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Structure of 3'-PO4/5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide.

ABSTRACT: RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that join 2',3'-cyclic phosphate ends to 5'-OH ends via RtcB-(histidinyl-N)-GMP, RNA 3'-phosphate, and RNA3'pp5'G intermediates. RtcB can ligate either 5'-OH RNA or 5'-OH DNA strands in vitro. The nucleic acid contacts of RtcB are uncharted. Here we report a 2.7 Å crystal structure of Pyrococcus horikoshii RtcB in complex with a 6-mer 5'-OH DNA oligonucleotide HOA1pT2pG3pT4pC5pC6, which reveals enzymic contacts of Asn202 to the terminal 5'-OH nucleophile; Arg238 to the A1pT2 and T2pG3 phosphates; Arg190 and Gln194 to the T2pG3 phosphate; and an Arg190 π-cation interaction with the G3 nucleobase. The structural insights affirm functional studies of E. coli RtcB that implicated the conserved counterpart of Arg238 in engagement of the 5'-OH strand for ligation. The essential active site Cys98 that coordinates two manganese ions is oxidized to cysteine sulfonic acid in our structure, raising the prospect that RtcB activity might be sensitive to modulation during oxidative stress.

SUBMITTER: Banerjee A

PROVIDER: S-EPMC8051266 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Structure of 3'-PO4/5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide.

Banerjee Ankan A Goldgur Yehuda Y Shuman Stewart S

RNA (New York, N.Y.) 20210416 5

RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that join 2',3'-cyclic phosphate ends to 5'-OH ends via RtcB-(histidinyl-N)-GMP, RNA 3'-phosphate, and RNA3'pp5'G intermediates. RtcB can ligate either 5'-OH RNA or 5'-OH DNA strands in vitro. The nucleic acid contacts of RtcB are uncharted. Here we report a 2.7 Å crystal structure of Pyrococcus horikoshii RtcB in complex with a 6-mer 5'-OH DNA oligonucleotide HO ...[more]

PMID: 33619169

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Project description:The opportunistic pathogen Pseudomonas aeruginosa is a significant cause of infection in immunocompromised individuals, cystic fibrosis patients, and burn victims. To benefit its survival, the bacterium adapt to either a motile or sessile lifestyle when infecting the host. The motile bacterium has an often activated type III secretion system (T3SS), which is virulent to the host, whereas the sessile bacterium harbors an active T6SS and lives in biofilms. Regulatory pathways involving Gac-Rsm or secondary messengers such as c-di-GMP determine which lifestyle is favorable for P. aeruginosa. Here, we introduce the RNA binding protein RtcB as a modulator of the switch between motile and sessile bacterial lifestyles. Using the wild-type P. aeruginosa PAO1, and a retS mutant PAO1(∆retS) in which T3SS is repressed and T6SS active, we show that deleting rtcB led to simultaneous expression of T3SS and T6SS in both PAO1(∆rtcB) and PAO1(∆rtcB∆retS). The deletion of rtcB also increased biofilm formation in PAO1(∆rtcB) and restored the motility of PAO1(∆rtcB∆retS). RNA-sequencing data suggested RtcB as a global modulator affecting multiple virulence factors, including bacterial secretion systems. Competitive killing and infection assays showed that the three T6SS systems (H1, H2, and H3) in PAO1(∆rtcB) were activated into a functional syringe, and could compete with Escherichia coli and effectively infect lettuce. Western blotting and RT-PCR results showed that RtcB probably exerted its function through RsmA in PAO1(∆rtcB∆retS). Quantification of c-di-GMP showed an elevated intracellular levels in PAO1(∆rtcB), which likely drove the switch between T6SS and T3SS, and contributed to the altered phenotypes and characteristics observed. Our data demonstrate a pivotal role of RtcB in the virulence of P. aeruginosa by controlling multiple virulence determinants, such as biofilm formation, motility, pyocyanin production, T3SS, and T6SS secretion systems towards eukaryotic and prokaryotic cells. These findings suggest RtcB as a potential target for controlling P. aeruginosa colonization, establishment, and pathogenicity.

Dataset Information

Structure of 3'-PO4/5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide.

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Structure of 3'-PO<sub>4</sub>/5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide.

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Dataset Information

Structure of 3'-PO4/5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide.

Publications

Structure of 3'-PO&lt;sub&gt;4&lt;/sub&gt;/5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide.

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OmicsDI is part of the ELIXIR infrastructure

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Structure of 3'-PO<sub>4</sub>/5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide.