Structure-based mechanistic insights into catalysis by tRNA thiolation enzymes
Introduction
tRNAs are key players in genetic code decoding, a fundamental process in all living organisms. All tRNAs feature post-transcriptional chemical modifications [1] that stabilize their tertiary structure and fine-tune the decoding process [2,3]. Sulfur, an essential element in life, is present in several cofactors and tRNAs: at positions 8, 9 in the core, 32, 33, 34, 37 around the anticodon and 54 in the T-loop (Figure 1a) [4,5]. The formation of 2-thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C) and 2-methylthioadenosine (ms2A) is catalyzed by specific enzymes called ThiI, TtcA, MnmA/Ctu1/Tuc1/Ncs6, MiaB and MtaB, TtuA [4,5] acting at positions 8, 32, 34, 37 and 54, respectively (Figure 1a). Because most thiI genes play no role in thiamine biosynthesis [6], ThiI is renamed here TtuI for tRNA thiouridine I.
There are two main classes of tRNA thiolation reactions. The insertion of sulfur within an inert CH bond is an [Fe–S]-dependent redox reaction catalyzed by the radical S-adenosyl-l-methionine (SAM) methylthiotransferases MiaB and MtaB. Because their structures remain unknown and their mechanisms have recently been reviewed [7,8], this class will not be discussed here. The non-redox substitution of oxygen for sulfur (Figure 1a) is catalyzed by ATP-dependent tRNA thiolases that share a pyrophosphatase (PPase) domain (Figure S1). We review here their crystal structures and catalytic mechanisms in light of research from the last two years showing that several of these enzymes are dependent on a [4Fe–4S] cluster (Table S1).
Section snippets
Formation of persulfides on reactive cysteines
The biosynthesis of sulfur-containing nucleosides involves several proteins that relay sulfur atoms originating from l-cysteine to tRNA [4,9,10]. In most cases, a pyridoxal-5′-phosphate-dependent cysteine desulfurase (IscS/Nsf1, YrvO, Nifz) first uses l-cysteine to form an enzyme-bound cysteine persulfide whose sulfur is next transferred to an acceptor protein [11, 12, 13, 14, 15]. This transfer is usually monitored by detecting, upon incubation with [35S]-l-cysteine, radioactive sulfur on the
U8-tRNA 4-sulfurtransferase TtuI (tRNA thiouridine I)
s4U at position 8 in the loop connecting the acceptor and D-stems of bacterial and archaeal tRNAs (Figure 1a) mediates cellular responses to UV stress [32]. In E. coli and Bacillus subtilis, TtuI and the cysteine desulfurase IscS [33,34] or NifZ [13], respectively, are required for s4U8-tRNA thiolation. TtuI enzymes have three conserved domains (Figures S1 and 2 a). Genomic analysis of the ttuI gene family identified two groups [9]: organisms like E. coli [35] that possess an additional
C32-tRNA 2-sulfurtransferase TtcA (tRNA-2-thiocytidine A)
TtcA enzymes target cytidine at position 32 near the anticodon in tRNAs (Figure 1a). The [Fe–S]-dependent TtcA/TtuA family was first identified following the characterization of E. coli and Salmonella thyphimurium strains deficient in s2C-modified tRNAs [44]. This class is characterized by a CXXC sequence motif in the central region (Figure S1). Analysis of tRNA from mutated strains indicated that the two cysteines in this motif are required for s2C formation [44] . Site-directed mutagenesis
Thiolation of U34
Sulfuration of U34 at the wobble position of the anticodon in Glu-tRNA, Gln-tRNA and Lys-tRNA (Figure 1a) is conserved in all organisms and guarantees fidelity of protein translation [46]. Lack of s2U34-tRNA results in severe growth reduction [12,15,18,47, 48, 49]. Two distinct enzyme families of the MnmA-types and Ncs6-types catalyze s2U34-tRNA formation (Figure S1). MnmA-like proteins operate in bacteria [22,50,51] and mitochondria [52], and Ncs6-like proteins in archaea and the eukaryotic
U54-tRNA 2-sulfurtransferase TtuA (tRNA-2-thiouridine A)
s2U at position 54 in the T-loop of tRNAs (Figure 1a) stabilizes its ternary structure in thermophilic bacteria and archaea for growth at high temperature [25]. Spectroscopic and biochemical analyses have shown that TtTtuA, PhTtuA and TtuA from T. maritima use a [4Fe–4S] cluster for U54-tRNA thiolation [28••,55••]. Thiolation did not occur in the absence of a sulfur source (Na2S [28••,55••] or TtTtuB-COSH [28••]), indicating that the sulfur atom incorporated into the nucleoside does not come
Conclusion
Although a general mechanism for tRNA thiolation was initially proposed, in which a persulfide attached to a catalytic cysteine is the sulfur donor for tRNA thiolation [16,51,57], there is increasing evidence that a sulfur-containing species bound to a [4Fe–4S] cluster, ligated to three cysteines only, can be the sulfurating agent [28••,31••,55••, Bimai, unpublished]. According to this finding, the tRNA thiolation enzymes for which a low in vitro activity has been detected and/or for which the
Conflict of interest statement
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank Marc Fontecave for sharing his enthusiasm for the iron–sulfur field and giving us competent advices all along this work.
This work was supported by the French State Program ‘Investissements d’Avenir’ (Grants ‘LABEX DYNAMO’, ANR-11-LABX-0011) and the CNRS.
References (57)
- et al.
Posttranscriptional RNA modifications: playing metabolic games in a cell’s chemical Legoland
Chem Biol
(2014) - et al.
Iron-sulfur proteins responsible for RNA modifications
Biochim Biophys Acta
(2015) - et al.
Shared-intermediates in the biosynthesis of thio-cofactors: mechanism and functions of cysteine desulfurases and sulfur acceptors
Biochim Biophys Acta
(2015) - et al.
Evidence for the transfer of sulfane sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA
J Biol Chem
(2000) - et al.
Biosynthesis of 4-thiouridine in tRNA in the methanogenic archaeon Methanococcus maripaludis
J Biol Chem
(2012) - et al.
The putative tRNA 2-thiouridine synthetase Ncs6 is an essential sulfur carrier in Methanococcus maripaludis
FEBS Lett
(2014) - et al.
Role of conserved cysteines in mediating sulfur transfer from IscS to IscU
FEBS Lett
(2005) - et al.
Iron-sulfur cluster biosynthesis in bacteria: mechanisms of cluster assembly and transfer
Arch Biochem Biophys
(2008) - et al.
Mechanistic insights into multiple sulfur mediators sulfur relay by involved in thiouridine biosynthesis at tRNA wobble positions
Mol Cell
(2006) - et al.
Identification of two tRNA thiolation genes required for cell growth at extremely high temperatures
J Biol Chem
(2006)
Posttranslational modification of cellular proteins by a ubiquitin-like protein in bacteria
J Biol Chem
The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA
J Biol Chem
The iscS gene in Escherichia coli is required for the biosynthesis of 4-thiouridine, thiamin, and NAD
J Biol Chem
Direct evidence that ThiI is an ATP pyrophosphatase for the adenylation of uridine in 4-thiouridine biosynthesis
ChemBioChem
A paradigm for biological sulfur transfers via persulfide groups: a persulfide-disulfide-thiol cycle in 4-thiouridine biosynthesis
Chem Commun
Crystal structure of a 4-thiouridine synthetase-RNA complex reveals specificity of tRNA U8 modification
Nucleic Acids Res
Purification, crystallization and preliminary crystallographic analysis of the putative thiamine-biosynthesis protein PH1313 from Pyrococcus horikoshii OT3
Acta Crystallogr Sect F Struct Biol Cryst Commun
MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli
Biochemistry
A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast
RNA
MODOMICS: a database of RNA modification pathways. 2017 update
Nucleic Acids Res
Biosynthesis and function of posttranscriptional modifications of transfer RNAs
Annu Rev Genet
Biosynthesis and functions of sulfur modifications in tRNA
Front Genet
Recent advances in our understanding of the biosynthesis of sulfur modifications in tRNAs
Front Microbiol
The danger of annotation by analogy: most "thiI" genes play no role in thiamine biosynthesis
J Bacteriol
On the role of additional [4Fe-4S] clusters with a free coordination site in radical-SAM enzymes
Front Chem
Comprehensive genomic analysis of sulfur-relay pathway genes
Genome Inform
Biosynthesis of sulfur-containing tRNA modifications: a comparison of bacterial, archaeal, and eukaryotic pathways
Biomolecules
Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli
J Bacteriol
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2022, Journal of Biological ChemistryCitation Excerpt :It is remarkable that the same overall protein architecture is used for two such distinct mechanisms for transferring sulfur into P2CMN to make P2TMN. Our demonstration of two distinct LarE sulfur-transfer mechanisms for the biosynthesis of the NPN cofactor has a clear parallel in the two reactions used for sulfur insertion during tRNA thionucleotide synthesis (11, 12). One thiobase-forming mechanism is exemplified by 4-thiouridine synthesis involving the initial activation of the precursor nucleotide using an adenylyltransferase followed by action of a sulfur transferase that catalyzes persulfide attack on the intermediate with release of AMP (30–32).
- 1
Present address: Department of Biochemistry and Biophysics, Stockholm University, Sweden.
- 2
Present address: IFP Energies nouvelles, 1 et 4 avenue de Bois-Préau, 92852 Rueil-Malmaison, France.
- 3
Equivalent contribution.