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Formation and function of wobble uridine modifications in transfer RNA of Saccharomyces cerevisiae Bo Huang Department of Molecular Biology Umeå University Umeå, Sweden 2007 1 Doctoral Dissertation 2007 Department of Molecular Biology Umeå University SE-901 87, Umeå, Sweden Copyright © 2007 by Bo Huang ISBN: 978-91-7264-450-2 Printed by Print & Media, Umeå University, Umeå, Sweden, 2007, 2003867 2 TABLE OF CONTENTS Papers in this thesis….…………….………….…….….........................................5 Abstract……………….…………….…….......…...................................................6 1 INTRODUCTION….……...……..............….………….......................... 7 1.1 Transfer RNA….……….……………..............….………….......................... 7 1.1.1 Biogenesis of tRNA………….….….….……............................................ 8 1.1.2 Decoding of messenger RNA (mRNA)..………....…….…………..……. 8 1.2 Modified nucleosides in tRNA……….....…..…......................................….... 10 1.3 Gene products required for tRNA modification…......................................…. 13 1.3.1 Multi-subunit tRNA modification enzymes......................................…..... 13 1.3.2 Multifunctional tRNA modification enzymes..……….............................. 15 1.4 Wobble uridine modifications………………...….......................................… 15 1.5 Phenotypes of tRNA modification mutants...................................................... 17 1.6 Translation……………..……….……….….................................................... 18 1.7 Effect of modified wobble uridines on tRNA decoding properties.................. 19 2 RESULTS AND DISCUSSION................................................................ 21 Conclusions…………………………………………........................................….. 32 Acknowledgements……………………………….............................................… 33 References………………….………………………...........................................… 35 Papers I - V 3 4 Papers in this thesis This thesis is based on the following papers, which are referred to in the text by their roman numerals. I An early step in wobble uridine tRNA modification requires the Elongator complex. Huang B, Johansson MJO, Byström AS. RNA. 2005; 11(4):424-36 II A genome-wide screen in Saccharomyces cerevisiae for mutants defective in formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in tRNA. Lu J*, Huang B*, Byström AS. *Contributed equally Manuscript III Elevated levels of two tRNA species bypass the requirement for Elongator complex in transcription and exocytosis. Esberg A, Huang B, Johansson MJO, Byström AS. Mol Cell. 2006; 24(1):139-48 IV A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast. Björk GR, Huang B, Persson OP, Byström AS. RNA. 2007; 13(8):1245-55 V Eukaryotic wobble uridine modifications promote a functionally redundant decoding system. Johansson MJO, Esberg A, Huang B, Björk GR, Byström AS. Manuscript Paper I, III, and IV are reproduced with permission from the publishers. 5 Abstract Transfer RNAs (tRNAs) act as adaptor molecules in decoding messenger RNA into protein. Frequently found in tRNAs are different modified nucleosides, which are derivatives of the four normal nucleosides, adenosine (A), guanosine (G), cytidine (C), and uridine (U). Although modified nucleosides are present at many positions in tRNAs, two positions in the anticodon region, position 34 (wobble position) and position 37, show the largest variety of modified nucleosides. In Saccharomyces cerevisiae, the xm5U type of modified uridines found at position 34 are 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2´-O-methyluridine, (ncm5Um), 5-methoxycarbonylmethyluridine (mcm5U), and 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Based on the complex structure of these nucleosides, it is likely that their formation requires several synthesis steps. The Elongator complex consisting of proteins Elp1p - Elp6p, and the proteins Kti11p - Kti14p, Sit4p, Sap185p, and Sap190p were shown to be involved in 5-carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) side-chain synthesis at position 34 in eleven tRNA species. The proteins Urm1p, Uba4p, Ncs2p, Ncs6p, and Yor251cp were also identified to be required for the 2-thio (s2) group formation of the modified nucleoside mcm5s2U at wobble position. Modified nucleosides in the anticodon region of tRNA influence the efficiency and fidelity of translation. The identification of mutants lacking ncm5-, mcm5-, or s2-group at the wobble position allowed the investigation of the in vivo role of these nucleosides in the tRNA decoding process. It was revealed that the presence of ncm5-, mcm5- or s2-group promotes reading of G-ending codons. The concurrent presence of the mcm5- and the s2-groups in the wobble nucleoside mcm5s2U improves reading of A- and G-ending codons, whereas absence of both groups is lethal to the yeast cell. The Elongator complex was previously proposed to regulate polarized exocytosis and to participate in elongation of RNA polymerase II transcription. The pleiotropic phenotypes observed in Elongator mutants were therefore suggested to be caused by defects in exocytosis and transcription of many genes. Here it is shown that elevated levels of hypomodified tRNA Lys and tRNA Gln can mcm 5 s 2 UUG mcm 5 s 2 UUU efficiently suppress these pleiotropic phenotypes, suggesting that the defects in transcription and exocytosis are indirectly caused by inefficient translation of mRNAs encoding proteins important in these processes. 6 1 INTRODUCTION 1.1 Transfer RNA Transfer RNAs (tRNAs) are the adaptor molecules that play a critical role in decoding messenger RNA (mRNA) into protein. The tRNA molecules are usually 75 to 90 nucleotides in length. In their cloverleaf-like secondary structures, there exist several base-paired “stem” regions, and unpaired “loop” regions. These include (1) acceptor stem, formed by the 5' and 3' ends of the tRNA which always terminates with CCA at the 3' end; (2) D stem and loop, containing the modified nucleoside dihydrouridine (D); (3) anticodon stem and loop, including the anticodon triplet, i. e., nucleotides at position 34 to 36; (4) variable loop, that depending on tRNA species differs in length; (5) TΨC stem and loop, where nearly all tRNAs have ribothymidine (T), pseudouridine (Ψ) and cytosine at position 54 to 56 (Fig. 1). Base stacking and hydrogen bonding between the D loop and TΨC loop bring the cloverleaf-like secondary structure into a reversed L-shaped tertiary structure (Kim et al., 1974; Robertus et al., 1974) (Fig. 1). 3' A C C CCA end 5' 1 TΨC loop 72 Acceptor stem D loop TΨC stem D stem TΨC loop D loop Anticodon stem 34 Anticodon loop Variable loop Anticodon loop Fig. 1 Secondary structure (left) and tertiary structure (Kim et al., 1974) (right) of tRNA 7 1.1.1 Biogenesis of tRNA In eukaryotes, tRNA genes are transcribed into precursor tRNAs by RNA polymerase III (Sprague, 1995). The precursor tRNAs undergo a series of posttranscriptional processing events to generate a mature form. These events include (1) cleavage of the 5' leader by Ribonuclease P (RNase P) (Darr et al., 1992; Altman et al, 1995); (2) removal of the 3' trailer sequence by either endo- or exo-nucleases (Engelke et al., 1985; Furter et al., 1992; Papadimitriou & Gross, 1996; Takaku et al., 2003; Dubrovsky et al., 2004); (3) addition of CCA at the 3' end by the ATP(CTP):tRNA nucleotidyltransferase (Aebi et al., 1990; Chen et al., 1990); (4) removal of intron in intron-containing tRNAs by the tRNA splicing endonuclease (Trotta et al., 1997), the tRNA ligase, and the phosphotransferase (Phizicky et al., 1986; Phizicky et al., 1992; Culver et al., 1997; Spinelli et al., 1997; Abelson et al., 1998); and (5) modifications of nucleosides by a number of tRNA modifying enzymes (Björk, 1995; Cermakian & Cedergren, 1998; Johansson & Bytröm, 2005). 1.1.2 Decoding of messenger RNA (mRNA) The genetic code is read in triplets of nucleotides. The combination of the four nucleotides in mRNA yields 64 possible codons. Of these, 61 code for amino acids and 3 signal termination of translation. This means that the genetic code is degenerated, i.e. for most of the 20 amino acids more than one codon exists (Fig. 2). Since cells always have less than 61 tRNA species, e.g. 42 tRNA species in yeast cells, some tRNAs must be able to read more than one codon. Therefore, Crick proposed the wobble hypothesis to give an explanation to extended decoding properties of tRNAs (Crick, 1966). In his hypothesis, base pairing of the first two nucleotides in the codon with nucleotides 36 and 35 of the anticodon in tRNA would be canonical Watson-Crick base pairings, i. e., A with U, U with A, C with G, or G with C. Position 34 of the anticodon in addition to canonical base pairing also has the ability to form certain non-canonical base pairings with the third position of the 8 codon. Position 34 was therefore called wobble position (Fig. 1 and 3). Based on the geometry of base pairing, Crick proposed that a uridine (U34) should pair with an A at the third position of the codon (A3), and wobble to G3, while a G34 should pair with C3, and wobble to U3. In addition, the modified wobble nucleoside inosine (I34) was proposed to pair with C3, and wobble to U3 and A3. Inosine was the only known modified wobble nucleoside at that time. Revised wobble hypotheses have since emerged to include the role of other modified nucleosides at the wobble position (see below). U U C A G UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG C Phe Leu Leu Ile Met Val UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG A Ser Pro Thr Ala UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG G Tyr STOP His Gln Asn Lys Asp Glu UGU Cys UGC UGA STOP Trp UGG CGU CGC Arg CGA CGG AGU Ser AGC AGA Arg AGG GGU GGC Gly GGA GGG U C A G U C A G U C A G U C A G Fig. 2 The codon table (modified from Crick, 1968) 9 Anticodon stem and loop (ASL) of tRNA 3’ 5’ 36 35 34 5’---------------------------------------- 3’ 1 2 3 1 2 3 mRNA 1 2 3 Fig. 3 Schematic diagram of the interaction between codon and anticodon. 1.2 Modified nucleosides in tRNA Transfer RNA contains modified nucleosides, which are derivatives of the four standard nucleosides, U, C, A, and G. To date, over 100 modified nucleosides have been identified in tRNAs from eukaryotes, archaea, and bacteria (DuninHorkawicz et al., 2006). Of the 50 different modified nucleosides found in eukaryotic tRNAs, 25 have been identified in the 30 sequenced cytoplasmic tRNA species from Saccharomyces cerevisiae (Limbach et al., 1994; Johansson & Bytröm, 2005; Sprinzl & Vassilenko, 2005; Dunin-Horkawicz et al., 2006). These modified nucleosides are distributed all over the tRNA molecule (Fig. 4). For example, 5-methyluridine (m5U) is present at position 54 in all sequenced tRNA species except initiator tRNA ( tRNA iMet ) (Sprinzl & Vassilenko, 2005). Dihydrouridine (D), is present in the D-loop in all tRNA species and sometimes at position 47. In contrast, the 2'-O-ribosyladenosine (phosphate), Ar(p), is only present at position 64 in the tRNA iMet from S. cerevisiae (Keith et al., 1990; Glasser et al., 1991). The nucleosides at the anticodon loop, particularly at position 34 (the wobble position) and position 37 (3'-adjacent to anticodon), show the largest variation of modified nucleosides (Fig. 4). These modified nucleosides are believed to improve the 10 efficiency and fidelity of mRNA decoding (Björk, 1995; Yokoyama & Nishimura, 1995; Urbonavicius et al., 2001). A C C Ψ Cm Am 1 4 Ψ Ar(p) Ψ 67 D 17 Gm D 1 ac4C m G 2 mG Ψ D 16 13 12 18 20 20A 20B D D I Gm Cm m5 C Ψ ncm5U ncm5Um mcm5U mcm5s2U m22G Ψ m2G Cm m 3C Ψ 10 9 m5C 49 m5C 48 26 Ψ Ψ Ψ m1A 58 65 64 27 47 D 46 54 55 m7G 44 Ψ m5U 28 Um m 5C 39 Ψ 38 Ψ 40 31 32 37 34 35 36 Ψ Ψ m1I t6A i6A m1G yW Fig. 4 Modified nucleosides in cytoplasmic tRNAs from S. cerevisiae (adapted from Johansson & Byström, 2005) Abbreviations: I, inosine; m1I, 1-methylinosine; m1A, 1-methyladenosine; t6A, N6Threonylcarbamoyl-adenosine; i6A, N6-isopentenyladenosine; Ar(p), 2´-O-ribosyladenosine (phosphate); Am, 2´-O-methyladenosine; m5C, 5-methylcytidine; ac4C, N4-acetylcytidine; m3C, 3-methylcytidine; Cm, 2´-O-methylcytidine; m1G, 1-methylguanosine; m2G, N2methylguanosine; m22G, N2, N2-dimethylguanosine; Gm, 2´-O-methylguanosine; m7G, 7methylguanosine; yW, wybutosine; Ψ, pseudouridine; D, dihydrouridine; m5U, 5methyluridine; Um, 2'-O-methyluridine; mcm5U, 5-methoxycarbonyl-methyluridine; mcm5s2U, 5-methoxycarbonylmethyl-2-thiouridine; ncm5U, 5-carbamoylmethyluridine; ncm5Um, 5-carbamoylmethyl-2'-O-methyluridine. 11 The biosynthesis of a modified nucleoside requires one or more catalytic reactions (Johansson & Byström, 2005). For example, the 1-methylguanosine (m1G9) synthesis requires a one-step methylation by using S-Adenosyl Methionine (AdoMet) as the methyl donor (Jackman et al., 2003) while formation of the 1-methylinosine (m1I37) requires two catalytic reactions, a deamination of A to I followed by a methylation of I (Grosjean et al., 1996). Formation of the wybutosine (yW37) is much more complex, proceeding through stepwise reactions (Fig. 5) (Noma et al., 2006). In tRNA biogenesis, a sequential formation of the different modified nucleosides is not required as inability to form one modified nucleoside does not influence the synthesis of other modified nucleosides at other positions of the tRNA (Johansson & Bystöm, 2005). O O H3 C N N N H2 N G N N Trm10 HN m1G 9 R O NH2 O H3C N N Tad1 HN N N N A N N I 37 N N N H2N R G N N CH 3 yW37 m I37 N N R R HO2CCHNH2 CH2 N 1 R Tyw1 N N H imG-14 N N N CH 3 N N N N CH3 R yW-58 N N H R yW-86 CH2 CH2 Tyw4 R Tyw3 HO2CCHNH2 N CH 3 N O CH2 Tyw4 Tyw2 N CH3 O CH2 N H3CO2CCHNH2 CH2 N H 3C N mG H3CO2CCHNHCO2CH3 CH2 O CH2 1 O Trm5 N HN N N R O H3C N N N Trm5 R O H2N N N H2 N R O N N CH 3 N N N CH3 R yW-72 Fig. 5 Examples of the synthesis of modified nucleosides in yeast (R represents ribose) 12 1.3 Gene products required for tRNA modification The advance in instrumentation and methodology has dramatically facilitated the identification of genes required for tRNA modification. So far, in S. cerevisiae 60 genes have been identified. These gene products include tRNA modifying enzymes as well as the proteins that in other ways are required for synthesis of the modified nucleosides (Table 1; Paper II). 1.3.1 Multi-subunit tRNA modification enzymes In most cases, one protein catalyzes the formation of one tRNA modification. However, there exist some examples that formation of modified nucleosides requires enzymes consisting of more than one polypeptide. For instance, the methylation of 1-methyladenosine (m1A58) is catalyzed by a nuclear complex consisting of Trm6p and Trm61p. It is likely that Trm6p directs the binding of tRNA, whereas Trm61p binds the methyl donor AdoMet (Calvo et al., 1999; Anderson et al., 2000; Kadaba et al., 2004). The two subunits of adenosine deaminase Tad2p/Tad3p complex share significant homology, and appear not to display clear split functions (Gerber & Keller, 1999). Although Tad2p is suggested to be the catalytic subunit, both subunits are required for I34 formation (Gerber & Keller, 1999). For synthesis of the complex modified nucleoside yW37, at least five proteins, including Trm5p, Tyw1p, Tyw2p, Tyw3p, and Tyw4p, are required. The Tyw2p, Tyw3p, and Tyw4p are suggested to be a multi-subunit yW-synthase (Fig. 5) (Noma et al., 2006). 13 Table1. Modified nucleosides in S. cerevisiae cytoplasmic tRNAs and gene products required for their a b fo rmations. Based on the sequence of 30 of the 42 tRNA species. Indicates that no gene product has c 2 2 Val been identified. The tRNA CAC s pecies has m G rather than m 2 G at position 26 (Gorbulev et al., 2 1977), the m G is presumably also catalyzed by Trm1p. (adapted from Johansson & Byström, 2005) M odified nucleoside Positiona Genes required for modification I m1I 34 37 TAD2, TAD3 TAD1, TRM5 Gerber & Keller 1999 m1A 58 TRM6, TRM61 Anderson et al. 1998; Anderson et al. 2000; Ozanick et al. 2005 t 6A 37 _b i6A Ar(p) Am m5C 37 64 4 34, 40, 48, 49 MOD5 RIT1 TRM13 TRM4 Laten et al. 1978; Dihanich et al 1987; Åström & Byström 1994; Åström et al. 1999 Wilkinson et al. 2007 ac4C 12 TAN1 Johansson & Byström 2004 m3C Cm 32 32, 34 4 _ TRM7 TRM13 Pintard et al. 2002 Wilkinson et al. 2007 m1G 9 37 10 26 TRM10 TRM5 TRM11, TRM112 _c Jackman et al. 2003 Björk et al. 2001 m22G Gm 26 18 34 TRM1 TRM3 TRM7 Ellis et al. 1986; Martin et al 1994 Cavaille et al. 1999 Pintard et al. 2002 m7G yW 46 37 TRM8, TRM82 TRM5, TYW1-TYW4 Alexandrov et al. 2002 Björk et al. 2001; Noma et al. 2006 ψ 1, 26, 27, 28, 34, 36, 65, 67 38, 39 55 31 13, 35 32 16, 17 20 47 20A, 20B 54 PUS1 Simos et al. 1996; Motorin et al. 1998; Behm-Ansmant et al. 2006 Lecointe et al. 1998 Becker et al. 1997; Grosshans et al. 2001 Ansmant et al. 2001 Behm-Ansmant et al. 2003 Behm-Ansmant et al. 2004 Xing et al. 2002; Xing et al. 2004 Xing et al. 2002; Xing et al. 2004 Xing et al. 2004 Xing et al. 2004 m2G D m5U PUS3 PUS4 PUS6 PUS7 PUS8, PUS9 DUS1 DUS2 DUS3 DUS4 TRM2 Reference Gerber et al. 1998; Björk et al. 2001 Motorin & Grosjean 1999 Purushothaman et al. 2005 Hopper et al. 1982; Nordlund et al. 2000; Johansson & Byström 2002 Um 44 _ mcm5U 34 TRM9 ELP1-ELP6, KTI11-KTI13 Kalhor & Clarke 2003 Paper I mcm5s 2U 34 TRM9, NFS1, CFD1, NBP35 CIA1, ISU1, ISU2 ELP1-ELP6, KTI11-KTI13 NCS2, TUC1 Kalhor & Clarke 2003 Nakai et al. 2004; Nakai et al. 2007 Paper I PaperIII; Paper IV ncm5U 34 ELP1-ELP6, KTI11-KTI13 Paper I ncm5Um 34 ELP1-ELP6, KTI11-KTI13 Paper I 14 1.3.2 Multifunctional tRNA modification enzymes Some tRNA modifying enzymes have been proposed to possess more than one function. For example, a deletion of the truB gene leading to the abolishment of Ψ55 in E. coli tRNAs does not significantly affect the exponential growth, but a growth disadvantage is revealed when the mutant is grown in competition with the isogenic wild-type strain. A point mutation in the truB gene does not restore formation of the Ψ55, but efficiently compensates the growth disadvantage of the truB deletion mutant. This observation indicates that TruB has a function independent of the tRNA modification activity, possibly a chaperone function in tRNA biogenesis (Gutgsell et al., 2000). Inactivation of the TRM2 gene, encoding the methyltransferase responsible for synthesis of the 5-methyluridine at position 54 (m5U54), does not cause obvious growth defect in yeast. However, the trm2 deletion is lethal if combined with the sup61-T51C allele, which codes for a mutant form of an essential tRNA species ( tRNASer CGA ). A catalytically inactive Trm2p can rescue the double mutant by stabilizing the mutant form of tRNASer CGA , showing that Trm2p has a role in tRNA biogenesis independent of its methyltransferase activity (Johansson & Byström, 2002). 1.4 Wobble uridine modifications In S. cerevisiae, 13 of the 42 cytoplasmic tRNA species have a U at the wobble position (Percudani et al., 1997). Of these 13 tRNAs, tRNA Leu UAG contains an unmodified U and tRNA Ile ΨΑΨ has a pseudouridine (Randerath et al., 1979; Szweykowska-Kulinska et al., 1994). The remaining 11 tRNA species contain the ncm5U, ncm5Um, mcm5U, or mcm5s2U residue at the wobble position (Johansson & Byström, 2005; Lu et al., 2005) (Fig. 6). Based on their structures, the formation of ncm5U, ncm5Um, mcm5U and mcm5s2U modified nucleosides are likely to involve multiple synthesis steps and 15 gene products. The Trm9p methyltransferase is responsible for the last step, formation of the esterified methyl constituent of mcm5U34 and mcm5s2U34 (Kalhor & Clarke, 2003). Formation of the 2-thio (s2) group in wobble uridines of both mitochondrial and cytoplasmic tRNAs requires the Nfs1p, a cysteine desulfurase, which is a sulfur donor for the iron-sulfur (Fe-S) clusters (Kispal et al., 1999; Li et al., 1999; Nakai et al., 2004). Recently, the mitochondrial and cytoplasmic Fe-S cluster assembly machineries in yeast, termed ISC and CIA, respectively, were reported to be involved in the thiolation of wobble uridines in tRNAs (Nakai et al., 2007). Depletion of any of the three CIA components (Cfd1, Nbp35, or Cia1), or depletion of the two ISC components (Isu1p and Isu2p), leads to loss of s2-group formation in cytosolic mcm5s2U34-containing tRNAs, but not in mitochondrial mnm5s2U34-containing tRNAs (Nakai et al., 2007). O O CH2CNH2 HN O HO OH ncm5U O HO OCH3 ncm5Um O HO OH mcm5U CH2COCH3 HN N O HO O O CH2COCH3 HN N O HO O O CH2CNH2 HN N O HO O O N S HO O HO OH mcm5s2U Fig. 6 Examples of wobble uridine modifications present in eukaryotic cytosolic tRNAs. 16 1.5 Phenotypes of tRNA modification mutants Most tRNA modification mutants do not display obvious phenotypes under normal laboratory conditions. Once a growth defect is observed in a mutant, it usually indicates that the absence of the modified nucleoside leads to either less functional tRNAs with respect to decoding capacity or a decreased amount of tRNAs available for translation. Below are some examples of phenotypes in tRNA modification mutants. The I34 adenosine deaminase Tad2p/Tad3p is essential for cell viability (Gerber & Keller, 1999). A point mutation in the TAD3 gene of fission yeast destabilizes the Tad2p/Tad3p complex and causes a reduced level of I34 in the mutant (Tsutsumi et al., 2007). The tad3 mutant exhibited a temperature sensitivity and irreversible cell cycle arrest at both G1 and G2 phases (Tsutsumi et al., 2007). I34-containing tRNAs have been proposed to translate codons ending with U, C, or A (Crick, 1966). Inability to deaminate A34 to I34 was suggested to result in an altered wobble capacity leading to the decreased reading of A-ending codons (Crick, 1966; Yokoyama & Nishimura, 1995). In addition, presence of I34 in tRNA Ile IAU is an important positive determinant for the isoleucyl-tRNA synthetase (Senger et al., 1997). A group of gene products including Nfs1p, Cfd1p, Nbp35p, Cia1p, Isu1p, and Isu2p are required for the s2-group formation in mcm5s2U34, and they are essential for cell survival (Li et al., 1999; Nakai et al., 2004; Nakai et al., 2007). All these proteins have also been shown to be involved in the maturation of mitochondrial and cytoplasmic iron-sulfur (Fe-S) cluster proteins (Roy et al., 2003; Gerber et al., 2004; Muhlenhoff et al., 2004; Balk et al., 2005; Hausmann et al., 2005). It has been revealed that the Fe-S cluster proteins are required for many biological processes, including electron transport, enzyme catalysis and regulation of gene expression (Lill & Muhlenhoff, 2005). 17 Deletion of the TRM9 gene, encoding the mcm5s2U34/mcm5U34 methyltransferase, leads to subtle phenotypes. A trm9Δ mutant is slightly temperature sensitive (Ts) on media containing the aminoglycoside antibiotic paramomycin, which impairs translation by increasing codon misreading (Chernoff et al., 1994; Kalhor & Clarke, 2003). Of those enzymes that are responsible for the synthesis of modified nucleosides outside the anticodon region, only m1A58-synthase, a complex consisting of Trm6p/Trm61p, is known to be essential (Anderson et al., 2000). It has been shown that both subunits of the m1A58-methyltransferase are required for maturation of the initiator tRNA iMet (Anderson et al., 2000). 1.6 Translation Transfer RNAs participate primarily in translation. Translation can be divided into initiation, elongation, and termination. In the cap-dependent initiation of translation pathway, the 40S ribosomal subunit, initiator Met - tRNA iMet and initiation factors bind to the 5' end of mRNA to form a pre-initiation complex. The pre-initiation complex scans the mRNA in the 5' to 3' direction to find the start codon. In the initiation phase, the initiator Met - tRNA iMet interacts with the start codon, and this interaction determines the reading frame of mRNA. When the start codon is recognized, the initiation factors dissociate and the 60S ribosomal subunit is recruited to form the mature 80S initiation complex. After that translation enters the elongation phase. The 80S ribosome has three tRNA-binding sites: the A-site, where the aminoacyl-tRNA binds; the P-site, where peptidyl-tRNA binds after translocation; and the E-site, where the deacetylated tRNA binds before it is rejected from the ribosome. During the elongation phase, the aminoacyl-tRNA, forming a ternary complex with elongation factor eEF1A and GTP, is selected to the A-site of the ribosome based on the codon-anticodon interaction. Following the hydrolysis of GTP and release of eEF1A·GDP, the peptide attached to peptidyl-tRNA in the P-site 18 is transferred and forms an ester bond with the amino acid attached on the aminoacyl-tRNA in the A-site. This reaction is catalyzed by the peptidyl transferase center in the ribosome. In the next step, elongation factor 2 (eEF2) translocates the peptidyl-tRNA in the A-site to the P-site of the ribosome. Consequently, the deacetylated tRNA in the P-site is moved to the E-site. A new aminoacyltRNA·eEF1A·GTP ternary complex is then accepted into the A-site. By repeating this cyclic process, the peptide chain is extended on the peptidyl-tRNA. When a stop codon is presented in the A-site, it signals to the termination phase of translation. Stop codons are recognized by eukaryotic release factor 1 (eRF1), that together with eRF3 (in yeast) promotes the polypeptide chain release from the peptidyl-tRNA (Merrick & Hershey, 1996). 1.7 Effect of modified wobble uridines on tRNA decoding properties During translation, the interaction between the anticodon of tRNA and the codon in mRNA determines whether the tRNA will be selected. Presence of modified wobble nucleosides significantly affects this interaction, and they are thereby important for the decoding of mRNA. It was not known at the time when Crick proposed the wobble hypothesis that almost all wobble uridines in tRNAs are modified. Thus, the wobble hypothesis has been revised to account for the role of the modified nucleosides in tRNA decoding (Yokoyama et al., 1985; Agris, 1991; Lim, 1994; Yokoyama & Nishimura, 1995; Takai & Yokoyama, 2003). One such modified wobble uridine is xm5U, in which x represents any of several different groups and m5 is the methylene carbon directly bonded to the C5 atom of tRNAs (Mizuno & Sundaralingam, 1978). The xm5U may be thiolated at position 2 (xm5s2U) or methylated at the ribose 2'-hydroxyl group (xm5Um) (Mizuno & Sundaralingam, 1978; Takai & Yokoyama, 2003). It has been proposed that the presence of an xm5U residue at the wobble position would prevent the tRNAs from pairing with pyrimidine-ending codons (Yokoyama et al., 1985; Lim, 1994; Yokoyama & Nishimura, 1995). For decoding purine-ending codons, two models are 19 proposed. The first proposes that the xm5U34 causes efficient pairing with A and simultaneously reduces pairing with G (Yokoyama et al., 1985; Yokoyama & Nishimura, 1995). The second proposes that the presence of the xm5U34 residue improves the decoding of both A- and G-ending codons (Lim, 1994; Kruger & Sorensen, 1998; Yarian et al., 2002; Takai & Yokoyama, 2003; Murphy et al., 2004). The xm5s2U type of modifications in eukaryotic cytoplasmic tRNAs is mcm5s2U34 (Mizuno & Sundaralingam, 1978; Takai & Yokoyama, 2003). Based on in vitro studies and structural analyses, this modification was suggested to prevent pairing with pyrimidine-ending codons, but allow pairing with A-ending, and reduce pairing with G-ending codons (Sekiya et al., 1969; Lustig et al., 1981; Yokoyama et al., 1985). Recently an mcm5s2U34 residue has been suggested to allow reading of both A- and G-ending codons (Murphy et al., 2004). However, the U34-G3 wobble pairing in eukaryotes has been questioned based on the fact that, in eukaryotes, e.g., C. elegans, each U34-containing tRNA always has a C34-containing isoacceptor (Percudani, 2001). Lack of defined mutants has hampered the understanding on the in vivo role of the xm5U derivatives in eukaryotes. Thus, the primary aims of this thesis were: (1) to identify the gene products responsible for the formation of modified nucleosides ncm5U34, mcm5U34, and mcm5s2U34 in S. cerevisiae tRNAs; (2) to investigate the in vivo roles of modified wobble nucleosides ncm5U34, mcm5U34, and mcm5s2U34 in tRNAs. 20 2 RESULTS AND DISCUSSION 2.1 An early step in wobble uridine tRNA modification requires the Elongator complex (paper I) A mutation in the S. pombe sin3 locus was shown to cause three phenotypes. First, the mutation induced an anti-suppression phenotype, i.e. it abolished the ability of an ochre suppressor tRNASer to read the premature stop codon in the ade7-413 mRNA (Thuriaux et al., 1976); Second, the sin3 mutant showed a reduced level of the modified wobble nucleoside mcm5s2U (Heyer et al., 1984; Grossenbacher et al., 1986); Third, cells with sin3 mutations became longer, indicating a cell cycle defect (Heyer et al., 1984; Grossenbacher et al., 1986). To identify the gene product required for mcm5s2U synthesis, a genomic library was introduced into the sin3 mutant and the clones complementing the anti-suppression phenotype were isolated. The sin3+ gene was identified as an uncharacterized open reading frame (ORF) SPAC29A4.20. It was found that a sin3 null allele caused lack of mcm5s2U34 in tRNA Glu , and of mcm5U34 in the ochre suppressor tRNASer . mcm 5 s 2 UUC The Sin3 protein displays 76% identity at the amino acid level to the budding yeast S. cerevisiae Elp3 protein, and 77% identity to the human Elp3p (Hawkes et al., 2002). Like the S. pombe sin3 mutant, an S. cerevisiae elp3Δ mutant also exhibits the anti-suppression phenotype due to loss of the modified nucleoside mcm5U in the suppressor tRNA Tyr . The S. cerevisiae Elp3p is a subunit of the Elongator complex consisting of proteins Elp1p to Elp6p (Otero et al., 1999; Wittschieben et al., 1999; Krogan & Greenblatt, 2001; Winkler et al., 2001). It was shown that in any of the elp1 - elp6 mutants, ncm5U, mcm5U, and mcm5s2U are absent in tRNA Pro , tRNA Arg , and tRNA Glu , respectively. In any of ncm 5 UGG mcm 5 s 2 UUC mcm5UCU contains s2U, demonstrating that the elp1 - elp6 single mutants, tRNA Glu mcm 5 s 2 UUC 21 these mutants completely lacked the mcm5 side-chain at position 5, but not the 2-thio (s2) group at position 2 of the mcm5s2U nucleoside. Therefore, all six Elongator subunits are involved in formation of the ncm5 and mcm5 side-chains at wobble uridines. Moreover, the specific coimmunoprecipitation of Elp1p and Elp3p with in vitro transcribed tRNA Glu UUC indicates a direct involvement of Elongator in wobble uridine modification. All elp mutants display pleiotropic phenotypes including resistance to Kluyveromyces lactis killer toxin (see paper II). Additionally, a set of mutants has been identified to be killer toxin insensitive (KTI). The kti11 - kti13 mutants show the same pleiotropic phenotypes as the elp mutants (Frohloff et al., 2001; Jablonowski et al., 2001b; Fichtner & Schaffrath, 2002; Petrakis et al., 2005). The tRNA modification defect in kti11 and kti12 mutants was found to be the same as in elp mutants, i.e., absence of ncm5U, mcm5U, and mcm5s2U. In the kti13 mutant, reduced amount of the ncm5U, mcm5U, and mcm5s2U was detected. Thus, Kti11p Kti13p also participate in the synthesis of ncm5 and mcm5 side-chains at wobble uridines. Since no any intermediate of ncm5U, mcm5U, and mcm5s2U (except for s2U) is detected in any of the elp and kti mutants, it can be concluded that the Elp and Kti proteins are required in an early step of ncm5 and mcm5 of side-chain formation at wobble uridines. 2.2 A genome-wide screen in Saccharomyces cerevisiae for mutants defective in formation of the wobble nucleoside 5-methoxycarbonyl-methyl-2thiouridine in tRNA (paper II) Killer strains of the dairy yeast Kluyveromyces lactis secrete a heterotrimeric toxin (zymocin), which causes an irreversible growth arrest of sensitive yeast cells, such as S. cerevisiae in the G1 phase of the cell cycle (Gunge & Sakaguchi, 1981; Sugisaki et al., 1983; White, 1989; Schaffrath, 2005). The killer toxin is composed 22 of the α, β, and γ subunits and its cytotoxicity resides within the γ subunit (γ- toxin). Intracellular expression of γ-toxin mimics the effect of extragenous killer toxin (Tokunaga et al., 1989; Butler et al., 1991). It was recently shown that the K. lactis killer toxin is a tRNA endonuclease that cleaves tRNA Glu , tRNA Lys , mcm 5 s 2 UUC mcm 5 s 2 UUU and tRNA Gln 3' of the wobble nucleoside mcm5s2U (Lu et al., 2005). The mcm 5 s 2 UUG mcm5 side-chain is important for efficient cleavage by γ-toxin, and mutants lacking the mcm5 side-chain are resistant to endogenous expression of γ-toxin (Lu et al., 2005). To identify additional mutants resistant to the toxin and thereby being candidates to have the defect in synthesis of the mcm5 side-chain, a genome-wide screen was performed by using the killer eclipse assay (Kishida et al., 1996). From a collection of 4826 S. cerevisiae strains with individual genes deleted, 63 killer toxinresistant mutants were identified. Based on HPLC analyses of the bulk tRNA from these mutants, it was revealed that, in addition to mutants already known to have a defect in synthesis of the modified nucleoside mcm5s2U, i.e., elp1 - elp4, elp6, kti12, kti13, trm9, ncs2, and ncs6 (Kalhor & Clarke, 2003; paper III and IV) (null strains of elp5 and kti11 are not present in the deletion collection), additional mutants were identified in which formation of the mcm5 side-chain (sit4) or the s2-group (urm1, uba4, and yor251c) was impaired. A sit4 mutant was in fact earlier shown to be resistant to γ-toxin (Jablonowski et al., 2001a). Sit4p is as a protein phosphatase which is regulated by four associated proteins Sap4p, Sap155p, Sap185p, and Sap190p (Luke et al., 1996). Deletion of SAP185 and SAP190, but not deletion of the SAP4 and SAP155 genes, generates resistance to γ-toxin (Jablonowski et al., 2001a; Jablonowski et al., 2004). Accordingly, ncm5U, mcm5U, and mcm5s2U are absent in a sap185Δ sap190Δ double mutant, suggesting that the role of Sit4p in the ncm5 and mcm5 side-chain synthesis is regulated by Sap185p and Sap190p. Analysis of another previously known killer toxin-resistant mutant showed that in a kti14 mutant, formation of ncm5 23 and mcm5 side-chains is impaired. KTI14 encodes a protein kinase (DeMaggio et al., 1992) and it has been shown that Kti14p physically interacts with the core Elongator complex (Elp1 - Elp3 proteins) and the Sit4-Sap185-Sap190 complex (Ho et al., 2002; Gavin et al., 2006). Elp1p is a phosphoprotein and its dephosphorylation depends on Sit4p and its associated proteins Sap185p and Sap190p (Jablonowski et al., 2004). Kti12p is also associated with core Elongator complex (Fichtner et al., 2002a; Fichtner et al., 2003), and Kti12p can antagonize Sit4-dependent dephosphorylation of Elp1p (Jablonowski et al., 2004). Absence of or overexpression of Kti12p generates a defect in mcm5s2U formation (paper I and data not shown).Therefore, it seems likely that formation of ncm5 and mcm5 side-chains is dependent on the phosphorylation status of Elp1p in core Elongator complex by the action of Sit4p, Sap185, Sap190, Kti12p and Kti14p. It has been shown that both Ncs2p and Ncs6p are responsible for 2-thio formation at wobble uridine (paper III and IV). In the screen, three additional proteins Urm1p, Uba4p, and Yor251cp were also identified to be involved in 2-thio modification of cytoplasmic tRNA. The Ncs6p is homologous to the putative bacterial Fe-S cluster protein TtcA (paper IV) (Shigi et al., 2006), and it is likely that the role of Ncs6p in tRNA modification is dependent on the mitochondrial and cytosolic Fe-S cluster assembly machineries ISC and CIA, which have been shown to be involved in the 2-thio formation in tRNA (Nakai et al., 2007). Urm1p is a ubiquitin-like modifier and Uba4p is the activation enzyme (E1) for urmylation in yeast (Furukawa et al., 2000). Ubiquitin and ubiquitin-like modifiers are covalently attached to target proteins, and the stability, function, or localization of the target proteins may therefore be altered (Hochstrasser, 2000; Schnell & Hicke, 2003). The activity of the putative 2-thio-synthase Ncs6p could possibly be modulated by urmylation. 24 2.3 Elevated levels of two tRNA species bypass the requirement for Elongator complex in transcription and exocytosis (paper III) In addition to tRNA modification (see paper I), the Elongator complex has been proposed to participate in two other cellular processes: transcriptional elongation (Otero et al., 1999) and polarized exocytosis (Rahl et al., 2005). Strains with a mutation in any of the six Elongator subunit genes display pleiotropic phenotypes, including transcription and exocytosis defects, slow growth, delay in G1 phase of the cell cycle, temperature-sensitivity (Ts), and sensitivity to sodium chloride and caffeine (Otero et al., 1999; Frohloff et al., 2001; Jablonowski et al., 2001b; Fichtner et al., 2002b). In an attempt to sort out the cause-effect relationships of the elp mutations, genes that in high dosage could suppress the Ts phenotype of an elp3 null mutant were screened for. In addition to the wild-type ELP3 gene, tRNA genes that all encoded tRNA Lys were identified. Since the ELP1 - ELP6 mcm 5 s 2 UUU genes are required for the formation of ncm5 and mcm5 side-chains in 10 additional tRNA species (paper I and V), it was investigated if elevated levels of any of these tRNA species would suppress the Ts phenotype of the elp3 null strain. Elevated levels of tRNA Gln generated a weak suppression of the Ts phenotype in the mcm 5 s 2 UUG elp3 mutant. Increased dosage of tRNA Lys and tRNA Gln genes mcm 5 s 2 UUG mcm 5 s 2 UUU resulted in a cooperative growth improvement in any of the elp1 - elp6 mutants or the kti11 - kti13 mutants at the restrictive temperature. In addition, other phenotypes of the elp3 mutant, such as a delayed G1/S cell cycle transition, sensitivity to caffeine were also suppressed by high dosage of the two tRNA species. However, the suppression was not attributed to the restoration of tRNA modification. Both Gcn5 and Elp3 proteins are suggested to catalyse the acetylation of lysine 14 in histone H3 (Kuo et al., 1996; Winkler et al., 2002). The elp3 and gcn5 double mutant shows a synergistic growth defect, which was proposed to be caused by chromatin-remodelling defects simultaneously affecting initiation (gcn5Δ) and 25 elongation (elp3Δ) of Pol II transcription (Sterner & Berger, 2000; Wittschieben et al., 2000). However, the growth defect of an elp3Δ gcn5Δ double mutant was suppressed by increased levels of the tRNA Lys and tRNA Gln . mcm 5 s 2 UUG mcm 5 s 2 UUU Moreover, increased expression of these tRNAs counteracted the histone H3 acetylation defect of an elp3Δ but not an gcn5Δ mutant. These data strongly question the identity of Elp3p as a histone acetyltransferase. The Elongator complex has also been proposed to regulate polarized exocytosis (Rahl et al., 2005). A guanine nucleotide exchange factor, Sec2p, is required to mediate the process of vesicle delivery to a specific region of the plasma membrane (Salminen & Novick, 1987; Walch-Solimena et al., 1997). A sec2-59 mutant is temperature sensitive (Ts) (Nair et al., 1990), and introduction of an elp1 null allele into the sec2-59 mutant strain suppresses the Ts (Rahl et al., 2005). Increased expression of the hypomodified tRNA Lys and tRNA Gln in mcm 5 s 2 UUG mcm 5 s 2 UUU the elp1Δ sec2-59 double mutant generates a Ts phenotype, as observed in the sec259 single mutant. Furthermore, a deletion of the ELP1 gene leads to a mislocalization of the Sec2p (Rahl et al., 2005), and elevated levels of the hypomodified tRNA Lys and tRNA Gln restore the Sec2p localization. mcm 5 s 2 UUG mcm 5 s 2 UUU It is believed that the mcm5- and s2-groups in mcm5s2U have similar functions with respect to their influence on decoding properties of tRNA (Weissenbach & Dirheimer, 1978; Yokoyama et al., 1985; Sierzputowska-Graz, 1987; Lim, 1994; Durant et al., 2005). This suggests that a mutant lacking s2-group at wobble uridines would show phenotypes similar to the elp mutants. Accordingly, an ncs2 null strain, which lacks s2- but not mcm5-group, displays slow growth, sodium chloride and caffeine sensitivity, delayed transcriptional activation, and defects in chromatin remodeling and exocytosis. Elevated levels of the hypomodified 26 tRNA Lys and tRNA Gln suppressed all those phenotypes of the ncs2Δ mcm 5 s 2 UUG mcm 5 s 2 UUU strain. The facts that an ncs2 mutant, with an intact Elongator complex generates the same phenotypes as an elp3 mutant, and both mutations impair the function of tRNA Lys and tRNA Gln strongly suggest that phenotypes of Elongatormcm 5 s 2 UUG mcm 5 s 2 UUU deficient cells are caused by impaired translation. 2.4 A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast (paper IV) The mcm5s2U34 modification is present in three tRNA species, tRNA Lys , tRNA Gln , and tRNA Glu . The identification of mcm 5 s 2 UUG mcm 5 s 2 UUC mcm 5 s 2 UUU mutants lacking the mcm5-group (paper I) or the s2-group (paper II, this paper) of mcm5s2U34 made it possible to investigate if an unmodified U34 was allowed in the tRNAs normally harboring this modification. The Ncs6p, one of the proteins required for synthesis of the s2-group, is homologous to Escherichia coli TtcA that is required for the s2C32 synthesis in tRNAs. In contrast to the Mtu1p, which is involved in synthesis of the s2-group of mnm5s2U34 in mitochondrial tRNAs (Umeda et al., 2005), the Ncs6p is responsible for thiolation of wobble uridines in cytoplasmic tRNAs, and the gene encoding Ncs6p was therefore renamed TUC1 (Thiolation of Uridine in Cytoplasmic tRNA). Deletion of the ELP3 gene, combined with disruption of the TUC1 gene leads to cell lethality. Over-expression of hypomodified tRNA Lys , mcm 5 s 2 UUU tRNA Gln , and tRNA Glu can rescue the elp3Δ tuc1Δ double mutant. mcm 5 s 2 UUG mcm 5 s 2 UUC Importantly, the mcm5s2U was still missing in the rescued mutant cells. The aminoacylation and stability of these tRNAs were not affected by the absence of mcm5s2U. These results suggest that simultaneous lack of the mcm5 side-chain and 27 s2-group impairs the function of mcm5s2U34-containing tRNAs in translation. The mcm5s2U-containing tRNA species tRNA Lys , tRNA Gln , and mcm 5 s 2 UUG mcm 5 s 2 UUU tRNA Glu decode split codon boxes where the U-/C- or A-/G-ending codons mcm 5 s 2 UUC specify different amino acids. It has been proposed that the mcm5s2U34 residue prevents pairing with U-/C-ending codons (Yokoyama et al., 1985; Lim, 1994; Yokoyama & Nishimura, 1995). If correct, absence of the modification would induce missense errors caused by reading the noncognate codons ending with U and C. However, over-expressed hypomodified tRNA Lys , rescues the elp3Δ tuc1Δ mcm 5 s 2 UUU double mutant and simultaneously over-expressed tRNA Lys mcm 5 s 2 UUU , tRNA Gln , and tRNA Glu stimulate the growth. This suggests that the mcm 5 s 2 UUG mcm 5 s 2 UUC primary effect of mcm5s2U34 is to improve the efficiency of reading the cognate codon ending with A (the G-ending codons are read by C34-containing isoacceptors), but not to prevent missense errors resulting from decoding of noncognate U-/Cending codons. 2.5 Eukaryotic wobble uridine modifications promote a functionally redundant decoding system (paper V) Since the wobble hypothesis was presented, numerous revised models have been put forth to explain the role of modified wobble nucleosides in tRNA decoding. Most of them are based on in vitro translational systems or structural analyses of nucleosides and/or anticodon stem-loops (Yokoyama et al., 1985; Agris, 1991; Lim, 1994; Yokoyama & Nishimura, 1995; Takai & Yokoyama, 2003). However, little is known about the in vivo roles of the modified wobble uridines found in eukaryotes. Identification of S. cerevisiae gene products responsible for the synthesis of wobble ncm5-, mcm5-, and s2-groups allowed the investigation of the role of these modifications in decoding. 28 Two tRNA species, tRNA Arg , and tRNA Gly contain mcm5U at mcm 5 UCU mcm 5 UCC the wobble position. The tRNA Arg species decodes the split codon box AGN mcm 5 UCU where AGA and AGG codons are for arginine while AGU and AGC codons are for serine. The tRNA Gly decodes the glycine family codon box GGN. To mcm 5 UCC investigate the role of mcm5 group at U34 for decoding A-ending codons, two of the were deleted. No three genes coding for the mcm5U34-containing tRNA Gly mcm 5 UCC synthetic growth defect was observed when elp3∆ allele was introduced into this does not affect decoding of strain, suggesting that the mcm5 group of tRNA Gly mcm 5 UCC Arg GGA codons. The C34-containing tRNA species tRNA CCU and tRNA Gly CCC decode the G-ending codons that are present in the AGN and GGN box, respectively. Arg Deletion of the genes encoding tRNA CCU or tRNA Gly CCC does not cause a growth defect. However, in combination with an elp3 deletion, the double mutants show synthetic growth defects, indicating that the relevant function of an mcm5U34 residue is to improve pairing with G-ending codons. The three mcm5s2U-containing tRNA species, tRNA Gln mcm 5 s 2 UUG , tRNA Lys , and tRNA Glu decode split codon boxes where the mcm 5 s 2 UUC mcm 5 s 2 UUU pyrimidine-ending codons specify other amino acids. For each of these codon boxes, a C34-containing tRNA species complementary to the G-ending codon is present Lys Glu ( tRNA Gln CUG , tRNA CUU , and tRNA CUC ). Mutants with deletion of the single copy Glu tRNA Gln CUG (Weiss & Friedberg, 1986) or the two tRNA CUC genes are nonviable, suggesting that tRNA Gln and tRNA Glu cannot read G-ending codons. mcm 5 s 2 UUG mcm 5 s 2 UUC Deletion of ELP3 or TUC1 gene to abolish the formation of mcm5 side-chain or s2group in mcm5s2U34 does not rescue the mutants, indicating that reading of G-ending codons by mcm5s2U-containing tRNAs is not restricted by the mcm5- or s2-group. In 29 addition, over-expression of tRNA Glu did not suppress the lethality of a strain mcm 5 s 2 UUC Gln lacking tRNA Glu CUC . However, over-expression of tRNA mcm 5 s 2 UUG suppressed the lethality of a strain having the single copy of tRNA Gln CUG gene deleted. This suppression was not observed if an elp3Δ or tuc1Δ allele was introduced into the strain. This suggests that the mcm5 side-chain and s2-group of mcm5s2U34 cooperatively improve pairing with G. The ncm5U34 residue is present in tRNA Val , tRNA Ser , ncm 5 UAC ncm 5 UGA Pro Ala tRNA ncm , tRNA Thr , and tRNA ncm . These tRNA species decode 5 5 ncm 5 UGU UGC UGG codon boxes where all four codons specify the same amino acid (GUN, UCN, CCN, ACN, and GCN). This means that the correct amino acid would be inserted even if the tRNA would read all four codons. To investigate the role of ncm5U34 in reading of A-ending codons, the genes encoding tRNA Val and tRNA Ser were ncm 5 UAC ncm 5 UGA reduced to one copy, followed by introduction of an elp3 null allele to remove the ncm5 side-chain. In none of the mutants a growth defect was observed, suggesting that the ncm5 side-chain in ncm5U34 is not required to read the cognate A-ending codons. To study the role of ncm5U34 in reading G-ending codons, the single-copy Thr gene encoding tRNA Ser CGA or tRNA CGU was deleted. These mutants are lethal, suggesting that tRNA Ser and tRNA Thr do not read G-ending codons. ncm 5 UGA ncm 5 UGU However, over-expression of tRNA Ser and tRNA Thr rescued the mutants, ncm 5 UGA ncm 5 UGU and introduction of an elp3 null allele in these mutants leads to lethality. Moreover, a Val mutant lacking of tRNA CAC is viable, showing that tRNA Val reads the GUG ncm 5 UAC codon. However, in combination with deletion of ELP3, the double mutant became nonviable. These results indicate that the presence of the ncm5 side-chain improves pairing with G-ending codons. 30 Taken together, the mcm5- and s2-groups cooperatively enhance the ability of mcm5s2U34-containing tRNAs to decode the A-ending codons. The presence of an ncm5U34, mcm5U34, or mcm5s2U34 residue improves the ability of tRNAs to read Gending codons. 31 Conclusions 1. Elongator complex, Kti11p - Kti14p, Sit4p, Sap185p and Sap190p are involved in the synthesis of ncm5 and mcm5 side-chains at wobble uridines. The Urm1p, Uba4p, Ncs2p, Ncs6p and Yor251cp are required for formation of the s2-group in the wobble nucleoside mcm5s2U. 2. Increased levels of the hypomodified tRNA Lys and tRNA Gln mcm 5 s 2 UUG mcm 5 s 2 UUU species bypass the requirement for the Elongator complex in transcription and exocytosis, implying that the major function of Elongator is tRNA modification. 3. The mcm5- and s2-groups cooperatively enhance the ability of mcm5s2U34containing tRNAs to decode A-ending codons, whereas absence of both groups leads to lethality of the yeast cell. The presence of an ncm5U34, mcm5U34, or mcm5s2U34 residue improves the ability of the tRNAs to read G-ending codons. 32 Acknowledgements First of all, I would like to express my sincere thanks and gratitude to Anders Byström, my supervisor who opened the door for me to enter molecular biology. I really admire his enthusiasm and genuine interest in scientific research. His patience and inspirational guidance make the study possible. Many thanks also to Anders’ wife Mona for many great dinners hospitably served during these years. I would like to thank the past and present members of group AB, Jian Lu, Anders Esberg, and Marcus J.O. Johansson for their contribution to this thesis; Monica E. Nordlund, Changchun Chen, Yue Shen, and Fredrik Hugosson for their assistance in the laboratory. Many thanks to Glenn Björk, Mikael Wikström, Olof Persson, Tord Hagervall and their past and present group members Jaunius Urbonavicius, Mattias Lövgren, Ramune Leipuviene, Peng Chen, Hans K. Lundgren, Joakim Näsvall, Jörgen Stenvall, and Stefan Nord for helping extend my knowledge in journal club and group meeting. Kerstin Jacobsson, Gunilla Jäger, and Kristina Nilsson provide the excellent HPLC analyses. Tord Hagervall gave a critical reading of the thesis. I really appreciate the Department of Molecular Biology, Umeå University, where I have been provided with a friendly and creative environment for the scientific research and teaching. Thanks to all colleagues there for their constructive criticism and questions in Wednesday seminars. My gratitude is also present to Marita Waern, Berit Nordh, P-A, and Johnny Stenman, as well as the dishwashing, and media personnel for their valuable services. Outside the scientific research, the fishing, poker, and badminton clubs give me a lot fun, especially in the long winters with cold and dark. Thousands of thanks to Jian Lu, Ming Yuan, Jufang Wu, Gangwei Ou, Changchun Chen, Jun Yu, Xinhe Lai, and Nicklas Kadeby for playing and rejoicing together; to Olena 33 Rzhepishevska, Sa Chen, Peng Chen, Chang Chen, and Junfa Liu for their friendship. I would like to thank my former supervisor Prof. Taiyi Jin for his never ending encouragement and support. 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