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Murray P. Deutscher - One of the best experts on this subject based on the ideXlab platform.
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RNase ii regulates RNase PH and is essential for cell survival during starvation and stationary PHase
RNA, 2017Co-Authors: Shaheen Sulthana, Ernesto Quesada, Murray P. DeutscherAbstract:RNase II is the most active exoribonuclease in Escherichia coli cell extracts. Yet, its removal appears to have no deleterious effect on growing cells. Here, we show that RNase II is required for cell survival during prolonged stationary PHase and upon starvation. The absence of RNase II leads to greatly increased rRNA degradation and to the accumulation of rRNA fragments, both of which lead to a decline in cell survival. The deleterious effects of RNase II removal can be completely reversed by the simultaneous absence of a second exoribonuclease, RNase PH, an enzyme known to be required to initiate ribosome degradation in starving cells. We have now found that the role of RNase II in this process is to regulate the amount of RNase PH present in starving cells, and it does so at the level of RNase PH stability. RNase PH normally decreases as much as 90% during starvation because the protein is unstable under these conditions; however, in the absence of RNase II the amount of RNase PH remains relatively unchanged. Based on these observations, we propose that in the presence of RNase II, nutrient deprivation leads to a dramatic reduction in the amount of RNase PH, thereby limiting the extent of rRNA degradation and ensuring cell survival during this stress. In the absence of RNase II, RNase PH levels remain high, leading to excessive ribosome loss and ultimately to cell death. These findings provide another example of RNase regulation in response to environmental stress.
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degradation of ribosomal rna during starvation comparison to quality control during steady state growth and a role for RNase PH
RNA, 2011Co-Authors: Georgeta N Basturea, Michael A Zundel, Murray P. DeutscherAbstract:Ribosomal RNAs are generally stable in growing Escherichia coli cells. However, their degradation increases dramatically under conditions that lead to slow cell growth. In addition, incomplete RNA molecules and molecules with defects in processing, folding, or assembly are also eliminated in growing cells in a process termed quality control. Here, we show that there are significant differences between the pathways of ribosomal RNA degradation during glucose starvation and quality control during steady-state growth. In both processes, endonucleolytic cleavage of rRNA in ribosome subunits is an early step, resulting in accumulation of large rRNA fragments when the processive exoribonucleases, RNase II, RNase R, and PNPase are absent. For 23S rRNA, cleavage is in the region of helix 71, but the exact position can differ in the two degradative processes. For 16S rRNA, degradation during starvation begins with shortening of its 39 end in a reaction catalyzed by RNase PH. In the absence of this RNase, there is no 39 end trimming of 16S rRNA and no accumulation of rRNA fragments, and total RNA degradation is greatly reduced. In contrast, the degradation pattern in quality control remains unchanged when RNase PH is absent. During starvation, the exoribonucleases RNase II and RNase R are important for fragment removal, whereas for quality control, RNase R and PNPase are more important. These data highlight the similarities and differences between rRNA degradation during starvation and quality control during steady-state growth and describe a role for RNase PH in the starvation degradative pathway.
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exoribonucleases and endoribonucleases
EcoSal Plus, 2004Co-Authors: Zhongwei Li, Murray P. DeutscherAbstract:This review provides a description of the known Escherichia coli ribonucleases (RNases), focusing on their structures, catalytic properties, genes, PHysiological roles, and possible regulation. Currently, eight E. coli exoribonucleases are known. These are RNases II, R, D, T, PH, BN, polynucleotide PHosPHorylase (PNPase), and oligoribonuclease (ORNase). Based on sequence analysis and catalytic properties, the eight exoribonucleases have been grouped into four families. These are the RNR family, including RNase II and RNase R; the DEDD family, including RNase D, RNase T, and ORNase; the RBN family, consisting of RNase BN; and the PDX family, including PNPase and RNase PH. Seven well-characterized endoribonucleases are known in E. coli. These are RNases I, III, P, E, G, HI, and HII. Homologues to most of these enzymes are also present in Salmonella. Most of the endoribonucleases cleave RNA in the presence of divalent cations, producing fragments with 3'-hydroxyl and 5'-PHosPHate termini. RNase H selectively hydrolyzes the RNA strand of RNA?DNA hybrids. Members of the RNase H family are widely distributed among prokaryotic and eukaryotic organisms in three distinct lineages, RNases HI, HII, and HIII. It is likely that E. coli contains additional endoribonucleases that have not yet been characterized. First of all, endonucleolytic activities are needed for certain known processes that cannot be attributed to any of the known enzymes. Second, homologues of known endoribonucleases are present in E. coli. Third, endonucleolytic activities have been observed in cell extracts that have different properties from known enzymes.
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an essential function for the PHosPHate dependent exoribonucleases RNase PH and polynucleotide PHosPHorylase
Journal of Bacteriology, 1997Co-Authors: Zhihua Zhou, Murray P. DeutscherAbstract:: Escherichia coli cells lacking both polynucleotide PHosPHorylase (PNPase) and RNase PH, the only known P(i)-dependent exoribonucleases, were previously shown to grow slowly at 37 degrees C and to display a dramatically reduced level of tRNA(Tyr)su3+ suppressor activity. Here we show that the RNase PH-negative, PNP-negative double-mutant strain actually displays a reversible cold-sensitive PHenotype and that tRNA biosynthesis is normal. In contrast, ribosome structure and function are severely affected, particularly at lower temperatures. At 31 degrees C, the amount of 50S subunit is dramatically reduced and 23S rRNA is degraded. Moreover, cells that had been incubated at 42 degrees C immediately cease growing and synthesizing protein upon a shift to 31 degrees C, suggesting that the ribosomes synthesized at the higher temperature are defective and unable to function at the lower temperature. These data indicate that RNase PH and PNPase play an essential role that affects ribosome metabolism and that this function cannot be taken over by any of the hydrolytic exoribonucleases present in the cell.
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multiple exoribonucleases are required for the 3 processing of escherichia coli trna precursors in vivo
The FASEB Journal, 1993Co-Authors: Nina Bacher Reuven, Murray P. DeutscherAbstract:Our knowledge of the 3' processing of tRNA precursors is severely limited. Although six exoribonucleases able to act on Escherichia coli tRNA precursors in vitro have been identified, their involvement in tRNA maturation in vivo has not been demonstrated. Here we show, using a wide range of multiple RNase-deficient strains and a quantitative suppression assay, that at least five of these enzymes--RNase II, RNase D, RNase BN, RNase T, and RNase PH--can participate in the synthesis of functional tRNA(Tyr)su+3 in vivo. Moreover, any one of the five RNases is sufficient to allow tRNA processing to proceed although with varying effectiveness. Examination of the level of aminoacylation of tRNA isolated from RNase-deficient strains suggested that tRNA precursors accumulate in the most defective cells. These data indicate that exoribonucleases are required for tRNA maturation in vivo and that there is a high degree of functional overlap among the enzymes. These studies contribute to the identification of all the ...
Graham C Walker - One of the best experts on this subject based on the ideXlab platform.
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elevated levels of era gtpase improve growth 16s rrna processing and 70s ribosome assembly of escherichia coli lacking highly conserved multifunctional ybey endoribonuclease
Journal of Bacteriology, 2018Co-Authors: Anubrata Ghosal, Vignesh M P Babu, Graham C WalkerAbstract:YbeY is a highly conserved, multifunctional endoribonuclease that plays a significant role in ribosome biogenesis and has several additional roles. Here we show that overexpression of the conserved GTPase Era in Escherichia coli partially suppresses the growth defect of a ΔybeY strain while improving 16S rRNA processing and 70S ribosome assembly. This suppression requires both the ability of Era to hydrolyze GTP and the function of three exoribonucleases, RNase II, RNase R, and RNase PH, suggesting a model for the action of Era. Overexpression of Vibrio cholerae Era similarly partially suppresses the defects of an E. coli ΔybeY strain, indicating that this property of Era is conserved in bacteria other than E. coliIMPORTANCE This work provides insight into the critical, but still incompletely understood, mechanism of processing of the E. coli 16S rRNA 3' terminus. The highly conserved GTPase Era is known to bind to the precursor of the 16S rRNA near its 3' end. Both the endoribonuclease YbeY, which binds to Era, and four exoribonucleases have been implicated in this 3'-end processing. The results reported here offer additional insights into the role of Era in 16S rRNA 3'-end maturation and into the relationship between the action of the endoribonuclease YbeY and that of the four exoribonucleases. This study also hints at why YbeY is essential only in some bacteria and suggests that YbeY could be a target for a new class of antibiotics in these bacteria.
Karl-peter Hopfner - One of the best experts on this subject based on the ideXlab platform.
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structural framework for the mechanism of archaeal exosomes in rna processing
Molecular Cell, 2005Co-Authors: Katharina Büttner, Katja Wenig, Karl-peter HopfnerAbstract:Summary Exosomes emerge as central 3′→5′ RNA processing and degradation machineries in eukaryotes and archaea. We determined crystal structures of two 230 kDa nine subunit archaeal exosome isoforms. Both exosome isoforms contain a hexameric ring of RNase PHosPHorolytic (PH) domain subunits with a central chamber. Tungstate soaks identified three PHosPHorolytic active sites in this processing chamber. A trimer of Csl4 or Rrp4 subunits forms a multidomain macromolecular interaction surface on the RNase-PH domain ring with central S1 domains and periPHeral KH and zinc-ribbon domains. Structural and mutational analyses suggest that the S1 domains and a subsequent neck in the RNase-PH domain ring form an RNA entry pore to the processing chamber that only allows access of unstructured RNA. This structural framework can mechanistically unify observed features of exosomes, including processive degradation of unstructured RNA, the requirement for regulatory factors to degrade structured RNA, and leftover tails in rRNA trimming.
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Structural Framework for the Mechanism of Archaeal Exosomes in RNA Processing
Molecular cell, 2005Co-Authors: Katharina Büttner, Katja Wenig, Karl-peter HopfnerAbstract:Summary Exosomes emerge as central 3′→5′ RNA processing and degradation machineries in eukaryotes and archaea. We determined crystal structures of two 230 kDa nine subunit archaeal exosome isoforms. Both exosome isoforms contain a hexameric ring of RNase PHosPHorolytic (PH) domain subunits with a central chamber. Tungstate soaks identified three PHosPHorolytic active sites in this processing chamber. A trimer of Csl4 or Rrp4 subunits forms a multidomain macromolecular interaction surface on the RNase-PH domain ring with central S1 domains and periPHeral KH and zinc-ribbon domains. Structural and mutational analyses suggest that the S1 domains and a subsequent neck in the RNase-PH domain ring form an RNA entry pore to the processing chamber that only allows access of unstructured RNA. This structural framework can mechanistically unify observed features of exosomes, including processive degradation of unstructured RNA, the requirement for regulatory factors to degrade structured RNA, and leftover tails in rRNA trimming.
Gadi Schuster - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of arabidopsis chloroplast polynucleotide PHosPHorylase reveals roles for both RNase PH core domains in polyadenylation rna 3 end maturation and intron degradation
Plant Journal, 2011Co-Authors: Arnaud Germain, Shira Herlich, Shirley Larom, Sang Hu Kim, Gadi Schuster, David B SternAbstract:*SUMMARY Polynucleotide PHosPHorylase (PNPase) catalyzes RNA polymerization and 3 ¢fi 5¢ PHosPHorolysis in vitro, but its roles in plant organelles are poorly understood. Here, we have used in vivo and in vitro mutagenesis to study Arabidopsis chloroplast PNPase (cpPNPase). In mutants lacking cpPNPase activity, unusual RNA patterns were broadly observed, implicating cpPNPase in rRNA and mRNA 3¢-end maturation, and RNA degradation. Intron-containing fragments also accumulated in mutants, and cpPNPase appears to be required for a degradation step following endonucleolytic cleavage of the excised lariat. Analysis of poly(A) tails, which destabilize chloroplast RNAs, indicated that PNPase and a poly(A) polymerase share the polymerization role in wild-type plants. We also studied two lines carrying mutations in the first PNPase core domain, which does not harbor the catalytic site. These mutants had gene-dependent and intermediate RNA PHenotypes, suggesting that reduced enzyme activity differentially affects chloroplast transcripts. The interpretations of in vivo results were confirmed by in vitro analysis of recombinant enzymes, and showed that the first core domain affects overall catalytic activity. In summary, cpPNPase has a major role in maturing mRNA and rRNA 3¢-ends, but also participates in RNA degradation through exonucleolytic digestion and polyadenylation. These functions depend absolutely on the catalytic site within the second duplicated RNase PH domain, and appear to be modulated by the first RNase PH domain.
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domain analysis of the chloroplast polynucleotide PHosPHorylase reveals discrete functions in rna degradation polyadenylation and sequence homology with exosome proteins
The Plant Cell, 2003Co-Authors: Shlomit Yehudairesheff, Victoria Portnoy, Sivan Yogev, Noam Adir, Gadi SchusterAbstract:The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events, including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation. In spinach chloroplasts, the latter two steps of polyadenylation and exonucleolytic degradation are performed by the same PHosPHorolytic and processive enzyme, polynucleotide PHosPHorylase (PNPase). An analysis of its amino acid sequence shows that the protein is composed of two core domains related to RNase PH, two RNA binding domains (KH and S1), and an α-helical domain. The amino acid sequence and domain structure is largely conserved between bacteria and organelles. To define the molecular mechanism that controls the two opposite activities of this protein in the chloroplast, the ribonuclease, polymerase, and RNA binding properties of each domain were analyzed. The first core domain, which was predicted to be inactive in the bacterial enzymes, was active in RNA degradation but not in polymerization. Surprisingly, the second core domain was found to be active in degrading polyadenylated RNA only, suggesting that nonpolyadenylated molecules can be degraded only if tails are added, apparently by the same protein. The poly(A) high-binding-affinity site was localized to the S1 domain. The complete spinach chloroplast PNPase, as well as versions containing the core domains, complemented the cold sensitivity of an Escherichia coli PNPase-less mutant. PHylogenetic analyses of the two core domains showed that the two domains separated very early, resulting in the evolution of the bacterial and organelle PNPases and the exosome proteins found in eukaryotes and some archaea.
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polynucleotide PHosPHorylase functions as both an exonuclease and a poly a polymerase in spinach chloroplasts
Molecular and Cellular Biology, 2001Co-Authors: Shlomit Yehudairesheff, Merav Hirsh, Gadi SchusterAbstract:The chloroplast is the site of PHotosynthesis and other essential biosynthetic activities in plant cells. The chloroplast's structural proteins and enzymes are encoded by both nuclear and chloroplast genomes. During chloroplast development, chloroplast gene expression is tightly regulated at many levels, including mRNA accumulation (reviewed in references 3, 19, and 39). RNA metabolism involves a series of steps that are dependent on RNA secondary structures, nucleases, and regulatory RNA-binding proteins. A 100-kDa RNA-binding protein that is homologous to the bacterial exoribonuclease polynucleotide PHosPHorylase (PNPase) was isolated from a chloroplast protein extract and found to be the protein responsible for most exoribonucleolytic activity. The homology of the chloroplast and the bacterial enzymes was observed both in amino acid sequences and in biochemical characteristics (20). PNPase is a PHosPHorolytic exonuclease involved in degrading prokaryotic and organelle RNAs (31, 40). It is a reversible enzyme that can degrade RNA by using inorganic PHosPHate (Pi) or synthesize RNA by using any nucleoside diPHosPHate. Until recently, it has always been assumed that due to the high concentration of Pi in bacteria and chloroplasts (about 10 mM), PNPase worked only degradatively (31). Indeed, PNPase has been shown to be an important component of the mRNA degradation system in bacteria and chloroplasts (6, 8, 17, 37). However, as described below, recent studies have shown that PNPase can also function in vivo as a polymerase in bacterial cells (33). PNPase is composed of several domains homologous to those in other RNA-binding proteins and ribonucleases. These include the KH and S1 domains, which are located at the C-terminal region, and two RNase PH domains that are also found in other PHosPHorolytic RNase enzymes (43). Structural and biochemical analyses have revealed a trimeric quaternary structure of PNPase (43). Part of the PNPase population in Escherichia coli is associated with the endoribonuclease RNase E, an RNA helicase, an enolase, and possibly several other proteins in a high-molecular-weight complex called a degradosome (6, 8, 17, 27, 37). In the chloroplast, PNPase was purified as a complex of about 600 kDa, composed only of this protein (20; Baginsky et al., submitted for publication). Therefore, a degradosome complex similar to that of E. coli does not exist in the chloroplast. The molecular mechanism of RNA degradation in the chloroplast has been elucidated during the past few years and was found to be very similar to that of bacteria (19, 39). In both bacteria and chloroplasts, the first event is endoribonuclease cleavage of the RNA molecule. The endoribonuclease cleavage is followed by the addition of a poly(A) tail in the bacteria and poly(A) (23) or a poly(A)-rich tail in the chloroplasts (28–30). The polyadenylated cleavage products are then directed to rapid exonucleolytic degradation by PNPase and RNase II in E. coli and by PNPase and possibly other exoribonucleases in the chloroplasts (30). Therefore, polyadenylation is part of the RNA degradation machinery in bacteria, chloroplasts, and possibly also in plant mitochondria (6, 8, 15, 19, 24, 32, 37–39, 41). In contrast to what is observed with bacteria, evidences for activity of a 5′ to 3′ exoribonuclease in the chloroplasts of the green alga Chlamydomonas reinhardtii were obtained (10, 11, 34). Whether or not such an enzyme exists in the chloroplasts of higher plants and what its function is in the mechanism of RNA degradation are still open questions. E. coli PAP I is responsible for 90 to 95% of the poly(A) tails, which are generally adenosine homopolymers. A smaller amount of nucleotides other than adenosine were reported in stationary-PHase cells and when PNPase was overexpressed (5, 21, 33). It was recently reported that the second RNA polyadenylation activity in E. coli, which takes over in the absence of PAP I, is carried out by PNPase (33). In this situation, the elongated tails were found to consist not only of adenosines but also of the three other nucleotides. In spinach chloroplasts, the polyadenylated tails could be several hundred nucleotides long and were found to consist of about 70% adenosines, 25% guanosines, and 5% cytosines and uridines (28). Heterologous poly(A)-rich tails were obtained when several chloroplast transcripts including mRNAs and rRNA were analyzed (28; Anbussi-AbuToami and Schuster, unpublished results). Therefore, the heteropolymeric tails obtained by reverse transcription-PCR from spinach chloroplast transcripts resemble the tails obtained from E. coli in the absence of PAP I when PNPase is the polyadenylation enzyme. In contrast to the situation in spinach chloroplasts, only polyadenylated tails were obtained when five chloroplast transcripts, including mRNAs, rRNAs, and tRNAs of C. reinhardtii, were analyzed (23). Therefore, the poly(A) tails of chloroplast transcripts in C. reinhardtii resemble those in E. coli when PAP I is active. In contrast to what occurs in bacteria, RNA polyadenylation in plants is expected to be of both eukaryotic and prokaryotic types. In the eukaryotic process, nucleus-derived RNA polymerase II transcripts are polyadenylated and this polyadenylated tract is important for translation initiation. The prokaryotic process occurring in the chloroplasts and possibly also in the mitochondria includes the polyadenylation of organelle-encoded transcripts as part of the RNA-degradation mechanism (39). In order to understand the molecular mechanism and the elements that modulate and control the RNA polyadenylation reaction in the chloroplast, we sought to identify the chloroplast polyadenylation enzyme(s). Since the mechanism of mRNA polyadenylation and degradation in the chloroplast is so similar to that in E. coli, the default hypothesis was that our target was a chloroplast homologue of PAP I. Surprisingly, however, a PAP I homologue could not be detected in chloroplasts from either spinach or pea. When in vitro polyadenylation activity from pea leaves was purified previously, a fraction containing two proteins was purified (26). Since one of these was identified as PNPase, it was proposed to be a component of the polyadenylation machinery in the chloroplast whose role was to bind the RNA to be polyadenylated but not the catalytic polyadenylating enzyme by itself (26). Here, we suggest that chloroplast PNPase performs both polyadenylation and exonucleolytic degradation. The observation that no putative PAP protein carrying a typical chloroplast transit peptide could be identified in the completely sequenced Arabidopsis genome is in agreement with this hypothesis.
Anubrata Ghosal - One of the best experts on this subject based on the ideXlab platform.
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elevated levels of era gtpase improve growth 16s rrna processing and 70s ribosome assembly of escherichia coli lacking highly conserved multifunctional ybey endoribonuclease
Journal of Bacteriology, 2018Co-Authors: Anubrata Ghosal, Vignesh M P Babu, Graham C WalkerAbstract:YbeY is a highly conserved, multifunctional endoribonuclease that plays a significant role in ribosome biogenesis and has several additional roles. Here we show that overexpression of the conserved GTPase Era in Escherichia coli partially suppresses the growth defect of a ΔybeY strain while improving 16S rRNA processing and 70S ribosome assembly. This suppression requires both the ability of Era to hydrolyze GTP and the function of three exoribonucleases, RNase II, RNase R, and RNase PH, suggesting a model for the action of Era. Overexpression of Vibrio cholerae Era similarly partially suppresses the defects of an E. coli ΔybeY strain, indicating that this property of Era is conserved in bacteria other than E. coliIMPORTANCE This work provides insight into the critical, but still incompletely understood, mechanism of processing of the E. coli 16S rRNA 3' terminus. The highly conserved GTPase Era is known to bind to the precursor of the 16S rRNA near its 3' end. Both the endoribonuclease YbeY, which binds to Era, and four exoribonucleases have been implicated in this 3'-end processing. The results reported here offer additional insights into the role of Era in 16S rRNA 3'-end maturation and into the relationship between the action of the endoribonuclease YbeY and that of the four exoribonucleases. This study also hints at why YbeY is essential only in some bacteria and suggests that YbeY could be a target for a new class of antibiotics in these bacteria.
