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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
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
Papers in this thesis….…………….………….…….….........................................5
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
RESULTS AND DISCUSSION................................................................ 21
Conclusions…………………………………………........................................….. 32
Acknowledgements……………………………….............................................… 33
References………………….………………………...........................................… 35
Papers I - V
Papers in this thesis
This thesis is based on the following papers, which are referred to in the text by their
roman numerals.
An early step in wobble uridine tRNA modification requires the Elongator
Huang B, Johansson MJO, Byström AS. RNA. 2005; 11(4):424-36
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
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;
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
Eukaryotic wobble uridine modifications promote a functionally redundant
decoding system. Johansson MJO, Esberg A, Huang B, Björk GR, Byström
Paper I, III, and IV are reproduced with permission from the publishers.
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
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.
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).
CCA end
TΨC loop
Acceptor stem
D loop
TΨC stem
D stem
TΨC loop
D loop
Anticodon stem
Anticodon loop
Variable loop
Anticodon loop
Fig. 1 Secondary structure (left) and tertiary structure (Kim et al., 1974) (right)
of tRNA
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).
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
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).
Fig. 2 The codon table (modified from Crick, 1968)
Anticodon stem and loop (ASL) of tRNA
3’ 5’
36 35 34
5’---------------------------------------- 3’
1 2 3
1 2 3
1 2 3
Fig. 3 Schematic diagram of the interaction between codon and anticodon.
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
efficiency and fidelity of mRNA decoding (Björk, 1995; Yokoyama & Nishimura,
1995; Urbonavicius et al., 2001).
Ψ Ar(p)
ac4C m G
13 12
20A 20B
m5 C
m 3C
10 9
m5C 49
m5C 48
65 64
54 55
m 5C
39 Ψ
38 Ψ
34 35 36
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.
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).
H3 C
H2 N
m1G 9
Tad1 HN
I 37
CH 3
m I37
CH 3
CH 3
H 3C
Trm5 N
H2 N
CH 3
Fig. 5 Examples of the synthesis of modified nucleosides in yeast (R represents
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).
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).
Table1. Modified nucleosides in S. cerevisiae cytoplasmic tRNAs and gene products required for their
fo rmations. Based on the sequence of 30 of the 42 tRNA species. Indicates that no gene product has
been identified. The tRNA CAC
s pecies has m G rather than m 2 G at position 26 (Gorbulev et al.,
1977), the m G is presumably also catalyzed by Trm1p. (adapted from Johansson & Byström, 2005)
M odified
Genes required
for modification
Gerber & Keller 1999
Anderson et al. 1998; Anderson et al. 2000;
Ozanick et al. 2005
t 6A
34, 40, 48, 49
Laten et al. 1978; Dihanich et al 1987;
Åström & Byström 1994; Åström et al. 1999
Wilkinson et al. 2007
Johansson & Byström 2004
32, 34
Pintard et al. 2002
Wilkinson et al. 2007
TRM11, TRM112
Jackman et al. 2003
Björk et al. 2001
Ellis et al. 1986; Martin et al 1994
Cavaille et al. 1999
Pintard et al. 2002
Alexandrov et al. 2002
Björk et al. 2001;
Noma et al. 2006
1, 26, 27, 28,
34, 36, 65, 67
38, 39
13, 35
16, 17
20A, 20B
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
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
Kalhor & Clarke 2003
Paper I
mcm5s 2U
Kalhor & Clarke 2003
Nakai et al. 2004; Nakai et al. 2007
Paper I
PaperIII; Paper IV
Paper I
Paper I
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).
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
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).
Fig. 6 Examples of wobble uridine modifications present in eukaryotic cytosolic
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.,
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).
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).
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
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).
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
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
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
contains s2U, demonstrating that
the elp1 - elp6 single mutants, tRNA Glu
mcm 5 s 2 UUC
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
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.
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
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
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
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
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
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
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
tRNA Lys
and tRNA Gln
suppressed all those phenotypes of the ncs2Δ
mcm 5 s 2 UUG
mcm 5 s 2 UUU
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.
A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is
required for viability in yeast (paper IV)
is present
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
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
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.
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.
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
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.
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.
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
( tRNA Gln
CUG , tRNA CUU , and tRNA CUC ). Mutants with deletion of the single copy
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
addition, over-expression of tRNA Glu
did not suppress the lethality of a strain
mcm 5 s 2 UUC
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
tRNA ncm
, tRNA Thr
, and tRNA ncm
. These tRNA species decode
ncm 5 UGU
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
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
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
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.
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.
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
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.
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
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
Rzhepishevska, Sa Chen, Peng Chen, Chang Chen, and Junfa Liu for their
I would like to thank my former supervisor Prof. Taiyi Jin for his never
ending encouragement and support. Many thanks also to Madam Gu for her kind
help in my life, especially at the beginning, in Umeå.
I am especially grateful to my parents, Bangji Huang and Shuli Lin; my
younger brothers Yongdong and Hai, as well as their families. Thanks for their
support and understanding at all times.
The National Science Research Council of Sweden and the Swedish Cancer
Foundation are acknowledged for financial support.
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