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Murray P. Deutscher - One of the best experts on this subject based on the ideXlab platform.
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how rnase r degrades structured rna role of the helicase activity and the s1 domain
Journal of Biological Chemistry, 2016Co-Authors: Sk Tofajjen Hossain, Arun Malhotra, Murray P. DeutscherAbstract:Abstract RNase R, a ubiquitous 3′ Exoribonuclease, plays an important role in many aspects of RNA metabolism. In contrast to other Exoribonucleases, RNase R can efficiently degrade highly structured RNAs, but the mechanism by which this is accomplished has remained elusive. It is known that RNase R contains an unusual, intrinsic RNA helicase activity that facilitates degradation of duplex RNA, but how it stimulates the nuclease activity has also been unclear. Here, we have made use of specifically designed substrates to compare the nuclease and helicase activities of RNase R. We have also identified and mutated several residues in the S1 RNA-binding domain that are important for interacting with duplex RNA and have measured intrinsic tryptophan fluorescence to analyze the conformational changes that occur upon binding of structured RNA. Using these approaches, we have determined the relation of the RNA helicase, ATP binding, and nuclease activities of RNase R. This information has been combined with a structural analysis of RNase R, based on its homology to RNase II, whose structure has been determined, to develop a detailed model that explains how RNase R digests structured RNA and how this differs from its action on single-stranded RNA.
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Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo
Journal of Biological Chemistry, 2012Co-Authors: Tanmay Dutta, Arun Malhotra, Murray P. DeutscherAbstract:Abstract Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an Exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks Exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of Exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of Exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the Exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the Exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its Exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.
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the roles of individual domains of rnase r in substrate binding and Exoribonuclease activity the nuclease domain is sufficient for digestion of structured rna
Journal of Biological Chemistry, 2009Co-Authors: Helen A Vincent, Murray P. DeutscherAbstract:RNase R and RNase II are the two representatives from the RNR family of processive, 3' to 5' Exoribonucleases in Escherichia coli. Although RNase II is specific for single-stranded RNA, RNase R readily degrades through structured RNA. Furthermore, RNase R appears to be the only known 3' to 5' Exoribonuclease that is able to degrade through double-stranded RNA without the aid of a helicase activity. Consequently, its functional domains and mechanism of action are of great interest. Using a series of truncated RNase R proteins we show that the cold-shock and S1 domains contribute to substrate binding. The cold-shock domains appear to play a role in substrate recruitment, whereas the S1 domain is most likely required to position substrates for efficient catalysis. Most importantly, the nuclease domain alone, devoid of the cold-shock and S1 domains, is sufficient for RNase R to bind and degrade structured RNAs. Moreover, this is a unique property of the nuclease domain of RNase R because this domain in RNase II stalls as it approaches a duplex. We also show that the nuclease domain of RNase R binds RNA more tightly than the nuclease domain of RNase II. This tighter binding may help to explain the difference in catalytic properties between RNase R and RNase II.
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Substrate recognition and catalysis by the Exoribonuclease RNase R.
Journal of Biological Chemistry, 2006Co-Authors: Helen A Vincent, Murray P. DeutscherAbstract:Abstract RNase R is a processive, 3′ to 5′ hydrolytic Exoribonuclease that together with polynucleotide phosphorylase plays an important role in the degradation of structured RNAs. However, RNase R differs from other Exoribonucleases in that it can by itself degrade RNAs with extensive secondary structure provided that a single-stranded 3′ overhang is present. Using a variety of specifically designed substrates, we show here that a 3′ overhang of at least 7 nucleotides is required for tight binding and activity, whereas optimum binding and activity are achieved when the overhang is 10 or more nucleotides in length. In contrast, duplex RNAs with no overhang or with a 4-nucleotide overhang bind extremely poorly to RNase R and are inactive as substrates. A duplex RNA with a 10-nucleotide 5′ overhang also is not a substrate. Interestingly, this molecule is bound only weakly, indicating that RNase R does not simply recognize single-stranded RNA, but the RNA must thread into the enzyme with 3′ to 5′ polarity. We also show that ribose moieties are required for recognition of the substrate as a whole since RNase R is unable to bind or degrade single-stranded DNA. However, RNA molecules with deoxyribose or dideoxyribose residues at their 3′ termini can be bound and degraded. Based on these data and a homology model of RNase R, derived from the structure of the closely related enzyme, RNase II, we present a model for how RNase R interacts with its substrates and degrades RNA.
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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.
Cecilia M Arraiano - One of the best experts on this subject based on the ideXlab platform.
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defining the impact of Exoribonucleases in the shift between exponential and stationary phases
Scientific Reports, 2019Co-Authors: Vânia Pobre, Susana Barahona, Tatiane Dobrzanski, M B R Steffens, Cecilia M ArraianoAbstract:: The transition between exponential and stationary phase is a natural phenomenon for all bacteria and requires a massive readjustment of the bacterial transcriptome. Exoribonucleases are key enzymes in the transition between the two growth phases. PNPase, RNase R and RNase II are the major degradative Exoribonucleases in Escherichia coli. We analysed the whole transcriptome of exponential and stationary phases from the WT and mutants lacking these Exoribonucleases (Δpnp, Δrnr, Δrnb, and ΔrnbΔrnr). When comparing the cells from exponential phase with the cells from stationary phase more than 1000 transcripts were differentially expressed, but only 491 core transcripts were common to all strains. There were some differences in the number and transcripts affected depending on the strain, suggesting that Exoribonucleases influence the transition between these two growth phases differently. Interestingly, we found that the double mutant RNase II/RNase R is similar to the RNase R single mutant in exponential phase while in stationary phase it seems to be closer to the RNase II single mutant. This is the first global transcriptomic work comparing the roles of Exoribonucleases in the transition between exponential and stationary phase.
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Biochemical characterization of Campylobacter jejuni PNPase, an Exoribonuclease important for bacterial pathogenicity.
Biochimie, 2018Co-Authors: Jorge Casinhas, Rute G Matos, Nabila Haddad, Cecilia M ArraianoAbstract:Abstract Bacteria need to promptly respond to environmental changes. Ribonucleases (RNases) are key factors in the adaptation to new environments by enabling a rapid adjustment in RNA levels. The Exoribonuclease polynucleotide phosphorylase (PNPase) is essential for low-temperature cell survival, affects the synthesis of proteins involved in virulence and has an important role in swimming, cell adhesion/invasion ability, and chick colonization in C. jejuni. However, the mechanism of action of this ribonuclease is not yet known. In this work we have characterized the biochemical activity of C. jejuni PNPase. Our results demonstrate that Cj-PNP is a processive 3′ to 5′ Exoribonuclease that degrades single-stranded RNAs. Its activity is regulated according to the temperature and divalent ions. We have also shown that the KH and S1 domains are important for trimerization, RNA binding, and, consequently, for the activity of Cj-PNP. These findings will be helpful to develop new strategies for fighting against C. jejuni and may be extrapolated to other foodborne pathogens.
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characterizing the role of Exoribonucleases in the control of microbial gene expression differential rna seq
Methods in Enzymology, 2018Co-Authors: Vânia Pobre, Cecilia M ArraianoAbstract:Abstract Differential RNA-Seq is a next-generation technology method to determine the significant transcriptomic differences between two and more samples. With this method it is possible to analyze the total RNA content of different samples making it the best global analysis method currently available to study the roles of Exoribonucleases in the cell. These enzymes are responsible for the RNA processing and degradation in the cells and therefore affect the total RNA pool in ways not yet fully understood. In Escherichia coli there are three main degradative Exoribonucleases RNase II, RNase R, and PNPase that degrade the RNA from the 3′ to the 5′-end. These enzymes have several roles in the cell and even though they are degradative enzymes RNase II and PNPase can also protect some RNAs from degradation and PNPase can also act as an RNA polymerase under some conditions. The multiplicity of roles of these Exoribonucleases leads to a very high number of transcripts that are affected by their absence in the cell. With the differential RNA-Seq it is possible to obtain a much deeper understanding of how these enzymes work and regulate the bacterial gene expression. In this chapter we have described a differential RNA-Seq data analysis protocol applied to the study of Exoribonucleases. We also included the protocol for experimental validation of the RNA-Seq data using qPCR and motility assays. Although the methods described in this chapter were applied to the study of the Exoribonucleases, they can also be used for other differential RNA-Seq studies.
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major 3 5 Exoribonucleases in the metabolism of coding and non coding rna
Progress in Molecular Biology and Translational Science, 2018Co-Authors: Ricardo Dos F Santos, Ana P Quendera, Sofia Boavida, Andre F Seixas, Cecilia M Arraiano, Jose M AndradeAbstract:Abstract 3′–5′ Exoribonucleases are key enzymes in the degradation of superfluous or aberrant RNAs and in the maturation of precursor RNAs into their functional forms. The major bacterial 3′–5′ Exoribonucleases responsible for both these activities are PNPase, RNase II and RNase R. These enzymes are of ancient nature with widespread distribution. In eukaryotes, PNPase and RNase II/RNase R enzymes can be found in the cytosol and in mitochondria and chloroplasts; RNase II/RNase R-like enzymes are also found in the nucleus. Humans express one PNPase (PNPT1) and three RNase II/RNase R family members (Dis3, Dis3L and Dis3L2). These enzymes take part in a multitude of RNA surveillance mechanisms that are critical for translation accuracy. Although active against a wide range of both coding and non-coding RNAs, the different 3′–5′ Exoribonucleases exhibit distinct substrate affinities. The latest studies on these RNA degradative enzymes have contributed to the identification of additional homologue proteins, the uncovering of novel RNA degradation pathways, and to a better comprehension of several disease-related processes and response to stress, amongst many other exciting findings. Here, we provide a comprehensive and up-to-date overview on the function, structure, regulation and substrate preference of the key 3′–5′ Exoribonucleases involved in RNA metabolism.
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next generation sequencing analysis reveals that the ribonucleases rnase ii rnase r and pnpase affect bacterial motility and biofilm formation in e coli
BMC Genomics, 2015Co-Authors: Vânia Pobre, Cecilia M ArraianoAbstract:Background The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main Exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these Exoribonucleases not much is known about their global effect in the transcriptome.
Jeffrey S. Kieft - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of a polerovirus Exoribonuclease resistant rna shows how diverse sequences are integrated into a conserved fold
RNA, 2020Co-Authors: Anna-lena Steckelberg, David A. Costantino, Quentin Vicens, Jay C Nix, Jeffrey S. KieftAbstract:Exoribonuclease-resistant RNAs (xrRNAs) are discrete elements that block the progression of 5' to 3' Exoribonucleases using specifically folded RNA structures. A recently discovered class of xrRNA is widespread in several genera of plant-infecting viruses, within both noncoding and protein-coding subgenomic RNAs. The structure of one such xrRNA from a dianthovirus revealed three-dimensional details of the resistant fold but did not answer all questions regarding the conservation and diversity of this xrRNA class. Here, we present the crystal structure of a representative polerovirus xrRNA that contains sequence elements that diverge from the previously solved structure. This new structure rationalizes previously unexplained sequence conservation patterns and shows interactions not present in the first structure. Together, the structures of these xrRNAs from dianthovirus and polerovirus genera support the idea that these plant virus xrRNAs fold through a defined pathway that includes a programmed intermediate conformation. This work deepens our knowledge of the structure-function relationship of xrRNAs and shows how evolution can craft similar RNA folds from divergent sequences.
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a folded viral noncoding rna blocks host cell Exoribonucleases through a conformationally dynamic rna structure
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Anna-lena Steckelberg, Benjamin M Akiyama, David A. Costantino, Jeffrey S. KieftAbstract:Folded RNA elements that block processive 5′ → 3′ cellular Exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping Exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5′ end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use “codegradational remodeling,” exploiting the Exoribonucleases’ degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent Exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.
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a folded viral noncoding rna blocks host cell Exoribonucleases through programmed remodeling of rna structure
bioRxiv, 2018Co-Authors: Anna-lena Steckelberg, Benjamin M Akiyama, David A. Costantino, Jeffrey S. KieftAbstract:Folded RNA elements that block processive 5'-->3' cellular Exoribonucleases (xrRNAs) to produce biologically active viral non-coding RNAs were discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, it was unknown if this RNA structure-dependent mechanism exists elsewhere and if so, whether a singular RNA fold is required. Here, we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These novel xrRNAs have no sequence similarity to known xrRNAs, thus we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping Exoribonucleases. By solving the structure of a dianthovirus xrRNAs by x-ray crystallography, we reveal a complex fold that is very different from the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature that is created by a different set of intramolecular contacts; this may be a defining structural feature of xrRNAs. Remarkably, the dianthovirus xrRNA can use 'co-degradational remodeling,' exploiting the Exoribonuclease's degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent Exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.
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mechanism and structural diversity of Exoribonuclease resistant rna structures in flaviviral rnas
Nature Communications, 2018Co-Authors: Andrea Macfadden, Erich G. Chapman, René C. L. Olsthoorn, Zoe Odonoghue, Patricia A G C Silva, Mark G Sterken, Gorben P Pijlman, Peter J Bredenbeek, Jeffrey S. KieftAbstract:Flaviviruses such as Yellow fever, Dengue, West Nile, and Zika generate disease-linked viral noncoding RNAs called subgenomic flavivirus RNAs. Subgenomic flavivirus RNAs result when the 5'-3' progression of cellular Exoribonuclease Xrn1 is blocked by RNA elements called Xrn1-resistant RNAs located within the viral genome's 3'-untranslated region that operate without protein co-factors. Here, we show that Xrn1-resistant RNAs can halt diverse Exoribonucleases, revealing a mechanism in which they act as general mechanical blocks that 'brace' against an enzyme's surface, presenting an unfolding problem that confounds further enzyme progression. Further, we directly demonstrate that Xrn1-resistant RNAs exist in a diverse set of flaviviruses, including some specific to insects or with no known arthropod vector. These Xrn1-resistant RNAs comprise two secondary structural classes that mirror previously reported phylogenic analysis. Our discoveries have implications for the evolution of Exoribonuclease resistance, the use of Xrn1-resistant RNAs in synthetic biology, and the development of new therapies.
Gwennaele Fichant - One of the best experts on this subject based on the ideXlab platform.
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rna processing machineries in archaea the 5 3 Exoribonuclease arnase j of the β casp family is engaged specifically with the helicase ash ski2 and the 3 5 exoribonucleolytic rna exosome machinery
Nucleic Acids Research, 2020Co-Authors: Duy Khanh Phung, Clarisse Etienne, Manon Batista, Yann Moalic, Sébastien Laurent, Sophie Liuu, Violette Morales, Mohamed Jebbar, Petra Langendijkgenevaux, Gwennaele FichantAbstract:A network of RNA helicases, endoribonucleases and Exoribonucleases regulates the quantity and quality of cellular RNAs. To date, mechanistic studies focussed on bacterial and eukaryal systems due to the challenge of identifying the main drivers of RNA decay and processing in Archaea. Here, our data support that aRNase J, a 5'-3' Exoribonuclease of the β-CASP family conserved in Euryarchaeota, engages specifically with a Ski2-like helicase and the RNA exosome to potentially exert control over RNA surveillance, at the vicinity of the ribosome. Proteomic landscapes and direct protein-protein interaction analyses, strengthened by comprehensive phylogenomic studies demonstrated that aRNase J interplay with ASH-Ski2 and a cap exosome subunit. Finally, Thermococcus barophilus whole-cell extract fractionation experiments provide evidences that an aRNase J/ASH-Ski2 complex might exist in vivo and hint at an association of aRNase J with the ribosome that is emphasised in absence of ASH-Ski2. Whilst aRNase J homologues are found among bacteria, the RNA exosome and the Ski2-like RNA helicase have eukaryotic homologues, underlining the mosaic aspect of archaeal RNA machines. Altogether, these results suggest a fundamental role of β-CASP RNase/helicase complex in archaeal RNA metabolism.
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RNA processing machineries in Archaea: the 5′-3′ Exoribonuclease aRNase J of the β-CASP family is engaged specifically with the helicase ASH-Ski2 and the 3′-5′ exoribonucleolytic RNA exosome machinery
Nucleic Acids Research, 2020Co-Authors: Duy Khanh Phung, Clarisse Etienne, Manon Batista, Petra Langendijk-genevaux, Yann Moalic, Sébastien Laurent, Sophie Liuu, Violette Morales, Mohamed Jebbar, Gwennaele FichantAbstract:A network of RNA helicases, endoribonucleases and Exoribonucleases regulates the quantity and quality of cellular RNAs. To date, mechanistic studies focussed on bacterial and eukaryal systems due to the challenge of identifying the main drivers of RNA decay and processing in Archaea. Here, our data support that aRNase J, a 5'-3' Exoribonuclease of the β-CASP family conserved in Euryarchaeota, engages specifically with a Ski2-like helicase and the RNA exosome to potentially exert control over RNA surveillance, at the vicinity of the ribosome. Proteomic landscapes and direct protein-protein interaction analyses, strengthened by comprehensive phylogenomic studies demonstrated that aRNase J interplay with ASH-Ski2 and a cap exosome subunit. Finally, Thermococcus barophilus whole-cell extract fractionation experiments provide evidences that an aRNase J/ASH-Ski2 complex might exist in vivo and hint at an association of aRNase J with the ribosome that is emphasised in absence of ASH-Ski2. Whilst aRNase J homologues are found among bacteria, the RNA exosome and the Ski2-like RNA helicase have eukaryotic homologues, underlining the mosaic aspect of archaeal RNA machines. Altogether, these results suggest a fundamental role of β-CASP RNase/helicase complex in archaeal RNA metabolism.
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rna processing machineries in archaea the 5 3 Exoribonuclease arnase j of the β casp family is engaged specifically with the helicase ash ski2 and the 3 5 exoribonucleolytic rna exosome machinery
bioRxiv, 2019Co-Authors: Duy Khanh Phung, Clarisse Etienne, Manon Batista, Yann Moalic, Sébastien Laurent, Violette Morales, Mohamed Jebbar, Gwennaele Fichant, Petra Langendijkgenevaux, Marie BouvierAbstract:A network of RNA helicases, endoribonucleases, and Exoribonucleases regulates the quantity and quality of cellular RNAs. To date, mechanistic studies focused on bacterial and eukaryal systems due to the challenge of identifying the main drivers of RNA decay and processing in Archaea. Here, our data support that aRNase J, a 5′-3′ Exoribonuclease of the β-CASP family conserved in Euryarchaea, engages specifically with a Ski2-like helicase and the RNA exosome to potentially exert control over RNA surveillance, and that this occurs in the vicinity of the ribosome. Proteomic landscapes and direct protein-protein interaction analyses demonstrated that aRNase J interplay with ASH-Ski2 and the Csl4 cap exosome subunit. These in vitro data are strengthened by our phylogenomic studies showing a taxonomic co-distribution of aRNase J and ASH-Ski2 among the archaeal phylogeny. Finally, our T. barophilus whole-cell extract fractionation experiments provide evidences that an aRNase J/ASH-Ski2 complex might exist in vivo and hint at an association of aRNase J with the ribosome or polysomes that is stressed in absence of ASH-Ski2. While aRNase J homologues are found among bacteria, the RNA exosome and the Ski2-like RNA helicase have eukaryotic homologues, underlining the mosaic aspect of archaeal RNA machines. Altogether, these results suggest, for the first time, a fundamental role of β-CASP RNase/helicase complex in archaeal RNA metabolism. Finally, our results position aRNase J at the junction of RNA surveillance and translation processes, thus opening new perspectives and evolutionary scenario on RNA processing players in Archaea.
Arun Malhotra - One of the best experts on this subject based on the ideXlab platform.
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how rnase r degrades structured rna role of the helicase activity and the s1 domain
Journal of Biological Chemistry, 2016Co-Authors: Sk Tofajjen Hossain, Arun Malhotra, Murray P. DeutscherAbstract:Abstract RNase R, a ubiquitous 3′ Exoribonuclease, plays an important role in many aspects of RNA metabolism. In contrast to other Exoribonucleases, RNase R can efficiently degrade highly structured RNAs, but the mechanism by which this is accomplished has remained elusive. It is known that RNase R contains an unusual, intrinsic RNA helicase activity that facilitates degradation of duplex RNA, but how it stimulates the nuclease activity has also been unclear. Here, we have made use of specifically designed substrates to compare the nuclease and helicase activities of RNase R. We have also identified and mutated several residues in the S1 RNA-binding domain that are important for interacting with duplex RNA and have measured intrinsic tryptophan fluorescence to analyze the conformational changes that occur upon binding of structured RNA. Using these approaches, we have determined the relation of the RNA helicase, ATP binding, and nuclease activities of RNase R. This information has been combined with a structural analysis of RNase R, based on its homology to RNase II, whose structure has been determined, to develop a detailed model that explains how RNase R digests structured RNA and how this differs from its action on single-stranded RNA.
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Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo
Journal of Biological Chemistry, 2012Co-Authors: Tanmay Dutta, Arun Malhotra, Murray P. DeutscherAbstract:Abstract Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an Exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks Exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of Exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of Exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the Exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the Exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its Exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.
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selective 2 hydroxyl acylation analyzed by protection from Exoribonuclease
Journal of the American Chemical Society, 2010Co-Authors: Kady Ann Steen, Arun Malhotra, Kevin M WeeksAbstract:Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a powerful approach for characterizing RNA structure and dynamics at single-nucleotide resolution. However, SHAPE technology is limited, sometimes severely, because primer extension detection obscures structural information for approximately 15 nts at the 5' end and 40-60 nts at the 3' end of the RNA. Moreover, detection by primer extension is more complex than the actual structure-selective chemical interrogation step. Here we quantify covalent adducts in RNA directly by adduct-inhibited Exoribonuclease degradation. RNA 2'-O-adducts block processivity of a 3'-->5' Exoribonuclease, RNase R, to produce fragments that terminate three nucleotides 3' of the modification site. We analyzed the structure of the native thiamine pyrophosphate (TPP) riboswitch aptamer domain and identified large changes in local nucleotide dynamics and global RNA structure upon ligand binding. In addition to numerous changes that can be attributed to ligand recognition, we identify a single nucleotide bulge register shift, distant from the binding site, that stabilizes the ligand-bound structure. Selective 2'-hydroxyl acylation analyzed by protection from Exoribonuclease (RNase-detected SHAPE) should prove broadly useful for facile structural analysis of small noncoding RNAs and for RNAs that have functionally critical structures at their 5' and 3' ends.
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Crystal structure of RNase T, an Exoribonuclease involved in tRNA maturation and end turnover.
Structure, 2007Co-Authors: Heping Zheng, Marcin Cymborowski, Maksymilian Chruszcz, Tatiana Skarina, Alexei Savchenko, Arun Malhotra, Yong Wang, Wladek MinorAbstract:Summary The 3′ processing of most bacterial precursor tRNAs involves exonucleolytic trimming to yield a mature CCA end. This step is carried out by RNase T, a member of the large DEDD family of exonucleases. We report the crystal structures of RNase T from Escherichia coli and Pseudomonas aeruginosa , which show that this enzyme adopts an opposing dimeric arrangement, with the catalytic DEDD residues from one monomer closely juxtaposed with a large basic patch on the other monomer. This arrangement suggests that RNase T has to be dimeric for substrate specificity, and agrees very well with prior site-directed mutagenesis studies. The dimeric architecture of RNase T is very similar to the arrangement seen in oligoribonuclease, another bacterial DEDD family Exoribonuclease. The catalytic residues in these two enzymes are organized very similarly to the catalytic domain of the third DEDD family Exoribonuclease in E. coli , RNase D, which is monomeric.
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Crystal Structure of Escherichia coli RNase D, an Exoribonuclease Involved in Structured RNA Processing
Structure, 2005Co-Authors: Yong Wang, Arun MalhotraAbstract:Summary RNase D (RND) is one of seven Exoribonucleases identified in Escherichia coli . RNase D has homologs in many eubacteria and eukaryotes, and has been shown to contribute to the 3′ maturation of several stable RNAs. Here, we report the 1.6 A resolution crystal structure of E. coli RNase D. The conserved DEDD residues of RNase D fold into an arrangement very similar to the Klenow fragment exonuclease domain. Besides the catalytic domain, RNase D also contains two structurally similar α-helical domains with no discernible sequence homology between them. These closely resemble the HRDC domain previously seen in RecQ-family helicases and several other proteins acting on nucleic acids. More interestingly, the DEDD catalytic domain and the two helical domains come together to form a ring-shaped structure. The ring-shaped architecture of E. coli RNase D and the HRDC domains likely play a major role in determining the substrate specificity of this Exoribonuclease.