scholarly journals Analysis of RNase P Protein (rnpA) Expression in Bacillus subtilis Utilizing Strains with Suppressible rnpA Expression

2006 ◽  
Vol 188 (19) ◽  
pp. 6816-6823 ◽  
Author(s):  
Markus Gößringer ◽  
Rosel Kretschmer-Kazemi Far ◽  
Roland K. Hartmann
Keyword(s):  
Bacillus Subtilis ◽  
Direct Proof ◽  
Rnase P ◽  
Protein Subunit ◽  
Cell Growth Arrest ◽  
P Protein ◽  
In The Wild ◽  
Arrest Growth ◽  
Protein Variants

ABSTRACT Bacterial RNase P is composed of an RNA subunit and a single protein subunit (encoded by the rnpB and rnpA genes, respectively). We constructed Bacillus subtilis mutant strains that conditionally express the RNase P protein under control of the xylose promoter (P xyl ). In one strain (d7), rnpA expression was efficiently repressed in the absence of the inducer xylose, leading to cell growth arrest. Growth could be restored by a second functional rnpA allele. This is the first RNase P protein knockdown strain, providing the first direct proof that the rnpA gene is essential in B. subtilis and, by inference, in other bacteria. We further show (i) that, in the wild-type context, rnpA expression is attenuated by transcriptional polarity and (ii) that translation of rnpA mRNA in B. subtilis can be initiated at two alternative start codons. His-tagged RNase P protein variants are functional in vivo and permit purification of in vivo-assembled holoenzymes by affinity chromatography. Simultaneous expression of plasmid-encoded RNase P RNA and His-tagged protein increased RNase P holoenzyme yields. Massive overproduction of RNase P protein in strain d7 is compatible with cell viability.

scholarly journals By Any Other Name: Heterologous Replacement of the Escherichia coli RNase P Protein Subunit Has In Vivo Fitness Consequences

PLoS ONE ◽  
2012 ◽  
Vol 7 (3) ◽  
pp. e32456 ◽  
Author(s):  
Paula C. G. Turrini ◽  
Jasmine L. Loveland ◽  
Robert L. Dorit
Keyword(s):  
Escherichia Coli ◽  
Rnase P ◽  
Protein Subunit ◽  
P Protein ◽  
Fitness Consequences

scholarly journals Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

2018 ◽  
Author(s):  
Agnes Karasik ◽  
Carol A. Fierke ◽  
Markos Koutmos
Keyword(s):  
Mitochondrial Diseases ◽  
Protein S ◽  
Rnase P ◽  
Ribonuclease P ◽  
Mitochondrial Rna ◽  
P Protein ◽  
Adenosyl Methionine ◽  
Methylation Activity

ABSTRACTHuman mitochondrial ribonuclease P (mtRNase P) is an essential three protein complex that catalyzes the 5’ end maturation of mitochondrial precursor tRNAs (pre-tRNAs). MRPP3 (Mitochondrial RNase P Protein 3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl methionine (SAM)-dependent methyltransferase MRPP1/2 sub-complex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAs in vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleaved in vivo. Consistent with this, we found that addition of the MRPP1 co-factor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs. Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process and methylate human mitochondrial pre-tRNAs.

scholarly journals Yeast mitochondrial RNase P RNA synthesis is altered in an RNase P protein subunit mutant: insights into the biogenesis of a mitochondrial RNA-processing enzyme.

1996 ◽  
Vol 16 (7) ◽  
pp. 3429-3436 ◽  
Author(s):  
V Stribinskis ◽  
G J Gao ◽  
P Sulo ◽  
Y L Dang ◽  
N C Martin
Keyword(s):  
Rna Processing ◽  
Mitochondrial Protein ◽  
Rnase P ◽  
Mitochondrial Rna ◽  
Wild Type ◽  
Protein Subunit ◽  
Mitochondrial Trna ◽  
P Deficiency ◽  
F Gene ◽  
P Protein

Rpm2p is a protein subunit of Saccharomyces cerevisiae yeast mitochondrial RNase P, an enzyme which removes 5' leader sequences from mitochondrial tRNA precursors. Precursor tRNAs accumulate in strains carrying a disrupted allele of RPM2. The resulting defect in mitochondrial protein synthesis causes petite mutants to form. We report here that alteration in the biogenesis of Rpm1r, the RNase P RNA subunit, is another consequence of disrupting RPM2. High-molecular-weight transcripts accumulate, and no mature Rpm1r is produced. Transcript mapping reveals that the smallest RNA accumulated is extended on both the 5' and 3' ends relative to mature Rpm1r. This intermediate and other longer transcripts which accumulate are also found as low-abundance RNAs in wild-type cells, allowing identification of processing events necessary for conversion of the primary transcript to final products. Our data demonstrate directly that Rpm1r is transcribed with its substrates, tRNA met f and tRNAPro, from a promoter located upstream of the tRNA met f gene and suggest that a portion also originates from a second promoter, located between the tRNA met f gene and RPM1. We tested the possibility that precursors accumulate because the RNase P deficiency prevents the removal of the downstream tRNAPro. Large RPM1 transcripts still accumulate in strains missing this tRNA. Thus, an inability to process cotranscribed tRNAs does not explain the precursor accumulation phenotype. Furthermore, strains with mutant RPM1 genes also accumulate precursor Rpm1r, suggesting that mutations in either gene can lead to similar biogenesis defects. Several models to explain precursor accumulation are presented.

scholarly journals RNase P protein subunit Rpp29 represses histone H3.3 nucleosome deposition

2016 ◽  
Vol 27 (7) ◽  
pp. 1154-1169 ◽  
Author(s):  
Alyshia Newhart ◽  
Sara Lawrence Powers ◽  
Prashanth Krishna Shastrula ◽  
Isabel Sierra ◽  
Lucy M. Joo ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Antisense Rna ◽  
Rnase P ◽  
Protein Subunit ◽  
P Protein ◽  
Histone H3.3 ◽  
Single Living Cells ◽  
Rna Complex ◽  
Nucleolar Rna ◽  
Single Living

In mammals, histone H3.3 is a critical regulator of transcription state change and heritability at both euchromatin and heterochromatin. The H3.3-specific chaperone, DAXX, together with the chromatin-remodeling factor, ATRX, regulates H3.3 deposition and transcriptional silencing at repetitive DNA, including pericentromeres and telomeres. However, the events that precede H3.3 nucleosome incorporation have not been fully elucidated. We previously showed that the DAXX-ATRX-H3.3 pathway regulates a multi-copy array of an inducible transgene that can be visualized in single living cells. When this pathway is impaired, the array can be robustly activated. H3.3 is strongly recruited to the site during activation where it accumulates in a complex with transcribed sense and antisense RNA, which is distinct from the DNA/chromatin. This suggests that transcriptional events regulate H3.3 recruited to its incorporation sites. Here we report that the nucleolar RNA proteins Rpp29, fibrillarin, and RPL23a are also components of this H3.3/RNA complex. Rpp29 is a protein subunit of RNase P. Of the other subunits, POP1 and Rpp21 are similarly recruited suggesting that a variant of RNase P regulates H3.3 chromatin assembly. Rpp29 knockdown increases H3.3 chromatin incorporation, which suggests that Rpp29 represses H3.3 nucleosome deposition, a finding with implications for epigenetic regulation.

scholarly journals Decay of ermC mRNA in a Polynucleotide Phosphorylase Mutant of Bacillus subtilis

1998 ◽  
Vol 180 (22) ◽  
pp. 5968-5977 ◽  
Author(s):  
David H. Bechhofer ◽  
Wei Wang
Keyword(s):  
Bacillus Subtilis ◽  
Rna Structure ◽  
Northern Blot Analysis ◽  
Polynucleotide Phosphorylase ◽  
Wild Type ◽  
Gene Coding ◽  
In The Wild ◽  
Rna Fragments ◽  
Specific Mrna

ABSTRACT ermC mRNA decay was examined in a mutant ofBacillus subtilis that has a deleted pnpA gene (coding for polynucleotide phosphorylase). 5′-proximal RNA fragments less than 400 nucleotides in length were abundant in thepnpA strain but barely detectable in the wild type. On the other hand, the patterns of 3′-proximal RNA fragments were similar in the wild-type and pnpA strains. Northern blot analysis with different probes showed that the 5′ end of the decay intermediates was the native ermC 5′ end. For one prominent ermCRNA fragment, in particular, it was shown that formation of its 3′ end was directly related to the presence of a stalled ribosome. 5′-proximal decay intermediates were also detected for transcripts encoded by theyybF gene. These results suggest that PNPase activity, which may be less sensitive to structures or sequences that block exonucleolytic decay, is required for efficient decay of specific mRNA fragments. However, it was shown that even PNPase activity could be blocked in vivo at a particular RNA structure.

scholarly journals Influence of Conformation of M. tuberculosis RNase P Protein Subunit on Its Function

PLoS ONE ◽  
2016 ◽  
Vol 11 (4) ◽  
pp. e0153798 ◽  
Author(s):  
Alla Singh ◽  
Shah Ubaid-ullah ◽  
Anup K. Ramteke ◽  
Janendra K Batra
Keyword(s):  
Rnase P ◽  
Protein Subunit ◽  
P Protein

scholarly journals Role of Spore Coat Proteins in the Resistance of Bacillus subtilis Spores to Caenorhabditis elegans Predation

2008 ◽  
Vol 190 (18) ◽  
pp. 6197-6203 ◽  
Author(s):  
Maria-Halima Laaberki ◽  
Jonathan Dworkin
Keyword(s):  
Bacillus Subtilis ◽  
Caenorhabditis Elegans ◽  
Spore Coat ◽  
Coat Proteins ◽  
Host Interaction ◽  
Wide Range ◽  
In The Wild

ABSTRACT Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also important effectors of innate immunity in a wide variety of hosts. Here, we use the bacteriovorous nematode Caenorhabditis elegans, a likely predator of Bacillus spores in the wild, to characterize the role of the spore coat in an ecologically relevant spore-host interaction. We found that ingested wild-type Bacillus subtilis spores were resistant to worm digestion, whereas vegetative forms of the bacterium were efficiently digested by the nematode. Using B. subtilis strains carrying mutations in spore coat genes, we observed a correlation between the degree of alteration of the spore coat assembly and the susceptibility to the worm degradation. Surprisingly, we found that the spores that were resistant to lysozyme in vitro can be sensitive to C. elegans digestion depending on the extent of the spore coat structure modifications.

scholarly journals In Vivo Display of a Multisubunit Enzyme Complex on Biogenic Magnetic Nanoparticles

2009 ◽  
Vol 75 (24) ◽  
pp. 7734-7738 ◽  
Author(s):  
Shoji Ohuchi ◽  
Dirk Schüler
Keyword(s):  
Model Experiment ◽  
Rnase P ◽  
Magnetotactic Bacteria ◽  
Functional Expression ◽  
Wild Type ◽  
Protein Subunit ◽  
Biological Complexes ◽  
First Time ◽  
Biogenic Magnetic Nanoparticles

ABSTRACT Magnetosomes are unique bacterial organelles comprising membrane-enveloped magnetic crystals produced by magnetotactic bacteria. Because of several desirable chemical and physical properties, magnetosomes would be ideal scaffolds on which to display highly complicated biological complexes artificially. As a model experiment for the functional expression of a multisubunit complex on magnetosomes, we examined the display of a chimeric bacterial RNase P enzyme composed of the protein subunit (C5) of Escherichia coli RNase P and the endogenous RNA subunit by expressing a translational fusion of C5 with MamC, a known magnetosome protein, in the magnetotactic bacterium Magnetospirillum gryphiswaldense. As intended, the purified C5 fusion magnetosomes, but not wild-type magnetosomes, showed apparent RNase P activity and the association of a typical bacterial RNase P RNA. Our results demonstrate for the first time that magnetosomes can be employed as scaffolds for the display of multisubunit complexes.

scholarly journals A functional RNase P protein subunit of bacterial origin in some eukaryotes

2011 ◽  
Vol 286 (5-6) ◽  
pp. 359-369 ◽  
Author(s):  
Lien B. Lai ◽  
Pilar Bernal-Bayard ◽  
Gireesha Mohannath ◽  
Stella M. Lai ◽  
Venkat Gopalan ◽  
...  
Keyword(s):  
Rnase P ◽  
Protein Subunit ◽  
Bacterial Origin ◽  
P Protein

scholarly journals Stability of Proteins Out of Service: the GapB Case of Bacillus subtilis

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Ulf Gerth ◽  
Eleonora Krieger ◽  
Daniela Zühlke ◽  
Alexander Reder ◽  
Uwe Völker ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Arginine Kinase ◽  
Adaptor Protein ◽  
Glycolytic Enzyme ◽  
Dependent Manner ◽  
Wild Type ◽  
Recognition Mechanism ◽  
Content Type ◽  
In The Wild

ABSTRACT Bacillus subtilis possesses two glyceraldehyde-3-phosphate dehydrogenases with opposite roles, the glycolytic NAD-dependent GapA and the NADP-dependent GapB enzyme, which is exclusively required during gluconeogenesis but not active under conditions promoting glycolysis. We propose that proteins that are no longer needed will be recognized and proteolyzed by Clp proteases and thereby recycled. To test this postulation, we analyzed the stability of the glycolytic enzyme GapA and the gluconeogenetic enzyme GapB in the presence and absence of glucose. It turned out that GapA remained rather stable under both glycolytic and gluconeogenetic conditions. In contrast, the gluconeogenetic enzyme GapB was degraded after a shift from malate to glucose (i.e., from gluconeogenesis to glycolysis), displaying an estimated half-life of approximately 3 h. Comparative in vivo pulse-chase labeling and immunoprecipitation experiments of the wild-type strain and isogenic mutants identified the ATP-dependent ClpCP protease as the enzyme responsible for the degradation of GapB. However, arginine protein phosphorylation, which was recently described as a general tagging mechanism for protein degradation, did not seem to play a role in GapB proteolysis, because GapB was also degraded in a mcsB mutant, lacking arginine kinase, in the same manner as in the wild type. IMPORTANCE GapB, the NADP-dependent glyceraldehyde-3-phosphosphate dehydrogenase, is essential for B. subtilis under gluconeogenetic conditions. However, after a shift to glycolytic conditions, GapB loses its physiological function within the cell and becomes susceptible to degradation, in contrast to GapA, the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, which remains stable under glycolytic and gluconeogenetic conditions. Subsequently, GapB is proteolyzed in a ClpCP-dependent manner. According to our data, the arginine kinase McsB is not involved as adaptor protein in this process. ClpCP appears to be in charge in the removal of inoperable enzymes in B. subtilis, which is a strictly regulated process in which the precise recognition mechanism(s) remains to be identified.

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