally by RNA processing in the 5' untranslated region (UTR) of rpoB(R) mRNA and that the endoribonuclease activity of Rid7C is responsible for mRNA processing, thereby overseeing several tracts of morphological and biochemical differentiation. We also provide evidence that Rid7C may be associated with RNase P M1 RNA, although M1 RNA is not required for rpoB(R) mRNA processing in vitro, and that Rid7C endoribonuclease activity is inhibited by A40926, suggesting the existence of a negative feedback loop in A40926 production and a role of the endogenous synthesis of A40926 in the modulation of biochemical differentiation in this microorganism. IMPORTANCE The YjgF/YER057c/UK114 family includes many proteins with diverse functions involved in detoxification, RNA maturation, and control of mRNA translation. We found that Rid7C is an endoribonuclease that is involved in processing of rpoB(R) mRNA, coding for a specialized RNA polymerase beta subunit that oversees morphological differentiation and A40926 antibiotic production in Nonomuraea gerenzanensis. Rid7C-mediated processing promotes rpoB(R) mRNA translation and antibiotic production, while Rid7C endoribonuclease activity is inhibited by A40926, suggesting a role of the endogenous synthesis of A40926 in modulation of biochemical differentiation in this microorganism. Finally, we show that recombinant Rid7C copurified with M1 RNA (the RNA subunit of RNase P) from Escherichia coli extract, suggesting a functional interaction between Rid7C and M1 RNA activities.

Understanding Catalysis of Phosphate‐Transfer Reactions by the Large Ribozymes

Large ribozymes are unique among catalytic RNA molecules in that their reactions involve intermolecular nucleophilic attack on an RNA phosphodiester linkage. Crystal structures of near-atomic resolution are now available for the group I and group II self-splicing introns and the RNA subunit of RNase P. The structural data agrees well with the earlier models proposed on the basis of biochemical studies and the evidence at hand suggests that all of the large ribozymes utilize a mechanism in which coordination of Mg(II) ions reduces the negative charge on the scissile phosphodiester linkage, as well as assists both the nucleophilic attack and the departure of the leaving group.

The Multiple Substrate Recognition Properties of Ribonuclease P: Achieving Uniformity in Processing Kinetics Despite Variation in Substrate Structure

Ribonuclease P is an essential enzyme that is responsible for catalyzing the maturation of the 5’ end of transfer RNA. In Bacteria, the RNase P holoenzyme is composed of a RNA subunit and a protein subunit. The RNA subunit of RNase P contains the enzyme active site and has the ability to process substrates in the absence of the protein component in vitro, but works as a holoenzyme in vivo. While nucleotide recognition elements adjacent to the site of processing have been identified, different tRNA sequences vary considerably. Remarkably, rates of pre-tRNA processing are uniform despite this variation in substrate sequence and structure. The mechanistic basis for multiple substrate recognition by the holoenzyme is the focus of this study, with the ultimate goal being a better understanding of uniformity in ptRNA processing and discriminates between cognate and non-cognate RNAs. We are determining kinetic schemes for a number of pre-tRNAs using fluorescence assays and standard discontinuous assays. Multiple turnover kinetics have been obtained for ptRNA608MET, a consensus pre-tRNA, and ptRNA605f-MET a non-consensus sequence. Both ptRNAs display a similar Vmax, and Km values. Neither tRNA displayed any burst nor lag phases in pre-steady state kinetics implying that the rate limiting step for our processing model is catalysis. Our initial hypothesis is that uniformity in substrate kcat/Km values results from differential 5’ leader sequence interactions with the protein that compensate for deviations from tRNA consensus recognition sequences. To obtain more insight into sequence and structure influence, we have set up a series of multiple turnover experiments with a consensus and non-consensus ptRNA competing against one another.

Inactivation of expression of two genes in Saccharomyces cerevisiae with the external guide sequence methodology.

The artificial inhibition of expression of genes in Saccharomyces cerevisiae is not a widespread, useful phenomenon. The external guide sequence (EGS) technology, which is well-proven in bacteria and mammalian cells in tissue culture and in mice, can also be utilized in yeast. The TOP2 and SRG1 genes can be inhibited by ∼30% with EGSs in vivo. Results in vitro also show convenient cleavage of the relevant transcripts by RNase P and appropriate EGSs. The feasible constructs shown to date have an EGS covalently linked to M1 RNA, the RNA subunit of RNase P from Escherichia coli. Greater efficiency in cleavage of transcripts can be fashioned using more than one EGS targeted to different sites in a transcript and stronger promoters controlling the EGS constructs.

Schizosaccharomyces cryophilus sp. nov., a new species of fission yeast.

The genus Schizosaccharomyces is presently comprised of three species, namely Schizosaccharomyces pombe, Schizosaccharomyces octosporus and Schizosaccharomyces japonicus. Here, we describe a hitherto unknown species, Schizosaccharomyces cryophilus, named for its preference for growth at lower temperatures than the other fission yeast species. Although morphologically similar to S. octosporus, analysis of several rapidly evolving sequences, including the D1/D2 divergent domain of the large subunit (LSU) rRNA gene, the RNA subunit of RNAse P and the internal transcribed spacer elements, revealed significant divergence from any previously characterized Schizosaccharomyces strain. Based on phylogenetic analysis of the D1/D2 domain of the LSU rRNA gene, S. octosporus is the closest known relative of S. cryophilus, with the sequences of the two species differing by 25 nucleotide substitutions (>4%). Sequencing of the S. cryophilus genome and phylogenetic analysis of all 1 : 1 protein orthologs confirmed this observation, and together with morphological and physiological characterization, supports the assignment of S. cryophilus as a new species within the genus Schizosaccharomyces. The type strain of the new species is NRRL Y-48691(T) (=NBRC 106824(T)=CBS 11777(T)).

The complete mitochondrial genome of the yeast Kluyveromyces thermotolerans

We report here the complete nucleotide sequence of the 23.5-kb mitochondrial genome from the yeast Kluyveromyces thermotolerans. It encodes, all on the same DNA strand, three subunits of cytochrome oxidase ( COX1, COX2 and COX3), three subunits of ATP synthetase ( ATP6, ATP8 and ATP9), the apocytochrome b ( COB), the ribosomal protein VAR1, 24 tRNAs, the small and large ribosomal RNAs, and the RNA subunit of RNase P. Three intronic ORFs are present within the COX1 gene group I introns. The K. thermotolerans mitochondrial genome is very similar to the Candida glabrata mitochondrial genome, as judged from clusters of gene order, gene transcription units and sequence similarities. Interestingly, the predicted secondary structure of the abnormal tRNA Thr1 contains 10 nucleotides in its anticodon loop. This sequence is available under EMBL Accession No. AJ634268.

Coordinate inhibition of expression of several genes for protein subunits of human nuclear RNase P.

The deliberate inhibition of expression of one of the protein subunits (Rpp38) of human nuclear RNase P is achievable by using external guide sequence (EGS) technology. Both the protein product and the mRNA are greatly reduced 24 h after transient transfection with a gene coding for an appropriate EGS. Control experiments indicated that four other protein subunits of RNase P and their RNAs are also inhibited with no external manipulation. The remaining RNase P proteins, their mRNAs, and the RNA subunit of RNase P all are unchanged. Several short nucleotide sequences adjacent to the ORFs for the inhibited genes are similar and could be targets for transcriptional repression. The explanation of coordinate inhibition of the expression of the product of one particular gene by the transfection of an EGS (or RNA interference) requires some care in terms of interpreting phenotypic effects because, in our case, several gene products that are not targeted are also inhibited.

The complete mitochondrial genome sequence of the pathogenic yeast Candida ( Torulopsis) glabrata

We report here the complete sequence of the mitochondrial (mt) genome of the pathogenic yeast Candida glabrata. This 20 kb mt genome is the smallest among sequenced hemiascomycetous yeasts. Despite its compaction, the mt genome contains the genes encoding the apocytochrome b ( COB), three subunits of ATP synthetase ( ATP6, 8 and 9), three subunits of cytochrome oxidase ( COX1, 2 and 3), the ribosomal protein VAR1, 23 tRNAs, small and large ribosomal RNAs and the RNA subunit of RNase P. Three group I introns each with an intronic open reading frame are present in the COX1 gene. This sequence is available under accession number AJ511533.

Essential roles of Bdp1, a subunit of RNA polymerase III initiation factor TFIIIB, in transcription and tRNA processing.

The essential Saccharomyces cerevisiae gene BDP1 encodes a subunit of RNA polymerase III (Pol III) transcription factor (TFIIIB); TATA box binding protein (TBP) and Brf1 are the other subunits of this three-protein complex. Deletion analysis defined three segments of Bdp1 that are essential for viability. A central segment, comprising amino acids 327 to 353, was found to be dispensable, and cells making Bdp1 that was split within this segment, at amino acid 352, are viable. Suppression of bdp1 conditional viability by overexpressing SPT15 and BRF1 identified functional interactions of specific Bdp1 segments with TBP and Brf1, respectively. A Bdp1 deletion near essential segment I was synthetically lethal with overexpression of PCF1-1, a dominant gain-of-function mutation in the second tetracopeptide repeat motif (out of 11) of the Tfc4 (tau(131)) subunit of TFIIIC. The analysis also identifies a connection between Bdp1 and posttranscriptional processing of Pol III transcripts. Yeast genomic library screening identified RPR1 as the specific overexpression suppressor of very slow growth at 37 degrees C due to deletion of Bdp1 amino acids 253 to 269. RPR1 RNA, a Pol III transcript, is the RNA subunit of RNase P, which trims pre-tRNA transcript 5' ends. Maturation of tRNA was found to be aberrant in bdp1-Delta 253-269 cells, and RPR1 transcription with the highly resolved Pol III transcription system in vitro was also diminished when recombinant Bdp1 Delta 253-269 replaced wild-type Bdp1. Physical interaction of RNase P with Bdp1 was demonstrated by coimmunoprecipitation and pull-down assays.

In vitro hyperprocessing of tRNAs by Bacillus subtilis ribonuclease P RNA

We have previously reported that the catalytic RNA subunit of ribonuclease P (RNase P) of Escherichia coli (M1 RNA) cleaves Drosophila initiator methionine tRNA (tRNA(i)Met), alanine tRNA (tRNA(Ala)) and histidine tRNA (tRNA(His)) within the mature tRNA sequences to produce specific fragments. We call this further cleavage hyperprocessing. These cleavages were dependent on the occurrence of altered conformations of the tRNAs. Here, we found that the RNase P RNA of Bacillus subtilis can hyperprocess these three tRNAs at the same sites as does M1 RNA. The hyperprocessing activity may probably be common feature for Bacterial RNase P RNAs.

9] Human ribonuclease P

Publisher Summary Ribonuclease P is a ribonucleoprotein nuclease required for the site-specific cleavage of the 5′ leader sequence of precursor tRNAs. In eubacteria, the RNA subunit of RNase P is the catalytic moiety and is capable of processing precursor tRNA in the presence of divalent metal ions. The RNA subunits of eukaryotic RNase P (human and yeast) do not exhibit enzymatic activity In Vitro, an indication that their protein components are required for the catalytic reaction. This chapter describes a purification procedure for human RNase P from HeLa cells that facilitated the characterization of nine of its protein subunits. This procedure also allows the separation of RNase P from the structurally related RNase MRP nuclease. The purification procedure shows that nuclear RNase P from HeLa cells has at least nine protein subunits in association with a single RNA species, H1 RNA. Nuclear RNase P in other eukaryotes, such as S. cerevisiae and Aspergillus nidulans, also consists of multiple protein subunits, some of which are evolutionarily conserved and an RNA subunit. The precise function of these subunits in tRNA processing is not yet understood. Reconstitution of the activity of RNase P in vitro may reveal the role of these subunits in catalysis.

Drosophila tRNAs hyperprocessed in vitro by ribonuclease P.

In transposon copia-related retrovirus-like particles of Drosophila, a 5' half fragment produced by the cleavage of mature initiator methionine tRNA is used as the primer for minus-strand reverse transcription. This cleavage is called hyperprocessing. We have previously reported that the catalytic RNA subunit of RNase P catalyzes this hyperprocessing in vitro and that this cleavage is dependent on the occurrence of an altered conformation of the tRNA substrate. Here, we found that other mature tRNAs of Drosophila were also hyperprocessed by M1 RNA in vitro and that some of such tRNAs were probably alanine and histidine tRNAs. Here we report these two tRNAs can also adopt their alternative conformations very similar to that of initiator methionine tRNA.

The RNA Subunit of Ribonuclease P from the Zebrafish, Danio rerio

A simple strategy has been devised to identify the gene encoding the RNA subunit of RNase P from the zebrafish, Danio rerio. The sequence obtained by amplification of genomic DNA with primers based on sequences common to two other vertebrates was confirmed by reverse transcription and amplification of RNA from a partially purified preparation of the holoenzyme. The 5' and 3' ends were determined by cyclizing the RNA, followed by reverse transcription and sequencing across the ligated RNA junction. The zebrafish sequence is 63% identical to that of Xenopus laevis nuclear RNase P RNA and 69% identical to the human RNase P RNA. A consensus secondary structure was constructed based on these nucleotide identities and on the many compensatory base changes in several regions among these three RNAs. The strategy used to obtain the zebrafish sequence should be useful in deriving analogous gene sequences from diverse classes of eukaryotes.

Synthesis and Self-Cleavage Reaction of a Chimeric Molecule between RNase P-RNA and Its Model Substrate1

We synthesized a chimeric RNA between the catalytic RNA subunit of RNase P from Escherichia coli (M1 RNA) and a model substrate of the enzyme. The model substrate is the smallest substrate of RNase P, having a simple stem-loop structure. This model substrate was added to the 3'-end of M1 RNA. This chimeric molecule, which we call M1 RNA-MS, is a self-cleaving RNA and is cleaved much more efficiently than the M1 RNA-pre-tRNA, an artificial self-cleaving RNA previously synthesized [Kikuchi et al. (1993) Nucleic Acids Res. 21, 4685-4689], that consists of a full-size tRNA precursor and the M1 RNA. The self-cleavage of M1 RNA-MS at 10 mM Mg2+ was an intramolecular reaction (cis-cleavage). Ca2+ supported the self-cleavage of M1 RNA-MS as effectively as Mg2+, although the self-cleavage of M1 RNA-pre-tRNA proceeded with low efficiency in the presence of Ca2+ as the only metal ion. Future application of the M1 RNA-MS molecule to the in vitro evolution of the M1 RNA and other experiments is proposed.

Multiple cis-acting elements are required for RNA polymerase III transcription of the gene encoding H1 RNA, the RNA component of human RNase P.

In humans, the H1 RNA, the RNA subunit of RNase P, is synthesized by RNA polymerase III. We have used block replacement mutagenesis to identify the sequences necessary for in vitro transcription of H1 RNA. We find that multiple cis-acting elements located in the H1 RNA 5'-flanking region are necessary for H1 RNA synthesis; no internal sequences are essential. Required cis-acting elements include sequences resembling proximal sequence element, distal sequence element, and TATA motifs. In this respect, the H1 RNA promoter is similar in structure to the promoters of the genes encoding the U6 snRNA, the 7 SK RNA and the MRP RNA. However, our mutational analysis indicates that the H1 promoter is unexpectedly complex, with several additional cis-acting elements spanning nearly 70 base pairs of the H1 RNA gene 5'-flanking sequence.

Analysis of the gene encoding the RNA subunit of ribonuclease P fromT.ThermophilusHB8

The gene for the RNA subunit of ribonuclease P from the extreme thermophilic eubacterium T. thermophilus HB8 was cloned using oligonucleotide probes complementary to conserved regions of RNase P RNA subunits from proteobacteria. The monocistronic gene and its flanking regions were sequenced. The gene is enclosed by a promoter and a rho-independent terminator. Nuclease S1 protection analyses showed that the primary transcript is identical with the mature RNA, i.e. no processing events are involved. The stem and loop structure of the terminator remains part of the mature molecule. In vitro transcription of the cloned gene with purified RNA polymerase from T. thermophilus yields the same RNA product as in vivo, indicating that no other components except RNA polymerase are involved in the synthesis of the RNA. RNase P RNA from T. thermophilus cleaved a pre-tRNA(Tyr) from E. coli with highest efficiency between 55 degrees C and 65 degrees C. The T. thermophilus RNA, which has a G-C content of 86% in helical regions, displays several structural idiosyncrasies, although its secondary structure is similar to that of proteobacteria. Numerous invariable nucleotides in the structural core of eubacterial RNase P RNAs are also conserved in the RNA from the extreme thermophilic eubacterium.

Catalysis by the RNA subunit of RNase P — a minireview

RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5′ termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate

Dependence of M1 RNA substrate specificity on magnesium ion concentration

We have constructed a plasmid expressing E. coli M1 RNA, the catalytic RNA subunit of ribonuclease P, under the control of a phage T7 promoter. The active M1 RNA species synthesized in vitro by T7 RNA polymerase from this vector was reacted with the tRNA(Gln) - tRNA(Leu) precursor RNA (Band K) encoded by phage T4. Only the tRNA(Leu) moiety of this dimeric precursor RNA contains the 3' terminal C-C-A sequence common to all tRNAs. We observed that protein-free M1 RNA was capable of processing the precursor RNA at the 5' ends of both tRNA tRNA sequences. The rate of cleavage of the tRNA(Gln) sequence was more strongly dependent on [Mg2+] than that of tRNA(Leu), increasing severalfold between 100 and 500 mM Mg2+, conditions under which the rate of cleavage at the tRNA(Leu) sequence was constant.

Metal ion requirements and other aspects of the reaction catalyzed by M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli

M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli, can under certain conditions catalytically cleave precursors to tRNA in the absence of C5, the protein moiety of RNase P. M1 RNA itself is not cleaved during the reaction, nor does it form any covalent bonds with its substrate. Only magnesium and, to a lesser extent, manganese ions can function at the catalytic center of M1 RNA. Several other ions either inhibit the binding of magnesium ion at the active site or function as structural counterions. The reaction rate of cleavage of precursors to tRNAs by M1 RNA is enhanced in the presence of poly-(ethylene glycol) or 2-methyl-2,4-pentanediol. Many aspects of the reaction catalyzed by M1 RNA are compatible with a mechanism in which phosphodiester bond cleavage is mediated by metal ion.