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Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs.

Chavarria NE, Hwang S, Cao S, Fu X, Holman M, Elbanna D, Rodriguez S, Arrington D, Englert M, Uthandi S, Söll D, Maupin-Furlow JA - PLoS ONE (2014)

Bottom Line: When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2.Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1).Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

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NcsA is required for growth of Hfx. volcanii at an elevated temperature (50°C).Hfx. volcanii H26 (wt, parent), Δsamp2, ΔncsA, and trans complemented ΔncsA strains were grown in ATCC 974 medium. Freshly isolated colonies were inoculated into 3 ml medium (in 13×100 mm culture tubes) and thrice subcultured at 42°C. Cells grown to logarithmic phase at 42°C from these subcultures were used as inoculum for monitoring growth at 50°C as presented in panel A. Cells grown to stationary-phase from these 50°C cultures were used as an inoculum for monitoring long-term growth at 50°C as presented in panel B. Inoculum was at 0.02 OD600 with rotary shaking (200 rpm) in 20 ml medium in 250 ml baffled flasks for the growth assays presented in panels A and B. For panel C, cell cultures, as indicated above each plate, were diluted to 0.1 OD600 and then spot-plated on solid agar ATCC 974 medium in serial dilutions as indicated. Plates were incubated at 50°C. Control experiments performed at 42°C are presented in Figure S4 in File S1. See Methods section for details.
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pone-0099104-g003: NcsA is required for growth of Hfx. volcanii at an elevated temperature (50°C).Hfx. volcanii H26 (wt, parent), Δsamp2, ΔncsA, and trans complemented ΔncsA strains were grown in ATCC 974 medium. Freshly isolated colonies were inoculated into 3 ml medium (in 13×100 mm culture tubes) and thrice subcultured at 42°C. Cells grown to logarithmic phase at 42°C from these subcultures were used as inoculum for monitoring growth at 50°C as presented in panel A. Cells grown to stationary-phase from these 50°C cultures were used as an inoculum for monitoring long-term growth at 50°C as presented in panel B. Inoculum was at 0.02 OD600 with rotary shaking (200 rpm) in 20 ml medium in 250 ml baffled flasks for the growth assays presented in panels A and B. For panel C, cell cultures, as indicated above each plate, were diluted to 0.1 OD600 and then spot-plated on solid agar ATCC 974 medium in serial dilutions as indicated. Plates were incubated at 50°C. Control experiments performed at 42°C are presented in Figure S4 in File S1. See Methods section for details.

Mentions: We next examined whether NcsA is necessary for optimal growth at elevated temperature similar to SAMP2 and UbaA [12]. Hfx. volcanii ΔncsA mutant and its trans complement were compared to H26 parent and Δsamp2 mutant for growth at 50°C. Growth at this elevated temperature was compared to growth at 42°C, a temperature within the range for optimal growth of Hfx. volcanii[20]. When cultured at 42°C, all four Hfx. volcanii strains were found to have comparable growth rates and cell yield under all conditions tested (Figure S4 in File S1). By contrast, when cells were grown at 42°C and transferred to 50°C, a slow-growth phenotype was observed for the ΔncsA and Δsamp2 mutant strains compared to the parent and ncsA trans-complement (Figure 3A). To examine whether this slow-growth phenotype may be attributed to suppressor mutation(s), the four Hfx. volcanii strains were grown to stationary phase at 50°C, inoculated into fresh medium, and monitored for growth at 50°C (Figure 3B). By this experimental approach, the ΔncsA and Δsamp2 mutant strains were found to display no detectable growth at 50°C compared to the robust growth and cell yield detected for the parent and trans-complement strains (Figure 3B). Similar results were observed by rich medium agar plate assay (Figure 3C). Thus, the slow growth phenotype of the ΔncsA and Δsamp2 strains was not due to a suppressor mutation but instead is likely due to a component(s) present in the 42°C inoculum that was relatively active for initial batch culture at 50°C but not functional for long-term growth at this elevated temperature.


Archaeal Tuc1/Ncs6 homolog required for wobble uridine tRNA thiolation is associated with ubiquitin-proteasome, translation, and RNA processing system homologs.

Chavarria NE, Hwang S, Cao S, Fu X, Holman M, Elbanna D, Rodriguez S, Arrington D, Englert M, Uthandi S, Söll D, Maupin-Furlow JA - PLoS ONE (2014)

NcsA is required for growth of Hfx. volcanii at an elevated temperature (50°C).Hfx. volcanii H26 (wt, parent), Δsamp2, ΔncsA, and trans complemented ΔncsA strains were grown in ATCC 974 medium. Freshly isolated colonies were inoculated into 3 ml medium (in 13×100 mm culture tubes) and thrice subcultured at 42°C. Cells grown to logarithmic phase at 42°C from these subcultures were used as inoculum for monitoring growth at 50°C as presented in panel A. Cells grown to stationary-phase from these 50°C cultures were used as an inoculum for monitoring long-term growth at 50°C as presented in panel B. Inoculum was at 0.02 OD600 with rotary shaking (200 rpm) in 20 ml medium in 250 ml baffled flasks for the growth assays presented in panels A and B. For panel C, cell cultures, as indicated above each plate, were diluted to 0.1 OD600 and then spot-plated on solid agar ATCC 974 medium in serial dilutions as indicated. Plates were incubated at 50°C. Control experiments performed at 42°C are presented in Figure S4 in File S1. See Methods section for details.
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Related In: Results  -  Collection

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pone-0099104-g003: NcsA is required for growth of Hfx. volcanii at an elevated temperature (50°C).Hfx. volcanii H26 (wt, parent), Δsamp2, ΔncsA, and trans complemented ΔncsA strains were grown in ATCC 974 medium. Freshly isolated colonies were inoculated into 3 ml medium (in 13×100 mm culture tubes) and thrice subcultured at 42°C. Cells grown to logarithmic phase at 42°C from these subcultures were used as inoculum for monitoring growth at 50°C as presented in panel A. Cells grown to stationary-phase from these 50°C cultures were used as an inoculum for monitoring long-term growth at 50°C as presented in panel B. Inoculum was at 0.02 OD600 with rotary shaking (200 rpm) in 20 ml medium in 250 ml baffled flasks for the growth assays presented in panels A and B. For panel C, cell cultures, as indicated above each plate, were diluted to 0.1 OD600 and then spot-plated on solid agar ATCC 974 medium in serial dilutions as indicated. Plates were incubated at 50°C. Control experiments performed at 42°C are presented in Figure S4 in File S1. See Methods section for details.
Mentions: We next examined whether NcsA is necessary for optimal growth at elevated temperature similar to SAMP2 and UbaA [12]. Hfx. volcanii ΔncsA mutant and its trans complement were compared to H26 parent and Δsamp2 mutant for growth at 50°C. Growth at this elevated temperature was compared to growth at 42°C, a temperature within the range for optimal growth of Hfx. volcanii[20]. When cultured at 42°C, all four Hfx. volcanii strains were found to have comparable growth rates and cell yield under all conditions tested (Figure S4 in File S1). By contrast, when cells were grown at 42°C and transferred to 50°C, a slow-growth phenotype was observed for the ΔncsA and Δsamp2 mutant strains compared to the parent and ncsA trans-complement (Figure 3A). To examine whether this slow-growth phenotype may be attributed to suppressor mutation(s), the four Hfx. volcanii strains were grown to stationary phase at 50°C, inoculated into fresh medium, and monitored for growth at 50°C (Figure 3B). By this experimental approach, the ΔncsA and Δsamp2 mutant strains were found to display no detectable growth at 50°C compared to the robust growth and cell yield detected for the parent and trans-complement strains (Figure 3B). Similar results were observed by rich medium agar plate assay (Figure 3C). Thus, the slow growth phenotype of the ΔncsA and Δsamp2 strains was not due to a suppressor mutation but instead is likely due to a component(s) present in the 42°C inoculum that was relatively active for initial batch culture at 50°C but not functional for long-term growth at this elevated temperature.

Bottom Line: When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2.Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1).Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
While cytoplasmic tRNA 2-thiolation protein 1 (Tuc1/Ncs6) and ubiquitin-related modifier-1 (Urm1) are important in the 2-thiolation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at wobble uridines of tRNAs in eukaryotes, the biocatalytic roles and properties of Ncs6/Tuc1 and its homologs are poorly understood. Here we present the first report of an Ncs6 homolog of archaea (NcsA of Haloferax volcanii) that is essential for maintaining cellular pools of thiolated tRNA(Lys)UUU and for growth at high temperature. When purified from Hfx. volcanii, NcsA was found to be modified at Lys204 by isopeptide linkage to polymeric chains of the ubiquitin-fold protein SAMP2. The ubiquitin-activating E1 enzyme homolog of archaea (UbaA) was required for this covalent modification. Non-covalent protein partners that specifically associated with NcsA were also identified including UbaA, SAMP2, proteasome activating nucleotidase (PAN)-A/1, translation elongation factor aEF-1α and a β-CASP ribonuclease homolog of the archaeal cleavage and polyadenylation specificity factor 1 family (aCPSF1). Together, our study reveals that NcsA is essential for growth at high temperature, required for formation of thiolated tRNA(Lys)UUU and intimately linked to homologs of ubiquitin-proteasome, translation and RNA processing systems.

Show MeSH