Characterization of 3′-Phosphate RNA Ligase Paralogs RtcB1, RtcB2, and RtcB3 from Myxococcus xanthus Highlights DNA and RNA 5′-Phosphate Capping Activity of RtcB3

Maughan, William P.; Shuman, Stewart

doi:10.1128/jb.00631-15

Cited by 14 publications

(10 citation statements)

References 38 publications

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“…Importantly, some antibiotics targeting the translational apparatus are more effective when Rtc is not functional, demonstrating that the Rtc system can be a part of the native resistome through its role in maintaining the integrity of rRNA. The existence of paralogues of RtcB with distinctive biochemical activities as seen in for example Myxococcus xanthus (28) suggests elaborations of RtcB functionalities will be important in some bacteria. Taken together, with the role that RtcB plays in tRNA maturation (5,6) and the unfolded protein response in higher systems (9,10), our findings suggest that RNA repair systems will support many key cellular processes ranging from maintaining the translational apparatus to control of antibiotic sensitivity and chemotactical behaviour in bacteria (this paper) to establishing neuronal networks in higher organisms (13,14).…”

Section: Resultsmentioning

confidence: 99%

Cellular and molecular phenotypes depending upon the RNA repair system RtcAB ofEscherichia coli

et al. 2016

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RNA ligases function pervasively across the three kingdoms of life for RNA repair, splicing and can be stress induced. The RtcB protein (also HSPC117, C22orf28, FAAP and D10Wsu52e) is one such conserved ligase, involved in tRNA and mRNA splicing. However, its physiological role is poorly described, especially in bacteria. We now show in Escherichia coli bacteria that the RtcR activated rtcAB genes function for ribosome homeostasis involving rRNA stability. Expression of rtcAB is activated by agents and genetic lesions which impair the translation apparatus or may cause oxidative damage in the cell. Rtc helps the cell to survive challenges to the translation apparatus, including ribosome targeting antibiotics. Further, loss of Rtc causes profound changes in chemotaxis and motility. Together, our data suggest that the Rtc system is part of a previously unrecognized adaptive response linking ribosome homeostasis with basic cell physiology and behaviour.

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Section: Resultsmentioning

confidence: 99%

Cellular and molecular phenotypes depending upon the RNA repair system RtcAB ofEscherichia coli

et al. 2016

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“…Mutational effects on DNA 3=-phosphate capping. Bacterial RtcB enzymes transfer GMP from GTP to a DNA 3=-phosphate end (DNAp) to form a DNAppG cap structure (8,12). To probe the enzymic requirements for DNA capping, we reacted the E. coli RtcB proteins (1 M) for 5 min at 37°C with 0.1 M 5=-32 Plabeled 12-mer pDNAp substrate (Fig.…”

Section: Effects Of Active-site Mutations On Ho Rnap Ligationmentioning

confidence: 99%

Distinct Contributions of Enzymic Functional Groups to the 2′,3′-Cyclic Phosphodiesterase, 3′-Phosphate Guanylylation, and 3′-ppG/5′-OH Ligation Steps of the Escherichia coli RtcB Nucleic Acid Splicing Pathway

Maughan

Shuman

2016

J Bacteriol

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Escherichia coli RtcB is a founding member of a family of manganese-dependent RNA repair enzymes that join RNA 2=,3=-cyclic phosphate (RNA>p) or RNA 3=-phosphate (RNAp) ends to 5=-OH RNA ( HO RNA) ends in a multistep pathway whereby RtcB (i) hydrolyzes RNA>p to RNAp, (ii) transfers GMP from GTP to RNAp to form to RNAppG, and (iii) directs the attack of 5=-OH on RNAppG to form a 3=-5= phosphodiester splice junction. The crystal structure of the homologous archaeal RtcB enzyme revealed an active site with two closely spaced manganese ions, Mn1 and Mn2, that interact with the GTP phosphates. By studying the reactions of wild-type E. coli RtcB and RtcB alanine mutants with 3=-phosphate-, 2=,3=-cyclic phosphate-, and 3=-ppG-terminated substrates, we found that enzymic constituents of the two metal coordination complexes (Cys78, His185, and His281 for Mn1 and Asp75, Cys78, and His168 for Mn2 in E. coli RtcB) play distinct catalytic roles. For example, whereas the C78A mutation abolished all steps assayed, the D75A mutation allowed cyclic phosphodiester hydrolysis but crippled 3=-phosphate guanylylation, and the H281A mutant was impaired in overall HO RNAp and HO RNA>p ligation but was able to seal a preguanylylated substrate. The archaeal counterpart of E. coli RtcB Arg189 coordinates a sulfate anion construed to mimic the position of an RNA phosphate. We propose that Arg189 coordinates a phosphodiester at the 5=-OH end, based on our findings that the R189A mutation slowed the step of RNAppG/ HO RNA sealing by a factor of 200 compared to that with wild-type RtcB while decreasing the rate of RNAppG formation by only 3-fold. IMPORTANCERtcB enzymes comprise a widely distributed family of manganese-and GTP-dependent RNA repair enzymes that ligate 2=,3=-cyclic phosphate ends to 5=-OH ends via RNA 3=-phosphate and RNA(3=)pp(5=)G intermediates. The RtcB active site includes two adjacent manganese ions that engage the GTP phosphates. Alanine scanning of Escherichia coli RtcB reveals distinct contributions of metal-binding residues Cys78, Asp75, and His281 at different steps of the RtcB pathway. The RNA contacts of RtcB are uncharted. Mutagenesis implicates Arg189 in engaging the 5=-OH RNA end. Escherichia coli RtcB exemplifies a novel family of RNA ligases implicated in tRNA splicing and RNA repair (1-7). Unlike classic RNA and DNA ligases, which join 3=-OH and 5=-phosphate ends, RtcB seals broken RNAs with 5=-OH ( HO RNA) and either 2=,3=-cyclic phosphate (RNAϾp) or 3=-phosphate (RNAp) ends. E. coli RtcB executes a multistep ligation pathway (5-7) entailing (i) reaction of the enzyme with GTP to form a covalent RtcB-(His337-N)-GMP intermediate, (ii) hydrolysis of RNAϾp to RNAp, (iii) transfer of guanylate from His337 to the polynucleotide 3=-phosphate to form a polynucleotide-(3=)pp(5=)G intermediate, and (iv) attack of a 5=-OH on the ϪNppG end to form the splice junction and liberate GMP (Fig. 1). The E. coli RtcB reaction pathway requires manganese as a divalent cation cofactor. The catalytic repertoire of E. coli ...

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“…Fungal Trl1 enzymes are potential therapeutic targets because their domain structures and biochemical mechanisms are unique compared to the RtcB-type tRNA repair systems elaborated by metazoans, archaea, and many bacteria (Popow et al 2011;Tanaka and Shuman 2011;Tanaka et al 2011a;Englert et al 2012;Desai et al 2013;Maughan and Shuman 2015). RtcB is a GTP-dependent RNA ligase that splices 3 ′ -PO 4 and 5 ′ -OH ends via a novel chemical mechanism entailing the formation of covalent RtcB-(histidinyl)-GMP and RNA 3 ′ pp 5 ′G intermediates.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis

Remus

Schwer

Shuman

2016

RNA

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Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that repairs RNA breaks with 2 ′ ,3 ′ -cyclic-PO 4 and 5 ′ -OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3 ′ -OH, 2 ′ -PO 4 , and 5 ′ -PO 4 termini required for sealing by an N-terminal ligase domain. Trl1 enzymes are found in all human fungal pathogens and they are promising targets for antifungal drug discovery because: (i) their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme; and (ii) there are no obvious homologs of the Trl1 ligase domain in mammalian proteomes. Here we characterize the tRNA ligases of two human fungal pathogens: Coccidioides immitis and Aspergillus fumigatus. The biological activity of CimTrl1 and AfuTrl1 was verified by showing that their expression complements a Saccharomyces cerevisiae trl1Δ mutant. Purified recombinant AfuTrl1 and CimTrl1 proteins were catalytically active in joining 2 ′ ,3 ′ -cyclic-PO 4 and 5 ′ -OH ends in vitro, either as full-length proteins or as a mixture of separately produced healing and sealing domains. The biochemical properties of CimTrl1 and AfuTrl1 are similar to those of budding yeast Trl1, particularly with respect to their preferential use of GTP as the phosphate donor for the polynucleotide kinase reaction. Our findings provide genetic and biochemical tools to screen for inhibitors of tRNA ligases from pathogenic fungi.

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Characterization of 3′-Phosphate RNA Ligase Paralogs RtcB1, RtcB2, and RtcB3 from Myxococcus xanthus Highlights DNA and RNA 5′-Phosphate Capping Activity of RtcB3

Cited by 14 publications

References 38 publications

Cellular and molecular phenotypes depending upon the RNA repair system RtcAB ofEscherichia coli

Cellular and molecular phenotypes depending upon the RNA repair system RtcAB ofEscherichia coli

Distinct Contributions of Enzymic Functional Groups to the 2′,3′-Cyclic Phosphodiesterase, 3′-Phosphate Guanylylation, and 3′-ppG/5′-OH Ligation Steps of the Escherichia coli RtcB Nucleic Acid Splicing Pathway

Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis

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