tRNA核苷修饰与细胞胁迫应答

细胞的生长和功能发挥需要特定的内部条件。当外界条件发生变化时,细胞要想保持这种特定的内部环境,需要许多过程的参与,其中最重要的一个部分是RNA代谢调节,其通常涉及一般翻译水平的下降和应激反应,以有利基因翻译的增加。tRNA是翻译机制的一个基本组成部分,在蛋白质合成过程中,它将氨基酸传递给核糖体。tRNA的显著特征之一是高度修饰,这些修饰有大量用途,包括确保翻译的准确性和高效性、维持tRNA折叠或稳定性等。细胞在逆境胁迫条件下,tRNA修饰水平会发生显著变化,并通过不同的途径影响细胞的翻译。本文阐述了tRNA核苷修饰与细胞胁迫之间的相互关系,描述了tRNA修饰响应胁迫应答的可能机制。     

The Levels of a Universally Conserved tRNA Modification Regulate Cell Growth

N⁶-Threonylcarbamoyl-adenosine (t⁶A) is a universal modification occurring at position 37 in nearly all tRNAs that decode A-starting codons, including the eukaryotic initiator tRNA (tRNA_i^Met). Yeast lacking central components of the t⁶A synthesis machinery, such as Tcs3p (Kae1p) or Tcs5p (Bud32p), show slow-growth phenotypes. In the present work, we show that loss of the Drosophila tcs3 homolog also leads to a severe reduction in size and demonstrate, for the first time in a non-microbe, that Tcs3 is required for t⁶A synthesis. In Drosophila and in mammals, tRNA_i^Met is a limiting factor for cell and animal growth. We report that the t⁶A-modified form of tRNA_i^Met is the actual limiting factor. We show that changing the proportion of t⁶A-modified tRNA_i^Met, by expression of an un-modifiable tRNA_i^Met or changing the levels of Tcs3, regulate target of rapamycin (TOR) kinase activity and influences cell and animal growth in vivo. These findings reveal an unprecedented relationship between the translation machinery and TOR, where translation efficiency, limited by the availability of t⁶A-modified tRNA, determines growth potential in eukaryotic cells.     

Classical molecular dynamics simulation of seryl tRNA synthetase and threonyl tRNA synthetase bound with tRNA and aminoacyl adenylate

Lacunae of understanding exist concerning the active site organization during the charging step of the aminoacylation reaction. We present here a molecular dynamics simulation study of the dynamics of the active site organization during charging step of subclass IIa dimeric SerRS from Thermus thermophilus (^ttSerRS) bound with ^tttRNA^Ser and dimeric ThrRS from Escherichia coli (^ecThrRS) bound with ^ectRNA^Thr. The interactions between the catalytically important loops and tRNA contribute to the change in dynamics of tRNA in free and bound states, respectively. These interactions help in the development of catalytically effective organization of the active site. The A76 end of the ^tttRNA^Ser exhibits fast dynamics in free State, which is significantly slowed down within the active site bound with adenylate. The loops change their conformation via multimodal dynamics (a slow diffusive mode of nanosecond time scale and fast librational mode of dynamics in picosecond time scale). The active site residues of the motif 2 loop approach the proximal bases of tRNA and adenylate by slow diffusive motion (in nanosecond time scale) and make conformational changes of the respective side chains via ultrafast librational motion to develop precise hydrogen bond geometry. Presence of bound Mg²⁺ ions around tRNA and dynamically slow bound water are other common features of both aaRSs. The presence of dynamically rigid Zinc ion coordination sphere and bipartite mode of recognition of ^ectRNA^Thr are observed.     

The structure of the TrmE GTP-binding protein and its implications for tRNA modification

TrmE is a 50 kDa guanine nucleotide-binding protein conserved between bacteria and man. It is involved in the modification of uridine bases (U34) at the first anticodon (wobble) position of tRNAs decoding two-family box triplets. The precise role of TrmE in the modification reaction is hitherto unknown. Here, we report the X-ray structure of TrmE from Thermotoga maritima. The structure reveals a three-domain protein comprising the N-terminal alpha/beta domain, the central helical domain and the G domain, responsible for GTP binding and hydrolysis. The N-terminal domain induces dimerization and is homologous to the tetrahydrofolate-binding domain of N,N-dimethylglycine oxidase. Biochemical and structural studies show that TrmE indeed binds formyl-tetrahydrofolate. A cysteine residue, necessary for modification of U34, is located close to the C1-group donor 5-formyl-tetrahydrofolate, suggesting a direct role of TrmE in the modification analogous to DNA modification enzymes. We propose a reaction mechanism whereby TrmE actively participates in the formylation reaction of uridine and regulates the ensuing hydrogenation reaction of a Schiff's base intermediate.     

RNA Ligation and the Origin of tRNA

A straightforward origin of transfer RNA,(tRNA), is difficult to envision because of the apparentlycomplex idiosyncratic interaction between the D-loop and T-loop. Recently, multiple examples of the T-loop structuralmotif have been identified in ribosomal RNA. These examplesshow that the long-range interactions between the T-loop andD-loops seen in tRNA are not an essential part of the motifbut rather are facilitated by it. Thus, the core T-loopstructure could already have existed in a small RNA prior tothe emergence of the tRNA. The tRNA might then have arisenby expansion of an RNA that carried the motif. With thisidea in mind, Di Giulio's earlier hypothesis that tRNAevolved by a simple duplication or ligation of a minihelixRNA was re-examined. It is shown that an essentially moderntRNA structure can in fact be generated by the ligation oftwo 38-nucleotide RNA minihelices of appropriate sequence.Although rare, such sequences occur with sufficientfrequency, (1 in 3 × 10⁷), that they could be found in astandard in vitro RNA selection experiment. Theresults demonstrate that a series of RNA duplications, aspreviously proposed, can in principal account for the originof tRNA. More generally, the results point out that RNAligation can be a powerful driving force for increasedcomplexity in the RNA World.     

Common thiolation mechanism in the biosynthesis of tRNA thiouridine and sulphur‐containing cofactors

2‐Thioribothymidine (s²T), a modified uridine, is found at position 54 in transfer RNAs (tRNAs) from several thermophiles; s²T stabilizes the L‐shaped structure of tRNA and is essential for growth at higher temperatures. Here, we identified an ATPase (tRNA‐two‐thiouridine C, TtuC) required for the 2‐thiolation of s²T in Thermus thermophilus and examined in vitro s²T formation by TtuC and previously identified s²T‐biosynthetic proteins (TtuA, TtuB, and cysteine desulphurases). The C‐terminal glycine of TtuB is first activated as an acyl‐adenylate by TtuC and then thiocarboxylated by cysteine desulphurases. The sulphur atom of thiocarboxylated TtuB is transferred to tRNA by TtuA. In a ttuC mutant of T. thermophilus, not only s²T, but also molybdenum cofactor and thiamin were not synthesized, suggesting that TtuC is shared among these biosynthetic pathways. Furthermore, we found that a TtuB—TtuC thioester was formed in vitro, which was similar to the ubiquitin‐E1 thioester, a key intermediate in the ubiquitin system. The results are discussed in relation to the mechanism and evolution of the eukaryotic ubiquitin system.     

tRNA Binding,Positioning, and Modification by the Pseudouridine Synthase Pus10

Pus10 is the most recently identified pseudouridine synthase found in archaea and higher eukaryotes. It modifies uridine 55 in the TΨC arm of tRNAs. Here, we report the first quantitative biochemical analysis of tRNA binding and pseudouridine formation by Pyrococcus furiosus Pus10. The affinity of Pus10 for both substrate and product tRNA is high (K_d of 30 nM), and product formation occurs with a K_m of 400 nM and a k_cat of 0.9 s^− 1. Site-directed mutagenesis was used to demonstrate that the thumb loop in the catalytic domain is important for efficient catalysis; we propose that the thumb loop positions the tRNA within the active site. Furthermore, a new catalytic arginine residue was identified (arginine 208), which is likely responsible for triggering flipping of the target uridine into the active site of Pus10. Lastly, our data support the proposal that the THUMP-containing domain, found in the N-terminus of Pus10, contributes to binding of tRNA. Together, our findings are consistent with the hypothesis that tRNA binding by Pus10 occurs through an induced-fit mechanism, which is a prerequisite for efficient pseudouridine formation.     

Combined tRNA modification defects impair protein homeostasis and synthesis of the yeast prion protein Rnq1

Modified nucleosides in tRNA anticodon loops such as 5-methoxy-carbonyl-methyl-2-thiouridine (mcm⁵s²U) and pseuduridine (Ψ) are thought to be required for an efficient decoding process. In Saccharomyces cerevisiae, the simultaneous presence of mcm⁵s²U and Ψ38 in tRNA^Gln_UUG was shown to mediate efficient synthesis of the Q/N rich [PIN⁺] prion forming protein Rnq1.¹ Klassen R, Ciftci A, Johanna Funk J, Bruch A, Butter F, Schaffrath R. tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast. Nucleic Acids Res 2016; 44(22):10946-959. pii: gkw705; PMID:27496282; http://dx.doi.org/10.1093/nar/gkw705[Crossref], [PubMed], [Web of Science ®] [Google Scholar] In the absence of these two tRNA modifications, higher than normal levels of hypomodified tRNA^Gln_UUG, but not its isoacceptor tRNA^Gln_CUG can restore Rnq1 synthesis. Moroever, tRNA overexpression rescues pleiotropic phenotypes that associate with loss of mcm⁵s²U and Ψ38 formation. Notably, combined absence of different tRNA modifications are shown to induce the formation of protein aggregates which likely mediate severe cytological abnormalities, including cytokinesis and nuclear segregation defects. In support of this, overexpression of the aggregating polyQ protein Htt103Q, but not its non-aggregating variant Htt25Q phenocopies these cytological abnormalities, most pronouncedly in deg1 single mutants lacking Ψ38 alone. It is concluded that slow decoding of particular codons induces defects in protein homeostasis that interfere with key steps in cytokinesis and nuclear segregation.     

Crystallographic and mutational studies on the tRNA thiouridine synthetase TtuA

In thermophilic bacteria, specific 2‐thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2‐thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur‐carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2‐thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 Å resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N‐ and C‐terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNA^Ile₂ lysidine synthetase), than to the other type of tRNA 2‐thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP‐binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA‐binding site. Proteins 2013; 81:1232–1244. © 2013 Wiley Periodicals, Inc.     

Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair

Breakage of tRNA(Lys(UUU)) by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection that is ultimately thwarted by a virus-encoded RNA repair system. PrrC homologs are prevalent in other bacteria, but their activities and substrates are not defined. We find that induced expression of EcoPrrC is toxic in Saccharomyces cerevisiae and E. coli, whereas the Neisseria meningitidis PrrC (NmePrrC) is not. PrrCs consist of an N-terminal NTPase module and a C-terminal nuclease module. Domain swaps identified the EcoPrrC nuclease domain as decisive for toxicity when linked to either the Eco or Nme NTPase. Indeed, a single arginine-to-tryptophan change in the NmePrrC nuclease domain (R316W) educed a gain-of-function and rendered NmePrrC toxic to yeast, with genetic evidence for tRNA(Lys(UUU)) being the relevant target. The reciprocal Trp-to-Arg change in EcoPrrC (W335R) abolished its toxicity. Further mutagenesis of the EcoPrrC nuclease domain highlighted an ensemble of 15 essential residues and distinguished between hypomorphic alleles and potential nuclease-nulls. We report that the RNA repair phase of the bacterial virus-host dynamic is also portable to yeast, where coexpression of the T4 enzymes Pnkp and Rnl1 ameliorated the toxicity of NmePrrC-R316W. Plant tRNA ligase AtRNL also countered NmePrrC-R316W toxicity, in a manner that depended on AtRNL's 5'-kinase and ligase functions.     

Mutations in Cytosine-5 tRNA Methyltransferases Impact Mobile Element Expression and Genome Stability at Specific DNA Repeats

1. Download high-res image (131KB)
2. Download full-size image

     

Mechanisms of the tRNA wobble cytidine modification essential for AUA codon decoding in prokaryotes

Bacteria and archaea have 2-lysylcytidine (L or lysidine) and 2-agmatinylcytidine (agm²C or agmatidine), respectively, at the first (wobble) position of the anticodon of the AUA codon-specific tRNA^Ile. These lysine- or agmatine-conjugated cytidine derivatives are crucial for the precise decoding of the genetic code. L is synthesized by tRNA^Ile-lysidine synthetase (TilS), which uses l-lysine and ATP as substrates. Agm²C formation is catalyzed by tRNA^Ile-agm²C synthetase (TiaS), which uses agmatine and ATP for the reaction. Despite the fact that TilS and TiaS synthesize structurally similar cytidine derivatives, these enzymes belong to non-related protein families. Therefore, these enzymes modify the wobble cytidine by distinct catalytic mechanisms, in which TilS activates the C2 carbon of the wobble cytidine by adenylation, while TiaS activates it by phosphorylation. In contrast, TilS and TiaS share similar tRNA recognition mechanisms, in which the enzymes recognize the tRNA acceptor stem to discriminate tRNA^Ile and tRNA^Met.     

Sequence-structure-function relationships of a tRNA (m7G46) methyltransferase studied by homology modeling and site-directed mutagenesis

The Escherichia coli TrmB protein and its Saccharomyces cerevisiae ortholog Trm8p catalyze the S-adenosyl-L-methionine-dependent formation of 7-methylguanosine at position 46 (m7G46) in tRNA. To learn more about the sequence-structure-function relationships of these enzymes we carried out a thorough bioinformatics analysis of the tRNA:m7G methyltransferase (MTase) family to predict sequence regions and individual amino acid residues that may be important for the interactions between the MTase and the tRNA substrate, in particular the target guanosine 46. We used site-directed mutagenesis to construct a series of alanine substitutions and tested the activity of the mutants to elucidate the catalytic and tRNA-recognition mechanism of TrmB. The functional analysis of the mutants, together with the homology model of the TrmB structure and the results of the phylogenetic analysis, revealed the crucial residues for the formation of the substrate-binding site and the catalytic center in tRNA:m7G MTases.