Limits...
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.

Show MeSH

Related in: MedlinePlus

Multiple amino acid sequence alignment of Hfx. volcanii NcsA (HVO_0580) with ANH superfamily members including proteins of Saccharomyces cerevisiae (ScNcs6, GI:50593215), Homo sapiens (HsNcs6, GI:74713747), Pyrococcus horikoshii (PH1680, GI:14591444; PH0300, GI:14590222), Thermus thermophilus (TTHA0477 or TtuA, GI: 55980446), Salmonella typhimurium (StTtcA, GI:16764998), and Escherichia coli (EcTtcA, GI:85674916).Conserved residues are highlighted in red, grey and black, with the conserved residues in red of the ATP pyrophosphatase signature PP-motif (SGGXDS) involved in ATP binding (Bork and Koonin, 1994) as well as motifs CXXC and GHXXDD (which act to recognize RNA) present in the TtcA protein family (Jager et al., 2004). Zinc fingers are highlighted in blue boxes, ubiquitin-fold modified lysine residues are in red boxes, and conserved catalytic cysteine residues are indicated by a star. Secondary structural elements predicted for HVO_0580 based on Phyre2 3D homology modeling are highlighted with blue arrows (β-sheets) and green cylinders (α-helices) above the amino acid sequence.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048286&req=5

pone-0099104-g001: Multiple amino acid sequence alignment of Hfx. volcanii NcsA (HVO_0580) with ANH superfamily members including proteins of Saccharomyces cerevisiae (ScNcs6, GI:50593215), Homo sapiens (HsNcs6, GI:74713747), Pyrococcus horikoshii (PH1680, GI:14591444; PH0300, GI:14590222), Thermus thermophilus (TTHA0477 or TtuA, GI: 55980446), Salmonella typhimurium (StTtcA, GI:16764998), and Escherichia coli (EcTtcA, GI:85674916).Conserved residues are highlighted in red, grey and black, with the conserved residues in red of the ATP pyrophosphatase signature PP-motif (SGGXDS) involved in ATP binding (Bork and Koonin, 1994) as well as motifs CXXC and GHXXDD (which act to recognize RNA) present in the TtcA protein family (Jager et al., 2004). Zinc fingers are highlighted in blue boxes, ubiquitin-fold modified lysine residues are in red boxes, and conserved catalytic cysteine residues are indicated by a star. Secondary structural elements predicted for HVO_0580 based on Phyre2 3D homology modeling are highlighted with blue arrows (β-sheets) and green cylinders (α-helices) above the amino acid sequence.

Mentions: We next determined whether Hfx. volcanii NcsA had conserved active site residues common to ANH superfamily members using Phyre2-based homology modeling and multiple amino acid sequence alignment (Figure 1, Figure S2 in File S1). By this approach, NcsA was found to have a conserved 3D-structural fold and residues common to Ncs6 and TtuA of the TtcA family group II including the five C-X2-[C/H] motifs and the PP motif (P-loop-like motif in a widespread ATP pyrophosphatase domain; SGGXDS, where X is any amino acid residue) [14], [17]–[19]. Based on recent study of TtuA by in vivo site-directed mutagenesis and x-ray crystallography, the first and second C-X2-[C/H] motifs form an N-terminal zinc finger (ZnF1), the third C-X2-C forms the putative catalytic active site and the C-terminal zinc finger (ZnF2) is formed by the fourth and fifth C-X2-C motifs [17]. Thus, Hfx. volcanii NcsA is predicted to have conserved residues of the cysteine-rich- and PP-motifs that mediate the binding, adenylation and thiolation of tRNA.


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)

Multiple amino acid sequence alignment of Hfx. volcanii NcsA (HVO_0580) with ANH superfamily members including proteins of Saccharomyces cerevisiae (ScNcs6, GI:50593215), Homo sapiens (HsNcs6, GI:74713747), Pyrococcus horikoshii (PH1680, GI:14591444; PH0300, GI:14590222), Thermus thermophilus (TTHA0477 or TtuA, GI: 55980446), Salmonella typhimurium (StTtcA, GI:16764998), and Escherichia coli (EcTtcA, GI:85674916).Conserved residues are highlighted in red, grey and black, with the conserved residues in red of the ATP pyrophosphatase signature PP-motif (SGGXDS) involved in ATP binding (Bork and Koonin, 1994) as well as motifs CXXC and GHXXDD (which act to recognize RNA) present in the TtcA protein family (Jager et al., 2004). Zinc fingers are highlighted in blue boxes, ubiquitin-fold modified lysine residues are in red boxes, and conserved catalytic cysteine residues are indicated by a star. Secondary structural elements predicted for HVO_0580 based on Phyre2 3D homology modeling are highlighted with blue arrows (β-sheets) and green cylinders (α-helices) above the amino acid sequence.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048286&req=5

pone-0099104-g001: Multiple amino acid sequence alignment of Hfx. volcanii NcsA (HVO_0580) with ANH superfamily members including proteins of Saccharomyces cerevisiae (ScNcs6, GI:50593215), Homo sapiens (HsNcs6, GI:74713747), Pyrococcus horikoshii (PH1680, GI:14591444; PH0300, GI:14590222), Thermus thermophilus (TTHA0477 or TtuA, GI: 55980446), Salmonella typhimurium (StTtcA, GI:16764998), and Escherichia coli (EcTtcA, GI:85674916).Conserved residues are highlighted in red, grey and black, with the conserved residues in red of the ATP pyrophosphatase signature PP-motif (SGGXDS) involved in ATP binding (Bork and Koonin, 1994) as well as motifs CXXC and GHXXDD (which act to recognize RNA) present in the TtcA protein family (Jager et al., 2004). Zinc fingers are highlighted in blue boxes, ubiquitin-fold modified lysine residues are in red boxes, and conserved catalytic cysteine residues are indicated by a star. Secondary structural elements predicted for HVO_0580 based on Phyre2 3D homology modeling are highlighted with blue arrows (β-sheets) and green cylinders (α-helices) above the amino acid sequence.
Mentions: We next determined whether Hfx. volcanii NcsA had conserved active site residues common to ANH superfamily members using Phyre2-based homology modeling and multiple amino acid sequence alignment (Figure 1, Figure S2 in File S1). By this approach, NcsA was found to have a conserved 3D-structural fold and residues common to Ncs6 and TtuA of the TtcA family group II including the five C-X2-[C/H] motifs and the PP motif (P-loop-like motif in a widespread ATP pyrophosphatase domain; SGGXDS, where X is any amino acid residue) [14], [17]–[19]. Based on recent study of TtuA by in vivo site-directed mutagenesis and x-ray crystallography, the first and second C-X2-[C/H] motifs form an N-terminal zinc finger (ZnF1), the third C-X2-C forms the putative catalytic active site and the C-terminal zinc finger (ZnF2) is formed by the fourth and fifth C-X2-C motifs [17]. Thus, Hfx. volcanii NcsA is predicted to have conserved residues of the cysteine-rich- and PP-motifs that mediate the binding, adenylation and thiolation of tRNA.

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
Related in: MedlinePlus