FUNCTIONAL EFFECT OF ALTERATIONS TO

E. coli METHIONYL-tRNA SYNTHETASE β-LINKER LENGTH

BY

YIYUAN XIA

A Thesis Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

MASTER OF SCIENCE

Chemistry

May, 2015

Winston-Salem, North Carolina

Approved By:

Rebecca W. Alexander, Ph.D., Advisor

Patricia C. Dos Santos, Ph.D., Chair

Lindsay R. Comstock-Ferguson, Ph.D.

ii

Acknowledgments

I would like to give my grateful acknowledgments to the following individuals

who have made this work possible:

My dear parents, Chunliang Xia and Lingying Xu, for raising and for being there

for me in my life.

My advisor, Dr. Rebecca W. Alexander, for giving me the opportunity to work in

her lab. I appreciate the guide she kindly provided for my science research career.

My committee members, Dr. Patricia C. Dos Santos, for introducing me the very

first biochemistry research experience. She and her lab have been a great help for this

work. Dr. Lindsay R. Comstock-Ferguson, for teaching me the first biochemistry course.

Dr. Mark E. Welker, for providing me research insights.

My friendly Alexander lab members, including Dr. Keng-ming Chang, Dr.

Sandhya Bharti Sharma, Adam S. Davidson, Lindsay Macnamara, and Alissa D.

Guarnaccia, for the help, knowledge, and encouragement during my research life. In

particular, Dr. Keng-ming Chang, for mentoring my research skills; Alissa D.

Guarnaccia, for prior work on project II.

All my loving friends appeared in my life, for supporting and encouraging me

through my ups and downs. They have made my life fulfilled and colorful.

Funding from the Department of Chemistry, Wake Forest University and the

National Science Foundation is gratefully acknowledged.

iii

Table of Contents

List of Tables v

List of Figures vi

Abstract vii

Chapter 1 - Introduction 1

1.1 tRNA structure and function 1

1.2 Aminoacyl-tRNA synthetase 3

1.3 Methionyl-tRNA synthetase 9

1.4 Molecular dynamics of wild-type MetRS 12

1.5 Hypothesis and goals 13

Chapter 2 - Experimental Procedures 15

2.1 Subcloning of B. burgdorferi metS 15

2.2 In vivo transcription of tRNAs 16

2.3 Site-directed mutagenesis of E. coli MetRS 18

2.4 Protein expression and purification 19

2.5 Aminoacylation assay 21

2.6 Methionyl adenylate assay 22

2.7 Circular dichroism spectroscopy 23

iv

Chapter 3 - Experimental analysis of B. burgdorferi tRNAMet discrimination 24

3.1 Introduction 24

3.2 Results 25

3.2.1 Cloning and expression of B. burgdorferi MetRS 25

3.2.2 Synthesis and purification of B. burgdorferi tRNAMet variants 28

3.3 Discussion 29

Chapter 4 - Experimental analysis of E. coli methionyl-tRNA synthetase β-linker length

4.1 Introduction 31

4.2 Results 33

4.2.1 Production of E. coli MetRS linker variants 33

4.2.2 Aminoacylation activity of linker variants 34

4.2.3 Methionyl adenylate synthesis activity of linker variants 37

4.2.4 Circular dichroism spectroscopy of linker variants 38

4.3 Discussion 39

Chapter 5 - Conclusion and discussion 41

References 44

Appendix 48

Curriculum Vitae 52

v

List of Tables

Table 1. Classification of aminoacyl-tRNA synthetases

Table 2. Crystal structure of MetRS with different ligands

vi

List of Figures

Figure 1. Secondary and tertiary structures of tRNA

Figure 2. The two-step tRNA aminoacylation reaction

Figure 3. Crystal structure of E. coli MetRS

Figure 4. E. coli MetRS β-strands connected the zinc knuckle to the enzyme body

Figure 5. Expression and purification of B. burgdorferi MetRS with pQE70-BbMetRS

plasmid

Figure 6. Agarose gel of pET28a-BbMetRS plasmid

Figure 7. Expression and solubility test of B. burgdorferi MetRS

Figure 8. Polyacrylamide gel electrophoresis of tRNA samples

Figure 9. E. coli MetRS β-linker motif

Figure 10. Protein purification of E. coli MetRS +124A/188A variant

Figure 11. 10 % SDS-PAGE of the wild-type E. coli MetRS and variants

Figure 12. Aminoacylation of tRNA by wild-type MetRS

Figure 13. Aminoacylation of tRNAMet by wild-type E. coli MetRS and β-linker variants

Figure 14. Methionyl adenylate formation by wild-type E. coli MetRS and β-linker

variants

Figure 15. Circular dichroism spectroscopy of wild-type E. coli MetRS and the variants

vii

Abstract

Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that catalyze the

attachment of amino acids to their cognate tRNAs for protein biosynthesis at the

ribosome. Methionyl-tRNA synthetase (MetRS), which belongs to AARS subclass Ia, is

especially interesting, since it recognizes two functionally distinct tRNA substrates, an

initiator tRNAfMet and an elongator tRNAMet. The zinc binding knuckle of the E. coli

MetRS plays an important role in methionine activation and amino acid transfer to the 3'

end of tRNAMet. But information on how the zinc knuckle affects tRNA binding or

catalysis is still limited. The goal of this project is to investigate the effect of

repositioning the zinc binding knuckle on MetRS catalytic activity. Since the zinc

binding knuckle is linked to the body of the MetRS by the β-strands, the distance

between them will change when length of the β-linker is changed.

After designing and expressing three variants with different β-linker length variants, we

tested their catalytic activities. All the variants decreased the methionine activation and

the tRNAMet aminoacylation activities to some extent. For the variant located at the end

of the β-linker, decreases were the most significant. The decrease of the tRNAMet

aminoacylation activity seemed to be due to the decrease of the methionine activation

activity for the variant with the shorter β-linker. The variant with the insertion located in

the middle of the β-linker retained high adenylate formation but exhibited low

aminoacylation. Further work is needed to determine whether tRNAMet binding or

methionine transfer (or both) is affected.

1

Chapter 1

Introduction

1.1 tRNA structure and function

Transfer RNAs (tRNAs) are a family of ribonucleic acids (RNAs) that fold into a

cloverleaf secondary structure and L-shaped tertiary structure; they are essential

components of the cellular protein synthesis machinery. Yeast tRNAAla was first

sequenced in 1965, and tRNAPhe was the first nucleic acid whose crystal structure was

solved in 1974.3 There are more than 5800 known tRNA sequences from more than 111

organisms including archaea, bacteria, higher and lower eukarya.4 Complete sets of

tRNAs include at least one isoacceptor species for each of the 20 standard amino acids.4

tRNA is one of the most abundant groups of nucleic acids and has a high degree of

structural conservation to preserve its function.5

Most tRNAs contain about 76 nucleotides, although their length can be up to 93

nucleotides.6 Their cloverleaf structure has four distinct helical segments and three loops

(Figure 1). The parts of the cloverleaf structure are the acceptor stem, the dihydrouridine

(D) stem-loop, the anticodon stem-loop, and the TΨC stem-loop.7 The 3' end of all

tRNAs ends with the single-stranded sequence N73CCAOH. Nucleotide 73 (N73) is called

the discriminator base and can be any base, although it is conserved among isoacceptors.

The role of this nucleotide is to enable aminoacyl-tRNA synthetases (AARSs) to partition

tRNAs into groups for recognition.8 The free 3' - hydroxyl group on the terminal

adenosine serves as the amino acid attachment site. The CCAOH sequence is essential for

aminoacylation and positioning on the ribosome.9 The dihydrouridine (D) stem-loop is a

4 base-pair stem ending in a loop that typically contains dihydrouridine. The anticodon

2

stem-loop is a 5 base-pair stem whose loop contains the trinucleotide anticodon that

decodes the mRNA codon on the ribosomal small subunit. In the TΨC stem-loop, there is

a conserved sequence that is transcribed as UUC. In this sequence, the first uridine is

enzymatically methylated to thymine (T) and the second uridine is enzymatically

modified to pseudouridine (Ψ).10

The helices of the tRNA cloverleaf secondary structure coaxially stack to form a

distinct L-shaped tertiary structure (Figure 1). The acceptor arm in L-shaped tRNA

consists of the coaxially stacked acceptor and TΨC stems, while the anticodon arm

consists of coaxially stacked anticodon and D stems. Nucleotides in the tRNA core make

complex hydrogen bond and stacking interactions.11 In protein translation, the L-shaped

tRNA interacts with its cognate AARS and with the ribosome. The acceptor arm interacts

Figure 1. Secondary and tertiary structures of tRNA. A. E. coli tRNAMet secondary

structure; B. yeast tRNAPhe tertiary structure.1

B

D loop

Acceptor stem

Discriminator

base

TΨC

loop

Anticodon

A

3

with the catalytic domain of its cognate AARS to accept the activated amino acid. In

peptide bond formation, it also contacts the peptidyl transferase center of the large

ribosomal subunit. The anticodon arm contacts the anticodon binding domain of its

cognate AARS and binds the messenger RNA (mRNA) at the decoding center of the

small ribosomal subunit.12

tRNA contains many modified nucleotides. Some of the nucleotides are common

in most species, such as the ribothymidine in the TΨC loop and the dihydrouridine in the

D loop. Others are only in specific tRNAs, such as the queuosine derivatives at the first

anticodon position of certain tRNAAsn, tRNAAsp, and tRNATyr species.13 Modified

nucleotides are found at 61 different positions in tRNAs, mainly in the loop regions, and

can make as many as 20% of nucleotides in tRNAs from higher organisms.14 Many of the

tRNAs with modified nucleotides retain their full aminoacylation affinity even without

the modifications present.15 But some modified residues are directly involved in the

aminoacylation reaction, such as a lysidine residue in the anticodon of a minor tRNAIle

species from E. coli.16

1.2 Aminoacyl-tRNA synthetase

Translation is the process organisms use to synthesize the sequence of amino

acids in proteins from the corresponding nucleotide instructions of mRNA.17 In

translating mRNA into protein, aminoacylated tRNAs are the substrates for the

ribosome’s peptidyl transferase activity. Aminoacylation of tRNAs is catalyzed by a set

of enzymes called aminoacyl-tRNA synthetases (AARSs) that help to maintain the

integrity of the genetic code.18 AARSs are modular enzymes, with at least two

4

polypeptide domains that are responsible for catalysis and substrate recognition.19 The

ancestral domain activates its cognate amino acid to the corresponding adenylate with

ATP and promotes aminoacylation of the tRNA 3' - terminus.19 A separate anticodon

domain binds its cognate tRNA’s anticodon in most aminoacylation systems. Many

tRNA anticodons act as allosteric activators of the aminoacylation reaction.20 For some

AARSs, aminoacyl adenylate formation is also dependent on cognate tRNA binding.18b

tRNA aminoacylation is a two-step reaction that occurs in a single active site of

the AARS enzyme. The reactions are shown (Figure 2) in which AA is an amino acid and

AARS is the cognate aminoacyl-tRNA synthetase to that amino acid. In the first step,

ATP and amino acid bind at the active site. The α-carboxylate of the amino acid attacks

the α-phosphate of the ATP by an in-line nucleophilic displacement mechanism. This

leads to the formation of an enzyme-bound aminoacyl-adenylate and a pyrophosphate

leaving group. The first step occurs in the absence of tRNA except for GlnRS, GluRS,

ArgRS and class I LysRS.20 In the second step, the 2' - hydroxyl or 3' - hydroxyl of the

tRNA’s terminal adenosine nucleophilically attacks the aminoacyl adenylate. This leads

to release of AMP as the leaving group and produces the aminoacyl-tRNA.21

The AARSs are divided into two unrelated classes (class I and class II) based on

mutually exclusive sequence motifs and structural similarities of their catalytic

domains.18a These individual classes are again divided into several subclasses based on

additional sequence and structure homologies (Table 1). The class organization is

conserved through evolution except for LysRS, which primarily exists as a class II AARS

5

Figure 2. The two-step tRNA aminoacylation reaction. Both amino acid activation

(adenylate synthesis) and amino acid transfer (esterification) are catalyzed at a single

active site.

6

but is a class I AARS in bacteria and archaea.22

Table 1. Classification of aminoacyl-tRNA synthetases. The structural organization is

indicated for each enzyme, for example α2 is a homodimer.18b

Class I Class II

Subclass a ArgRS α GylRS α2

CysRS α HisRS α2

IleRS α ProRS α2

LeuRS α ThrRS α2

ValRS α SerRS α2

LysRS I α

MetRS α, α2

Subclass b Gln RS α AsnRS α2

GluRS α AspRS α2

LysRS II α2

Subclass c TryRS α2 AlaRS α, α4

TyrRS α2 PheRS α2β2

PheRS α, α2β2

The enzymes now identified as belonging to class I were first noted by common

sequence motifs. The catalytic domain of class I enzymes contains an 11-residue

signature sequence ending in HIGH and also contains the motif KMSKS.20 The KMSKS

signature motif always accompanies the HIGH sequence in a given AARS.18b These

sequence motifs are not found in class II enzymes, which instead have three highly

variable sequence motifs in their catalytic domains.

The major difference between the two AARS classes is the structure of their

active sites. The active site in class I AARSs contains a Rossmann dinucleotide-binding

7

domain, while this domain is absent in the active site of class II AARSs. Instead, class II

active sites contain a novel antiparallel β-fold. As a result, class I AARSs bind ATP in an

extended conformation, while class II enzymes bind ATP in a bent conformation.19 The

other major difference between these two classes is in their binding of tRNA, which is

found by crystallographic studies of synthetase:tRNA complexes. Class I AARSs

approach the acceptor stem of tRNA from the minor groove side with the variable loop

facing the solvent, while class II AARSs approach the major groove side of the acceptor

stem and the variable loop faces the synthetase.23

The first crystal structure of an AARS solved at high resolution was Bacillus

stearothermophilus TyrRS.24 Analyses of this crystal structure provided insight into the

mechanism of amino acid recognition in class I AARS, which showed that the reaction

takes place by using the binding energy produced from the enzyme-substrate interaction

to stabilize the transition state. High resolution crystal structures of other class I AARSs

helped increase understanding of the mechanism of amino acid activation. The TrpRS

crystal structure showed an open AARS that also contained a distinctive anticodon

binding domain, which was proposed to be added later in evolution and recognizes the

cognate tRNA anticodon.25 In some cases, one domain of an AARS can be removed

without affecting the activity of the other domains. The E. coli AlaRS is the largest

AARS and exists in vivo as a tetramer of 875 amino acid monomers. Deletion of the C-

terminus after residue Gly-699 results in an active monomeric protein, while a protein

truncated after the first 461 residues was able to catalyze the aminoacylation reaction. 20

In another study, the deletion of 11 amino acids after residue Trp-461 in E. coli MetRS

reduced the efficiency of the aminoacylation reaction but adenylate formation and

8

microhelix aminoacylation were not reduced.26 The crystal structure of the E. coli MetRS

monomer shows two major domains responsible for tRNA anticodon binding: N-terminal

active site domain and the C-terminal domain. 2 The research based on the E. coli MetRS

variants truncated Arg-537 showed that the contacts between the N- and C-terminal

domains are important for the proper folding. These conformational changes happened

because of the movement of a small domain which include the KMSKS and HIGH

motifs.27 A similar role of KMSKS and HIGH motifs has been found in GlnRS, though

there is a distinct amino acid activation difference between MetRS and GluRS.28

Crystal structures have also been solved for class II AARSs. These crystal

structures showed that the enzyme active sites are rigid templates for amino acid and

ATP substrates.29 The specificity of class II AARSs is determined by the contacts

between the side chains of amino acid substrates and active sites which connect to the

cognate amino acid. For some of the class II AARSs such as SerRS and AspRS, this is a

local effect, while it is the more global domain rearrangement for other AARSs such as

LysRS and HisRS.28, 30

Numerous evidence suggests that defects of AARS function can contribute to

human disease. The first human genetic diseases associated with AARS were Charcot-

Marie-Tooth disease type 2D (CMT2D) and distal spinal muscular atrophy type V

(dSMA-V). Both of these diseases are inherited in an autosomal dominant fashion,

caused by mutations in the GlyRS gene.31 Mutations in the mitochondrial TyrRS gene

cause the mitochondrial respiratory chain disorder MLASA Syndrome (Myopathy, Lactic

Acidosis, and Sideroblastic Anemia).32 Mutants in AlaRS cause infantile cardiomyopathy

which is a disease characterized by large, hypertrophic heart and fatality within the first

9

1–2 years of life.33 A mutation in the editing domain of AlaRS leads to a 2-fold decrease

of editing noncognate Ser-tRNA, which may lead to the development of ataxia in

mouse.34

1.3 Methionyl-tRNA synthetase

Methionyl-tRNA synthetase (MetRS) belongs to AARS subclass Ia (Table 1),

together with ArgRS, CysRS, IleRS, LeuRS, ValRS and LysRS I. Among the AARSs of

its class, MetRS is especially interesting, since it recognizes two functionally distinct

tRNA substrates, an initiator tRNAfMet and an elongator tRNAMet. The tRNA delivers

methionine for elongation of the protein chain. The initiator tRNA is used for initiation of

protein synthesis, while the elongator tRNA is used for inserting methionine into internal

peptide linkages.35 MetRSs isolated from numbers of species show greater structural

diversity compared to other AARSs, suggesting the ability to interact with other proteins

to form functional complexes.36

E. coli MetRS is a homodimer of 76 kDa monomers. The genetically engineered

monomeric version lacking the terminal 121-residue dimerization domain is composed of

547 residues (64 kDa) and has been crystallized.2 Monomeric E. coli MetRS has four

major domains (Figure 3). The N-terminal domain (red, residues 1-99 and 251-325) folds

into the class I Rossmann fold and includes the HIGH motif, which activates and

transfers methionine to the acceptor stem of tRNAMet. The connective polypeptide (CP)

domain (green, residues 100- 250) is between the halves of the Rossmann fold. The CP

domain contains a zinc knuckle structure which coordinates a zinc (II) ion liganded by

four cysteine residues.20, 37 The stem contact fold (yellow, residues 325-387) contains the

10

KMSKS sequence that stabilizes and orients the ATP for the adenylate reaction. Finally,

the C-terminal anticodon domain (blue, residues 388-547) recognizes the tRNAMet CAU

anticodon. It is an α-helical bundle motif and contributes significantly to the specificity

between MetRS and tRNAMet. In particular the highly conserved Trp-461 is essential for

recognition of the CAU anticodon of methionine-accepting tRNAs.38 A crystal structure

of the Aquifex aeolicus MetRS:tRNAMet complex showed the ring of Trp-422

(corresponding to Trp-461 in E. coli MetRS) stacked on the C34 and A35 nucleotides of

Connective

polypeptide

domain

Catalytic

domain

Stem

contact

domain

Anticodon

binding

domain

Figure 3. Crystal structure of E. coli MetRS. PDB id: 1QQT2 Major structural

domains are indicated.

11

the CAU anticodon.11

After the high resolution crystal structure of E. coli MetRS was determined in

1991, other MetRS structures were solved including complexes with tRNA or small

molecules (Table 2). These available MetRS structures can be grouped into four families

according to the organization of their CP domains. The CP domains of MetRS in

eukaryotes, archaea, and spirochetes carry two metal ions in two knuckle motifs, while

the other families have one zinc ion held in one or two knuckle motifs. The fourth family

lacks any metal ions. Prior experimental work showed that the zinc binding domain plays

an important role in methionine activation and amino acid transfer to the 3' end of

tRNA.37, 39

Table 2. Crystal structure of MetRS with different ligands.

Organism Ligand PDB id Reference

E. coli No ligand 1QQT 2

E. coli Methionine 1F4L 40

E. coli Methionine phosphonate 1P7P 41

E. coli Methioninyl adenylate 1PGO 41

A. aeolicus tRNAMet 2CSX 11

B. melitensis Selenomethionine 4DLP 42

M. smegmatis Methionine, adenosine 2X1L 43

P. abyssi No ligand 1RQG 44

T. thermophilus No ligand 1A8H 45

T. thermophilus Methioninyl adenylate 3VU8 46

12

1.4 Molecular dynamics of wild-type MetRS

In previous molecular dynamics simulations of E. coli MetRS carried out by our

lab, the defined regions of high mobility are in both the catalytic domain (Rossmann fold

and CP domain) and the anticodon domain.47 The study showed the most mobile region is

the CP domain, especially in the zinc binding motif. The zinc binding motif of the CP

domain has also been shown to be important for tRNA aminoacylation, as mutagenesis to

remove the zinc cation decreased the aminoacylation ability.48

In E. coli MetRS, residue

Trp-461 in the anticodon binding

domain is essential for anticodon

binding.2 Mutagenesis studies

showed that substitution of this

Trp residue with alanine

decreases the catalytic efficiency

of tRNAMet aminoacylation by

nearly five orders of magnitude.49

Another study with a more

conservative phenylalanine

substitution showed a 50-fold

decrease.38 Our simulations

showed positive dynamic

correlations between Trp-461 and residues in the two long β-strands of the CP domain.45

These β-strands (residues 118-129 and 182-192) resemble a “stalk” and link the zinc

Figure 4. E. coli MetRS β-strands (highlighted

in red) connected the zinc knuckle to the

enzyme body. PDB id: 1QQT

13

binding motif to the body of MetRS (Figure 4). Another prior study demonstrated the

connections between these regions since mutations in the zinc biding motif decrease the

affinity of tRNA binding.48 This affinity decrease may suggest direct interactions

between the zinc binding motif and tRNA acceptor stem binding or other structural or

functional importance of the β-strands in tRNAMet aminoacylation.

1.5 Hypothesis and goals

The goal of project I is to investigate substrate specificity of Borrelia burgdorferi

methionyl-tRNA synthetase (BbMetRS). In our previous work, BbMetRS exhibited only

a 2-fold difference in aminoacylation efficiency between its cognate tRNA Met

CAU and near-

cognate tRNA Ile

CAU transcripts, which is much lower than the other bacterial MetRS

enzymes tested.50 We mutated the 3-70 base pair in B. burgdorferi tRNA Met

CAU, which is

different from other tRNAMet substrates. We hypothesize that this base pair contributes to

B. burgdorferi selectivity and prevents mis-insertion of methionine at isoleucine codons.

The mutated B. burgdorferi tRNAMet

CAU will be compared to activities of the wild-type

tRNAMet

CAU and the tRNAIle

CAU of B. burgdorferi.

The goal of project II is to investigate the effect of repositioning the zinc binding

knuckle on MetRS catalytic activity. Prior work indicated that this zinc binding knuckle

plays an important role in methionine activation and amino acid transfer to the 3' end of

tRNAMet.37 But information on how the zinc knuckle affects tRNA binding or catalysis is

still limited. Since the zinc binding knuckle is linked to the body of the MetRS by β-

strands, the distance between them will change when the length of the β-linker is

changed. By comparing catalytic activities of enzymes containing different β-linker

14

lengths, the effect of the zinc binding knuckle position will be defined. We hypothesize

that the effect of each variant on MetRS function will be different depending on whether

the β-linker is shortened or lengthened. We propose that a shorter linker might have a

more negative effect on catalysis than a longer linker. To test these hypotheses, amino

acid positions were chosen on the β-linker to be opposite each other on the two strands.

The structure of the linker is known to be primarily antiparallel β-strands, and we sought

to shorten or lengthen the strands by one residue on each strand without disrupting the

rest of the protein structure.51 Functional assays were used to determine the impact of

altering the linker length on amino acid activation and tRNAMet aminoacylation.

15

Chapter 2

Experimental Procedures

2.1 Subcloning of B. burgdorferi metS

The B. burgdorferi metS gene was previously cloned into a pQE-70 plasmid

(pQE70/BbMetRS).50 In order to increase expression of the encoded protein, we sought

to subclone the metS gene into the pET28a expression plasmid. Two primers with

XhoI/NdeI restriction sites were designed (see Appendix for sequences). To produce the

BbMetRS fragment sequence, PCR reactions (50 µL) contained 250 ng each of the

forward and reverse primers, 100 ng pQE70/BbMetRS parent plasmid, 0.2 mM each

dNTP, 1 µL Pyrococcus furiosus (Pfu) Ultra DNA polymerase (Agilent), and 5 µL 10×

Pfu Ultra reaction buffer. Reactions were incubated in a thermocycler (BioRad iCycle) as

follows: 95 ºC for 4 minutes, 30 cycles of denaturation at 95 ºC for 1 minute, annealing at

62 ºC for 1 minute and extension at 72 ºC for 10 minutes and 72 ºC for 10 minutes.

Successful PCR products were verified by agarose gel electrophoresis. pET28a

plasmid to be used as the vector (100 µM) was digested with 1 µL each of XhoI and NdeI

restriction enzymes (Promega) and 2 µL of 10× NEbuffer 4 in 20 µL final volume. The

reaction was incubated at 37 ºC for one hour and analyzed by agarose gel electrophoresis.

Both the BbMetRS fragment and the pET28a fragment were isolated and purified from

the agarose gel using the QIAquick gel extraction kit (Qiagen). The ligation reactions (30

µL) contained 10 µL each BbMetRS gene insert and pET28a vector, 1 µL 10 mM ATP, 1

µL DNA ligase (Agilent) and 3 µL 10× ligase buffer supplied by the manufacturer. The

reaction was incubated overnight at 16 ºC to produce the pET28a/BbMetRS plasmid.

16

The ligation product was transformed into Top 10 E. coli competent cells

(Invitrogen) by heat shocking 50 µL of chemically competent cells with 2 µL of plasmid

DNA. The cells were incubated on ice for 5 minutes and heat shocked at 42 ºC for 45

seconds followed by incubation on ice for 2 minutes. Cells were combined with 250 µL

of Luria Broth (LB) for 1 hour at 37 ºC with shaking and plated on 35 µg/mL kanamycin

(Kan)/LB agar plates. Incubation at 37 ºC overnight yielded individual colonies that were

picked from the plate and used to inoculate 5 mL LB media containing 35 µg/mL Kan;

cultures grew at 37 ºC with shaking overnight. Plasmid DNA was isolated using the

Qiaprep Miniprep Kit (Qiagen). A 10 µL portion of the isolated plasmid was digested

with 1 µL of XhoI and NdeI restriction enzyme (Promega) and 2 µL of the 10× NEbuffer

4 in a 20 µL total reaction. The reaction was incubated at 37 ºC for one hour and

analyzed by agarose gel electrophoresis, which revealed two pieces of around 3500 kb

and 3000 kb. After confirming correct plasmid size, the samples were sequenced by

Genewiz (South Plainfield, NJ) to verify incorporation of the B. burgdorferi metS gene.

Sequence results are shown in the Appendix.

2.2 In vitro transcription of tRNAs

All tRNAs used were produced by in vitro transcription of blunt duplex DNAs

using T7 RNA polymerase according to the method of Sherlin and coworkers.52 The mG

and mU represent the 2-O-methyl nucleotides that have been shown to produce clean 3'

ends of tRNA transcripts. The Klenow DNA polymerase reaction was used to produce a

blunt duplex DNA template corresponding to the tRNA gene behind a T7 promoter.

Primers for Klenow were designed such that the 3' end of the sense strand had a 10 to 15

17

bp overlap with the 3' end of the antisense stand. The primers for B. burgdorferi tRNAMet

(U3G-A70C) were designed such that the uracil at nucleotide 3 was changed to guanine

and the adenine at nucleotide 70 was changed to cytosine. All primer sequences are

shown in the Appendix.

The Klenow reaction contained 4 µM each primer, 4 µM dNTPs, 50 U/mL

Klenow fragment DNA polymerase (New England Biolabs Inc.) and 10 µL NEB 2 buffer

(New England Biolabs Inc.) for in a total of 400 µL. The reaction was incubated in a

thermocycler (BioRad iCycle) for 8 cycles of 10 ºC for 30 sec and 37 ºC for 30 sec.

Each 1 mL in vitro transcription reaction contained 400 µL Klenow generated

template DNA described above, 5 mM each NTP, 250 mM HEPES·KOH (pH 7.5), 2 mM

spermidine, 40 mM DTT, 30 mM MgCl2, 0.1 mg/mL BSA and 40 µg/mL T7 RNA

polymerase. The reaction was incubated overnight at 37 ºC and digested by 10 U/mL

RNase-free DNase I for 1.5 hours. The reaction was centrifuged to remove the Mg2+·PPi

precipitate that forms upon nucleotide incorporation. The supernatant was ethanol

precipitated overnight by adding 1/10 reaction volume of 3 M NaOAc (pH 5.2) and 2.5

reaction volume of ice-cold ethanol at -20 ºC.

A 20 cm height, 3 mm thickness 16 % denaturing polyacrylamide gel was used to

purify the ethanol precipitated tRNA. Each tRNA pellet was resuspended in 20 mM

HEPES·KOH (pH 7.5) combined with 90% formamide, and boiled for 3 minutes. A 200

µL portion of each tRNA solution was loaded into each of four wells and a formamide

dye (bromophenol blue/Xylene cyanol) marker was loaded in a separate well. The gel

was electrophoresed at 8 W for 12 to 18 hours until 2 hours after the second marker

passed the bottom of the gel. The band corresponding to each tRNA was visualized by

18

UV shadowing and cut from the gel. The gel piece was crushed though a 5 mL syringe

barrel and soaked in 3 mL extraction buffer (0.5 M NH4Ac (pH 7.4), 1 mM EDTA) at 37

ºC in a rotator for 2 hours to extract each tRNA. The solution was passed through a 20

µm syringe filter (Fisher Brand) to separate the gel pieces from the buffer containing

tRNA. The supernatant was ethanol precipitated overnight by adding 4/15× 10 M NH4Ac

and 2.5× ice cold ethanol at -20 ºC. The gel extraction was repeated another two times for

overnight and another 2 hours. All the resulting ethanol precipitates were centrifuged and

washed with 70% ethanol. Pellets were dissolved in 20 mM HEPES (pH 7.5) and stored

in -20 ºC.

Each tRNA concentration was measured by absorbance at 260 nm using a

Nanodrop spectrophotometer (Thermo Scientific). The molar extinction coefficients (ε)

of E. coli tRNAMet, B. burgdorferi tRNAMet, B. burgdorferi tRNAMet U3G-A70C, and B.

burgdorferi tRNA Ile

CAU are 1002.6 mM-1·cm-1, 945.6 mM-1·cm-1, 946.7 mM-1·cm-1, and

960.8 mM-1·cm-1, respectively, at 260 nm. Before use in an aminoacylation assay, the

tRNA was annealed by heating at 80 ºC and cooling to 60 ºC at which time MgCl2 was

added to a final concentration of 1 mM. The reaction was allowed to cool slowly to room

temperature and was then ready for the aminoacylation assay.

2.3 Site-directed mutagenesis of E. coli MetRS

Alterations of the length of the β-linker in E. coli MetRS were accomplished

using the QuikChange mutagenesis method (Invitrogen). Primers were designed based on

the pSW101 parent plasmid that encodes E. coli MetRS behind a T7 promoter and N-

terminal His6-tag. Primer sequences are given in the Appendix. Reactions (50 µL)

19

contained 250 ng each of the forward and reverse primers, 100 ng pSW101 parent

plasmid, 0.2 mM dNTP, 1 µL Pfu Ultra DNA polymerase (Agilent), and 5 µL 10× Pfu

Ultra reaction buffer. Reactions were incubated in a thermocycler (BioRad iCycler) as

follows: 95 ºC for 4 minutes before cycles, denaturation at 95 ºC for 1 minute, annealing

at 55 ºC for 1 minute and extension at 72 ºC for 14 minutes (15 cycles), 72 ºC for 10

minutes after cycles. Successful PCR products were verified by agarose gel

electrophoresis. Reactions were incubated at 37 ºC for 1 hour after adding 1 µL of DpnI

enzyme.

Plasmids were transformed into Top 10 E. coli competent cells (Invitrogen) by

heat shocking 50 µL of competent cells with 2 µL of plasmid DNA. The cells were

incubated on ice for 5 minutes and heat shocked at 42 ºC for 45 seconds followed by

incubation on ice for 2 minutes. Cells were combined with 250 µL of Luria Broth (LB)

for 1 hour at 37 ºC with shaking and plated on 35 µg/mL kanamycin (Kan)/LB agar

plates. Incubation at 37 ºC overnight yielded individual colonies that were picked from

the plate and used to inoculate 5 mL LB media containing 35 µg/mL Kan; cultures grew

at 37 ºC with shaking overnight. Plasmid DNA was isolated using the Qiaprep Miniprep

Kit (Qiagen). A 10 µL portion of the isolated plasmid was digested with 1 µL of HindШ

restriction enzyme (Promega) and 2 µL of the 10× NEbuffer 2 for 20 µL total reaction.

The reaction was incubated in 37 ºC for one hour and analyzed by agarose gel

electrophoresis, which revealed two pieces of around 6000 kb and 1000 kb. After

confirming correct plasmid size, the samples were sequenced by Genewiz, Inc. (South

Plainfield, NJ) to verify the addition and deletion mutants. Sequence results are shown in

the Appendix.

20

2.4 Protein expression and purification

Plasmid DNA was transformed into Rosetta DE3 E. coli cells (Novagen Inc.) by

heat shocking 100 µL of competent cells with 2 µL of the plasmid DNA (100 µM). The

cells were heat shocked at 42 ºC for 45 seconds and incubated on ice for 2 minutes.

Transformed cells were incubated with 500 µL of Luria Broth (LB) for 1 hour at 37 ºC

with shaking. The cells were plated on 35 µg/mL kanamycin (Kan) LB agar plates and

incubated at 37 ºC overnight. A single colony was picked from the plate and incubated in

5 mL LB media containing 35 µg/mL Kan at 37 ºC with shaking overnight. The small

culture was transferred into 1 L LB-Kan (35 µg/mL) media, incubating at 37 ºC with

shaking. When the absorbance at 600 nm reached 0.5 to 0.6, the protein expression was

induced with isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1

mM.

Wild-type MetRS induction proceeded for 3 hours, variants for 6 hours prior to

harvesting cells by centrifuging at 6300 rpm for 10 min in an Avanti J-E series centrifuge

(Beckman-Coulter). The cell pellets were resuspended in 30 mL lysis buffer A (50 mM

Tris·HCl (pH 7.5), 500 mM NaCl and 10 mM imidazole). Phenylmethylsulfonyl fluoride

(PMSF) protease inhibitor was added into the resuspended cells to a final concentration

of 1 mM. Cells were lysed using an Emulsiflex C5 high-pressure homogenizer (Avestin)

and centrifuged at 15,000 rpm for 30 minutes to remove cell debris.

Protein was purified by fast protein liquid chromatography (FPLC) on a Biologic

DuoFlow (Bio-Rad) with QuadTec detector (Bio-Rad). The supernatant was loaded on a

1 mL His-Trap column (GE Healthcare) equilibrated with Ni2+ to bind the His-tagged

21

MetRS at a flow rate of 1 mL/min. The column was washed with 50 mL of lysis buffer A

at a flow rate of 2 mL/min. The column was washed by 20 mL of 96% lysis buffer A, 4%

elution buffer B (50 mM Tris·HCl (pH 7.5), 500 mM NaCl and 500 mM imidazole) at a

flow rate of 2 mL/min. Bound His-tagged MetRS was eluted by an imidazole gradient in

which elution buffer B was increased from 4% to 100% over 30 mL. Protein elution was

monitored at 280 nM. Selected fractions were analyzed by 10 % SDS-PAGE gel to check

protein purity.

Fractions containing MetRS were dialyzed against an imidazole-free buffer (40

mM HEPES·KOH (pH 7.5), 200 mM NaCl, 20 mM MgCl2, 20 mM KCl) overnight at 4

ºC. Purified protein was concentrated in 30 kDa MWCO Amicon Ultra centrifugal filters

(Millipore) spinning at 4000 rpm until the sample volume was reduced to 500 µL.

Concentrated proteins were frozen and stored at -20 ºC in 40 % glycerol, 30 mM

HEPES·KOH (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 10 mM KCl.

Concentrations of the proteins were measured by the absorbance at 280 nm using

a NanoDrop spectrophotometer (Thermo Scientific). The molar coefficient (ε) of E. coli

MetRS is 94,770 M-1·cm-1 at 280 nm and the molecular mass is 64,690 Da.

2.5 Aminoacylation assay

Aminoacylation reactions contained 0.1 mM methionine, 20 mM HEPES (pH

7.5), 4 mM ATP (pH 7.0), 150 mM NH4Cl, 0.1 mM EDTA, 10 mM MgCl2, trace [35S]

methionine (Perkin-Elmer) and annealed tRNAMet. To prepare for quenching the

reactions, 2.3 cm filter paper (Whatman) were soaked in 5% trichloroacetic acid (TCA)

22

solution containing 1 mM methionine and dried under a heat lamp. The aminoacylation

reactions were initiated by introducing of MetRS enzyme to the prepared reaction.

Reaction aliquots (5 µL) were removed every 30 seconds for 150 seconds and quenched

on the TCA-soaked filter papers. The filter papers were immediately dropped into an ice

cold TCA-methionine solution. All filter papers were washed five times for 10 minutes

each in fresh TCA-methionine solution to remove all 35S-methionine which was not

incorporated into tRNAMet. Finally, the filter papers were washed in 95% ethanol for

drying and counted by scintillation using a LS6500 Multipurpose Scintillation Counter

(Beckman). Final concentrations of 2 µM tRNA and 10 µM wild-type MetRS were used

in reactions to check the aminoacylation capacity of tRNA. Final concentrations of 3 µM

tRNA and 100 nM MetRS were used in reactions to determine aminoacylation activities

of wild-type MetRS and variant enzymes.

2.6 Methionyl adenylate assay

The activity of MetRS in methionyl adenylate formation, which is the first step of

aminoacylation, was determined by an adenosine triphosphate-pyrophosphate (ATP-PPi)

exchange assay. This assay quantifies the incorporation of 32P into ATP from labeled

pyrophosphate by the back reaction of the adenylate formation. Reactions contained 100

mM Tris-HCl (pH 7.5), 2 mM NaPPi, 10 mM 2-mercaptoethanol, 0.1 mg/mL BSA, 5

mM MgCl2, 10 mM KF, 100–1000 µM methionine, 2 mM ATP and [32P] NaPPi (Perkin-

Elmer). The reactions were initiated by introducing enzyme to the prepared reaction for a

final concentration of 100 nM. Reaction aliquots (10 µL) were removed every minute for

5 minutes and quenched in a microcentrifuge tube containing 450 µL charcoal slurry

23

quench reaction buffer (7% HClO4, 3% charcoal, 0.2 M NaPPi). The charcoal was

washed three times with wash buffer (10 mM PPi and 0.5% HClO4). The tubes were

centrifuged for 30 seconds after each wash to recover the charcoal. The tubes with

charcoal-adsorbed [32P] ATP were counted by scintillation using a LS6500 Multipurpose

Scintillation Counter (Beckman).

2.7 Circular dichroism spectroscopy

Circular dichroism spectroscopy was performed using an AVIV CD spectrometer

(Model 215, AVIV Biomedical Inc.) with a 1 mm quartz cuvette (Hellma Analytic Inc.)

and a bandwidth of 1 nm. Enzyme samples (500 µL of 1 µM) were prepared in 10 mM

phosphate buffer (pH 7.4). A low pressure nitrogen tank was connected to the CD

spectrometer. The parameters for the experiment were configured to measure the

wavelength by every nanometer from 190 nm to 250 nm. Each sample was scanned five

times. Phosphate buffer was used as the blank. The five runs for each sample were

averaged and the average blank spectrum was subtracted.

24

Chapter 3

Experimental analysis of B. burgdorferi tRNAMet discrimination

3.1 Introduction

The protein:tRNA interactions that drive tRNA aminoacylation specificity were

selected very early in the evolution of the genetic code. In general, there are 20

aminoacyl-tRNA synthetases, one for each standard amino acid. 53 Methionyl-tRNA

synthetase (MetRS) recognizes two functionally distinct tRNA substrates, an initiator

tRNAfMet and an elongator tRNAMet, for decoding the single methionine AUG codon. The

initiator tRNA is used for initiation of protein synthesis at the AUG start codon, while the

elongator tRNA is used for inserting methionine into internal peptide linkages. 35

However, the single methionine codon is similar to one of the isoleucine codons (AUA).

The only difference between the two trinucleotides is in the type of purine in the

anticodon wobble (3rd) position. Solutions to this potential discrimination problem vary

widely across the tree of life. In most organisms, the C34 wobble position of tRNAIle is

modified to prevent mis-aminoacylation by MetRS.50 Bacteria use a tRNAIle

CAU modified to

tRNAIle

LAU to specifically decode the AUA anticodon, where L is the C-modified lysidine

nucleotide.54 Eukaryotes use a tRNAIle

ΨAU whose U34 is post-transcriptionally modified to

pseudouridine, allowing accurate decoding of the AUA anticodon.55 Archaea use

agmatidine instead of the lysidine and pseudouridine above.56 Mitochondria use a

modified genetic code that assigns both AUA and AUG codons to methionine.57

From our previous work, the abilities of bacterial MetRSs from different clades to

differentiate cognate tRNAMet

CAU from near-cognate tRNAIle

CAU, have been investigated to

explore the actual distribution of modification-independent tRNAIle rejection by MetRS.50

25

We wanted to see whether the CAU anticodon was a dominant identity element for all

tRNAMet as previously determined for E. coli MetRS.58 Our prior data indicates that

tRNAIle identity elements are established late and independently in different bacterial

groups. From the bacterial clade, the examined species include MetRS from the

proteobacterium Helicobacter pylori (HpMetRS), the GC-rich Mycobacterium smegmatis

(MsMetRS), the firmicute Streptococcus pneumoniae (SpMetRS1), and the opportunistic

pathogen Mycoplasma penetrans (MpMetRS). From the archaeal clade, the MetRS

selected are from Escherichia coli (EcMetRS), the spirochete Borrelia burgdorferi

(BbMetRS), and the obligate anaerobe Bacteroides fragilis (BfMetRS). The BbMetRS

exhibited only a 2-fold difference in aminoacylation efficiency between its cognate and

near-cognate transcripts, which is much lower than the others. For example, the BfMetRS

exhibited a 2000-fold difference. BbMRS is the only example of a nondiscriminating

MetRS identified in this earlier work. We remain interested in how B. burgdorferi

prevents mis-insertion of methionine at isoleucine codons. The use of the 3-70 base pair

is a strong determinant for many MetRSs.50 The tRNAIle

CAU and tRNAMet

CAU of B. burgdorferi

contain different 3-70 base pairs, and the 3-70 base pair in B. burgdorferi tRNAMet

CAU is

different from the other MetRSs selected. We decided to mutate the U3-A70 of B.

burgdorferi tRNAMet

CAU into C3-G70 in order to compare the activities to the wild-type

tRNAIle

CAU and tRNAMet

CAU of B. burgdorferi.

3.2 Results

3.2.1 Cloning and expression of B. burgdorferi MetRS

Plasmid pQE70-BbMetRS, which expresses the B. burgdorferi metS gene in front

26

of a C-terminal His6 tag, was constructed by a previous member in our lab. We sought to

overexpress BbMetRS from this plasmid in E. coli XL-10 Gold cells. Unfortunately, the

expression level is low (note minimal difference in expression following the IPTG

induction) (Figure 5). Also perhaps because of low expression, there is a small-size

protein that co-purifies with the B. burgdorferi MetRS upon Ni+ affinity chromatography

Figure 5. Expression and purification of B. burgdorferi MetRS with pQE70-

BbMetRS plasmid. A. FPLC trace of BbMetRS affinity purification; B. 10% SDS-

PAGE gel after FPLC. Lane 1, EcMetRS standard (64 kDa); lane 2-3, before and after

IPTG induction; lane 4-8, fractions after FPLC. C. 10% SDS-PAGE gel after

ammonium sulfate treatment. Lane 1, EcMetRS standard; lane 2-4, precipitation at

15%, 40% and 55% ammonium sulfate. (Expected BbMetRS size: 85 kDa.)

A

B C

1 2 3 4 5 6 7 8 1 2 3 4

64

kDa

85

kDa 64

kDa

85

kDa

27

(Figure 5B, lane 4-8). The protein impurity remained after ammonium sulfate treatment.

Different growing temperatures and different concentrations of IPTG were tried in an

attempt to increase expression and purity. Both E. coli M15 cells and E. coli SG13009

cells were used to express BbMetRS from the pQE70 vector, but these purification

problems still existed.

In order to increase protein expression and purity, we decided to subclone the B.

burgdorferi metS gene into a pET28a expression vector. After digestion of pQE70-

BbMetRS and pET28a with XhoI and

NdeI, both the metS insert and the

pET28a vector were isolated from an

agarose gel and ligated with T7 DNA

ligase. The ligation reaction was

transformed into E. coli Top10

competent cells and the resulting

plasmids were isolated. Agarose gel

electrophoresis indicated fragments of

the inserted gene (~2.9 Kb) and the

vector (~5.4 Kb), confirming successful

ligation (Figure 6).

The B. burgdorferi MetRS from the new pET28a-BbMetRS plasmid was tested

for expression and solubility (Figure 7). Several different kinds of cells were tried and

two representative attempts (NovaBlue cells and Arctic Cells) are shown (Figure 7). The

Figure 6. Agarose gel of pET28a-

BbMetRS plasmid. Lane 1, 10 KBp

DNA ladder; lane 2, plasmid without

digestion; lane 3, plasmid digested with

XhoI; lane 4, plasmid digested with

XhoI and NdeI.

1 2 3 4

28

expression of B. burgdorferi MetRS increased significantly, although after cell lysis, we

found that most of the desired protein was in the pellet instead of the supernatant. We

tried to use FPLC to purify the lysis, but nothing was obtained. Purification of BbMetRS

was not pursued further.

3.2.2. Synthesis and purification of B. burgdorferi tRNAMet variants

B. burgdorferi tRNAMet, tRNAMet U3G-A70C and tRNAIle were transcribed from

blunt duplex DNA and analyzed by denaturing polyacrylamide gel electrophoresis

(Figure 8A). The tRNAIle had two different sizes bands and the smaller one was at the

right size. A preparative 16 % denaturing polyacrylamide gel was used to purify the three

ethanol precipitated tRNA samples. They were analyzed by the polyacrylamide

Figure 7. Expression and solubility test of B. burgdorferi MetRS. A. Transformation

into NovaBlue cells. Lane 1, before induction; lane 2, after induction, lane3,

supernatant after cell lysis; lane 4, pellet after cell lysis. B. Transformation into Arctic

cells. Lane 1, before induction; lane 2, after induction, lane3, supernatant after cell

lysis; lane 4, pellet after cell lysis. Expected BbMetRS size is 85 kDa.

A B 1 2 3 4 1 2 3 4

Expected

BbMetRS

29

denaturing gel again (Figure 8B). Though only the smaller tRNAIle band was cut and

extracted from the big gel, the larger one still appeared on the gel.

3.3 Discussion

During all the work on this project, most of the time was spent on two challenges.

These are trying to find the optimal conditions to increase the expression and purity of

the B. burgdorferi MetRS. We tried different growing temperatures (10 ºC, 30 ºC, and 37

ºC), IPTG concentrations (0.1 mM, 0.25 mM, 0.5 mM, and 1 mM), cells types (NovaBlue

cells, Rosetta cells, and Arctic cells), lysis buffer (with or without Tween 20), induction

OD (0.5, 0.6, 0.8, 1, and 1.2 ), times (1 h, 3 h, 6 h, overnight, and 24 h) and temperatures

(10 ºC, 16 ºC, 30 ºC, and 37 ºC). None of these conditions contributed to improved

protein expression level or protein solubility. We thought the best way to increase the

Figure 8. Polyacrylamide gel electrophoresis of tRNA samples. A. tRNA transcripts

before purification. Lane 1, tRNAMet standard; lane 2, tRNAMet; lane 3, tRNAMet U3G-

A70C; lane 4, tRNAIle; B. tRNA transcripts after purification. Lane 1, tRNAMet; lane 2,

tRNAMet U3G-A70C; lane 3, tRNAIle.

1 2 3 4 1 2 3

A B

30

protein expression was to use a better expression vector, for example pET28a, for which

we needed to subclone to the B. burgdorferi metS gene. After many ligation attempts in

which the vector religated itself, we did succeed in generating the pET28a/BbMetRS

plasmid by using the QIAquick gel extraction kit (Qiagen) to isolate and purify the

fragments of insert gene and vector from the agarose gel. Unfortunately, although the

expression of BbMetRS increased, initial efforts indicated that the protein was insoluble

and this project was set aside.

31

Chapter 4

Experimental analysis of E. coli methionyl-tRNA synthetase β-linker length

4.1 Introduction

The β-strands composed of residues 118-129 and 182-192 resemble a “stalk” that

links the zinc binding knuckle to the body of MetRS. An earlier mutagenesis study

demonstrated a functional connection between these regions, since mutations in the zinc

biding motif decrease tRNA aminoacylation efficiency.48 This activity loss may suggest

direct interactions between the zinc binding motif and the tRNA acceptor stem and the

importance of the β-strands in the tRNA aminoacylation. What is not known is wheather

the zinc cation directly stabilizes acceptor stem binding or if there is another structural or

functional role for this motif.

The structure of the linker is known to be primarily antiparallel β-strands (Figure

9A), and we sought to shorten or lengthen the strands by one residue on each strand

without disrupting the rest of the protein structure.51 We considered small glycine or

alanine residues; glycine residues may cause too much flexibility, so alanine was chosen

as the insertion residue to lengthen the β-linker. Alanine residues were initially inserted

after residues Lys-127 and Pro-182. The resulting variant (+128A/184A) has alanine at

positions 128 and 184 (Figure 9B). These inserted residues are located near the end of the

β-linker which may change the structure of the β-linker instead of just changing its

length. In order to avoid that, we designed another addition mutant in which the inserted

residues are located in the middle of the β-linker. For the second addition mutant, two

alanine residues were inserted after residues Trp-123 and Ser-187 (variant named

32

+124A/188A) (Figure 9C). To shorten the β-linker, Thr-123 and Ser-187 were deleted

(Figure 9D). This variant we called ΔT123/S187.

Wild-type MetRS and linker variants were overexpressed in E. coli and purified

by affinity chromatography. The proteins were monitored by circular dichroism

spectroscopy. Enzyme activities were measured using an aminoacylation assay with 35S-

radiolabeled methionine and a pyrophosphate exchange assay with 32P-radiolabeled PPi.

Activity levels for each variant were compared to wild-type E. coli MetRS.

Figure 9. E. coli MetRS β-linker motif. A. Wild-type E. coli MetRS, B:

+128A/183A insertion variant, C. +124A/188A insertion variant, D. ΔT123/S187

deletion variant. The β-linker is colored red and location of deletions or insertions are

colored yellow.

A B

C D

33

4.2 Results

4.2.1 Production of E. coli MetRS linker variants

We designed variants of the CP domain β-linker by inserting or deleting residues

in each β strand of the linker. Nucleotide substitutions were made in His6-MetRS using

the QuikChange mutagenesis strategy (Invitrogen) on the pET28-cloned MetRS

Figure 10. Protein purification of E. coli MetRS +124A/188A variant. A. FPLC

trace of EcMetRS affinity purification (1mL His-Trap column; 2 mL/min flow rate).

Red: absorbance at 280 nm; Blue: percentage of buffer B (50 mM Tris·HCl (pH 7.5),

500 mM NaCl and 500 mM imidazole) used. B. 10 % SDS-PAGE gel analysis. Lane

1-2, before and after induction; lane 3, cell lysate before purification; lane 4-6, FPLC

fraction #10-12; lane 7, Kaleidoscope protein standards (Bio-Rad).

A

B 1 2 3 4 5 6 7 kDa

100

75

50

37

25

20

34

monomer plasmid (pSW101). All mutations were sequence verified (Genewiz, Inc.).

Wild-type and variants were expressed in E. coli chemically competent strains Rosetta-2

or NovaBlue (DE3). The His6-tag on the protein N-terminus enabled purification by Ni-

affinity chromatography. All the proteins were purified from the cell lysate with a yield

approximating that of

the wild-type MetRS

monomer. This

observation suggested

that the substitutions

introduced didn’t cause

significant instabilities

which would lead to

protein degradation or

aggregation. A

representative FPLC trace and SDS-PAGE for purification of the +124A/188A variant is

shown (Figure 10). Wild-type MetRS and all the variants were analyzed by SDS-PAGE

(Figure 11).

4.2.2 Aminoacylation activity of linker variants

To determine the effect of the β-linker substitutions on E. coli MetRS activity, we

used the established TCA filter assay for tRNA aminoacylation.59 This assay measures

the overall extent of tRNA aminoacylation, including both aminoacyl adenylate

formation and amino acid transfer. From this assay, we can compare the aminoacylation

Figure 11. 10 % SDS-PAGE of the wild-type E. coli

MetRS and variants. Lane 1, Kaleidoscope protein

standards (Bio-Rad); lane 2, wild-type MetRS; lane 3,

ΔT123/S187 variant; lane 4, +128A/183A variant; lane

5, +124A/188A variant.

. 1 2 3 4 5 kDa

100

75

50

37

25

35

activities of each variant to the wild-type enzyme.

The E. coli tRNAMet

used in this assay was

generated by in vitro

transcription and gel

purified, as described in

Chapter 2. In order to

determine the percentage of

tRNAMet able to be

aminoacylated, a “plateau”

aminoacylation assay was

performed using an excess

of wild-type MetRS (10

μM) compared to tRNAMet (2 μM). Each reaction aliquot (5 μL) contained 10 pmol

tRNAMet so the aminoacylation capacity is about 80 % (Figure 12). This represents a high

quality tRNAMet sample, which was used as the substrate for the rest of the

aminoacylation assays described here.

Each MetRS variant with altered β-linker length appeared to decrease the

aminoacylation of tRNAMet compared to the wild-type MetRS, whether the linker was

lengthened or shortened (Figure 13). Surprisingly, the variant with a shorter linker was

least affected, while the addition variants were affected more. Enzyme efficiency

(kcat/KM) can be approximated from the slopes of the linear portions of the activity curves.

This suggests that the deletion variant ΔT123/S187 retains 61% of wild-type activity,

Figure 12. Aminoacylation of tRNA by wild-type

MetRS. This “plateau” assay contained 2 μM

tRNAMet and 10 μM E. coli MetRS in standard

aminoacylation buffer. The reaction was carried out

at 25 ºC.

36

while the addition variants +124A/188A and +128A/183A retain 31% and 6% of wild-

type activity, respectively.

As the aminoacylation assay measures the overall reaction including adenylate

formation and amino acid transfer, we wanted to know at what step the defect occurred.

We therefore used a pyrophosphate exchange assay to measure the methionine activation

activity of the enzyme variants in the absence of tRNA.

Figure 13. Aminoacylation of tRNAMet by wild-type E. coli MetRS and β-linker

variants. Inset shows +128A/183A variant aminoacylation activity. Reactions are

carried out in the aminoacylation assay buffer, as described in Chapter 2, with 3 μM

tRNAMet and 100 nm each enzyme. Error bars represent the standard deviation from at

least 4 trials.

Wild-type

ΔT123/S187 variant

+124A/188A variant

+128A/183A variant

+128A/183A variant

Time (sec)

[35S

] M

et-t

RN

AM

et (

pm

ol)

37

4.2.3 Methionyl adenylate synthesis activity of linker variants

To determine the methionine activation activity of the each variant and the wild-

type E. coli MetRS, we used the established pyrophosphate exchange assay.60 In this

assay, formation of methionyl adenylate is measured by an ATP-pyrophosphate exchange

reaction in which the adenylate synthesis in the absence of tRNA permits exchange of the

32P label between pyrophosphate and ATP. This assay essentially measures the back

reaction of the 32P label from pyrophosphate into ATP.

Figure 14. Methionyl adenylate formation by wild-type E. coli MetRS and β-

linker variants. Inset shows +128A/183A variant adenylate activity. Reactions are

carried out in ATP-PPi exchange assay buffer, as described in Chapter 2, using 100

nm each enzyme. Error bars represent the standard deviation from at least 4 trials.

+128A/183A variant

+124A/188A variant

ΔT123/S187 variant

Wild-type +128A/183A variant

Time (min)

[32P

] A

TP

(n

mo

l)

38

All the MetRS mutants with altered β-linker lengths showed a decrease in

methionine activation activity compared to wild-type MetRS (Figure 14). Only the

addition variant +128A/183A, however, exhibited significantly reduced activity

(approximately 5% of wild-type MetRS). The addition variant +124A/188A and deletion

variant ΔT123/S187 were only modestly reduced in adenylate formation (70% and 76%

of wild-type activity, respectively).

4.2.4 Circular dichroism spectroscopy of linker variants

In order to determine any impact on protein structure from these substitutions,

wild-type E. coli MetRS and all the variants were analyzed by circular dichroism (CD)

Figure 15. Circular dichroism spectroscopy of wild-type E. coli MetRS and the

variants. Blue: wild-type MetRS; green: ΔT123/S187 variant; red: +128A/183A

variant; pink: +124A/188A variant. Enzyme samples (500 µL of 1 µM) were prepared

in 10 mM phosphate buffer (pH 7.4). The parameters for the experiment were

configured to measure the wavelength by every nanometer from 190 nm to 250 nm.

The five runs for each sample were averaged and the average blank spectrum was

subtracted.

39

spectroscopy. This approach is a tool for rapid determination of the secondary structure

and folding properties of proteins, although the specific location of any changes in

structure are not identified.61 All the proteins exhibit nearly identical CD spectra,

indicating that they are of very similar structure (Figure 15). The spectra are

characteristic of a highly α-helical protein. The small discrepancies between these curves

are likely due to small differences in protein concentration. The overall structures of these

enzymes were not significantly affected by deletion or addition of residues on the β-

linker.

4.3 Discussion

In the absence of a high resolution structure showing how the tRNAMet acceptor

stem interacts with the MetRS active site, the function of the Zn2+ binding knuckle

remains unclear. The experiments described here demonstrate that the length of the β-

linker is important for both methionyl adenylate synthesis and amino acid transfer.

Each of three E. coli MetRS variants showed a decrease in the total tRNAMet

aminoacylation activity compared to wild-type MetRS, with the +128A/183A variant

most reduced. For methionyl adenylate synthesis, the +128A/183A variant was greatly

reduced but the other two exhibited near wild-type activity. For the ΔT123/S187 variant,

the activity ratio was similar between these two functions (61% and 76%), which

suggested that the decrease in tRNAMet aminoacylation activity was due to the decrease in

methionine activation. Similar results are observed for the +128A/183A variant, although

the residual activity was much lower (6% and 5%). The +124A/188A variant exhibited an

interesting activity profile. For this insertion, methionine activation was quite robust

40

(70% of wild-type activity) while tRNA aminoacylation was reduced to 31% of wild-type

activity. This finding suggested that this variant is defective in either tRNA binding or

transfer of activated methionine to the end of tRNAMet.

We can begin to rationalize these observations based on the E. coli MetRS

structure (Figure 9). Both the ΔT123/S187 variant and the +124A/188A variant had

deletion or insertions in the middle of the β-linker, while the +128A/183A variant had

amino acids inserted at the end of the β-linker connected to the zinc binding motif. Based

on these designs, our results suggest that the +128A/183A variant may not only change

the length of the β-linker but also change the structure of the zinc knuckle. The

observation that methionine activation of the +128A/183A variant was also much lower

than the others is consistent with a local structure defect.

Any structural defect is likely to be subtle since the circular dichroism

spectroscopy indicated that the overall structures of these variants were not significantly

affected by residues deleted or added into the β-linker. Despite the near identical CD

spectra, we have previously shown that even minor local structural changes can impact

MetRS function drastically.

In general, the length of the β-linker does affect MetRS function at both adenylate

formation and overall tRNAMet aminoacylation. We don’t yet know, however, whether

tRNAMet binding or methionine transfer (or both) is affected.

41

Chapter 5

Conclusion and discussion

In project I, both the B. burgdorferi tRNAMet and tRNAMet U3G-A70C were

successfully purified. The B. burgdorferi tRNAIle would need to be further purified in any

future work. The pQE70-BbMetRS plasmid and the pET28a-BbMetRs plasmid have

been constructed and their sequences are confirmed. In future work, BbMetRS protein

expression and purification needs to be optimized. A solution to the co-purification

problem using the pQE70-BbMetRS plasmid or the solution to solubility problem using

the pET28a-BbMetRs plasmid should be found. After all the enzymes and tRNAs are

obtained, the activities between all the three tRNAs can be compared. A careful analysis

of tRNA aminoacylation activities will help reveal how B. burgdorferi prevents mis-

insertion of methionine at isoleucine codons.

In project II, all the variants with altered β-linker length decreased the methionine

activation activities and the tRNAMet aminoacylation activities. For the variant located at

the end instead of in the middle of the β-linker, the decreases were much more

significant. The decrease of the tRNAMet aminoacylation activity was affected by the

decrease of the methionine activation activity for the variant with the shorter β-linker.

The most interesting variant is the +124A/188A insertion, which retained high adenylate

formation but exhibited low aminoacylation. In future work, kinetic assays are needed to

measure kcat and KM values of the methionine activation and tRNAMet aminoacylation

reactions. With the kinetic assays, more accurate and reliable data will be measured to

understand the role of the linker region in MetRS activity. Furthermore, more variants

altering the length of the β-linker could be informative to confirm the findings here. More

42

variants in the middle of the β-linker with different lengths will be helpful to the find

under which conditions the second step of the reaction will be affected. In order to

determine whether the altered function is due to tRNAMet binding or transfer,

fluorescence quenching can be used to measure the tRNAMet binding affinity and pre-

steady state kinetics can be used to determine the ktrans, the single turnover transfer rate

constant. Furthermore, molecular dynamics simulations of these variants will be used to

compare positioning of the zinc ion compared to wild-type MetRS. All these further

experiments should show that the preliminary studies described here have demonstrated

an effect of the β-linker length and the position of the zinc cation on E. coli MetRS

catalytic activity.

43

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48

Appendix

E. coli methionyl–tRNA synthetase

TQVAKKILVTCALPYANGSIHLGHMLEHIQADVWVRYQRMRGHEVNFICADDA

HGTPIMLKAQQLGITPEQMIGEMSQEHQTDFAGFNISYDNYHSTHSEENRQLSELI

YSRLKENGFIKNRTISQLYDPEKGMFLPDRFVKGTCPKCKSPDQYGDNCEVCGA

TYSPTELIEPKSVVSGATPVMRDSEHFFFDLPSFSEMLQAWTRSGALQEQVANK

MQEWFESGLQQWDISRDAPYFGFEIPNAPGKYFYVWLDAPIGYMGSFKNLCDK

RGDSVSFDEYWKKDSTAELYHFIGKDIVYFHSLFWPAMLEGSNFRKPSNLFVHG

YVTVNGAKMSKSRGTFIKASTWLNHFDADSLRYYYTAKLSSRIDDIDLNLEDFV

QRVNADIVNKVVNLASRNAGFINKRFDGVLASELADPALYKTFTDAAEVIGEAW

ESREFGKAVREIMALADLANRYVDEQAPWVVAKQAGRDADLQAICSMGINLFR

VLMTYLKPVLPKLTERAEAFLNTELTWDGIQQPLLGHKVNPFKALYNRIDMRQV

EALVEASKEEVK

B. burgdorferi methionyl–tRNA synthetase

MKKMNLVTAALPYVNNIPHLGNLVQVLSADAFARYSKMSGIETLYVCGTDEYG

TATETKALIENTTPLELCNKYYEIHKSIYKWFNIEFDIFGRTTNKNHQDIVQNFFLQ

LEKNGYIKERETEQFYCNKDSMFLADRYVIGECPECQSMAKGDQCDNCSKLLNP

TDLINPKCIICKNKPILKKTNHLYLDLPKIKTKLEKWIKNPDTSKNWNTNALKMT

KAFLRDGLKERAITRDLKWGIPVPKKGFENKVFYVWFDAPIGYISITKNIIKNWES

WWKNNDQVNLVQFIGKDNILFHTIIFPCIEIGSEENWTILNQLSSSEYLNYENLKFS

KSEGTGIFGNDAITTGIPSDIWRFYIYYNRPEKSDFQFMWQDLMERVNTELIDNFS

NLVNRVLTFQRKFFGDVIETIEIQNKFWKQITPKYNKILNLFKKTELKSALKEILKI

SSLGNKIFQDNEPWKRKNNSPQETKELISNLIYLIRDLSILMMPFIPETSKKIQQFFG

NSYQFSTKILGTKSGIKKIEFTEILFNKLEQKKINNLKLKYSGDKNMKENEQAENL

PIAKEQPENLFREKVLLRVVKINKIERNPEAKNLFILKLDDGTNKDKQIVSGLEGY

YTEEELLGKHIIIVDNLKPAKFRGIKSEGMLIAAEDKNKNFKVIIVEDSIQNPIAGE

RIILENDQNKDLACPPKIDINKFLKANIVAENGELKINGINLILENSKNKILSKDIPN

GTVC

Primer sequences

Addition of Alanine 128

Forward 5 – CGCACCATCTCTCAGCTGGCATACGATTCCGGAAAAAG – 3

Reverse 5 – CTTTTTCCGGATCGTATGCCAGCTGGAGATGGTGCG – 3

Addition of Alanine 183

Forward 5 – CTGGCGTACGCCGGCAGTAATGCGTGATTCTG – 3

49

Reverse 5 – CAGAATCACGCATTACTGCCGGCGTAGCGCCAG – 3

Deletion of Tyrosine 123

Forward 5 – CGGTTTTATTAAAAACCGCATCTCTCAGCTGTACGATCCGG – 3

Reverse 5 – CCGGATCGTACAGCTGAGAGATACGGTTTTTAATAAAACCG – 3

Deletion of Serine 187

Forward 5 – GCTACGCCGGTAATGCGTGATGAACACTTCTTCTTTGATCTG – 3

Reverse 5 – CAGATCAAAGAAGAAGTGTTCATCACGCATTACCGGCGTAGC – 3

Addition of Alanine 124

Forward 5 – GTTTTATTAAAAACCGCACCAGCATCTCTCAGCTGTACGATCCGG

– 3

Reverse 5 – CCGGATCGTACAGCTGAGAGATGCTGGTGCGGTTTTTAATAAAAC

– 3

Addition of Alanine 188

Forward 5 – ACGCCGGTAATGCGTGATTCTGCAGAACACTTCTTCTTTGATCTG

– 3

Reverse 5 – CAGATCAAAGAAGAAGTGTTCTGCAGAATCACGCATTACCGGCGT

– 3

B. burgdorferi tRNAMet

5 – AATTCCTGCAGTAATACGACTCACTATAGCTGATGTAGCTCAGTTGGTTAG

AGCACTCGGC TCATATCCGAGTTGTCGTGGG – 3

5 – TGGTGCTGATGGAGGGACTTGAACCCACGACAACTCGGATATG – 3

B. burgdorferi tRNAMet U3G–A70C

5 – AATTCCTGCAGTAATACGACTCACTATAGCCGATGTAGCTCAGTTGGTTA

GAGCACTCGGC TCATATCCGAGTTGTCGTGGG – 3

5 – TGGTGCCGATGGAGGGACTTGAACCCACGACAACTCGGATATG – 3

B. burgdorferi tRNAIle

5 – AATTCCTGCAGTAATACGACTCACTATAGGGCCCATAGCTCAGTTGGTTA

GAGCACCCGAC TCATAATCGGTAGGTCCCAGG – 3

5 – TGGTGGGCCCACCAGGACTTGAACCTGGGACCTACCGATTATG – 3

Sequencing results

A124/A188 addition variant

50

NTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATC

ATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGACTCAAGTCGC

GAAGAAAATTCTGGTGACGTGCGCACTGCCGTACGCTAACGGCTCAATCCAC

CTCGGCCACATGCTGGAGCACATCCAGGCTGATGTCTGGGTCCGTTACCAGC

GAATGCGCGGCCACGAGGTCAACTTCATCTGCGCCGACGATGCCCACGGTAC

ACCGATCATGCTGAAAGCTCAGCAGCTTGGTATCACCCCGGAGCAGATGATT

GGCGAAATGAGTCAGGAGCATCAGACTGATTTCGCAGGCTTTAACATCAGCT

ATGACAACTATCACTCGACGCACAGCGAAGAGAACCGCCAGTTGTCAGAACT

TATCTACTCTCGCCTGAAAGAAAACGGTTTTATTAAAAACCGCACCAGCATCT

CTCAGCTGTACGATCCGGAAAAAGGCATGTTCCTGCCGGACCGTTTTGTGAA

AGGCACCTGCCCGAAATGTAAATCCCCGGATCAATACGGCGATAACTGCGAA

GTCTGCGGCGCGACCTACAGCCCGACTGAACTGATCGAGCCGAAATCGGTGG

TTTCTGGCGCTACGCCGGTAATGCGTGATTCTGCAGAACACTTCTTCTTTGAT

CTGCCCTCTTTCAGCGAAATGTTGCAGGCATGGACCCGCAGCGGTGCGTTGC

AGGAGCAGGTGGCAAATAAAATGCAGGAGTGGTTTGAATCTGGCCTGCAACA

GTGGGATATCTCCCGCGACGCCCCTTACTTCGGTTTTGAAATTCCGAACGCGC

CGGGCAAATATTTCTACGTCTGGCTGGACGCACCGATTGGNTACATGGGTTCT

TTCAAGAATCTGTGCGACAAGCGCGGCGACAGCGTAAGCTTCGATGANACTG

GAAGAAAGANTNCACCGCCGAGCTGTACCACTTCATCGGTAAAGATATGTTT

ACTTCNCAGCCTN

A128/A183 addition variant

NGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGG

CAGCCATATGACTCAAGTCGCGAAGAAAATTCTGGTGACGTGCGCACTGCCG

TACGCTAACGGCTCAATCCACCTCGGCCACATGCTGGAGCACATCCAGGCTG

ATGTCTGGGTCCGTTACCAGCGAATGCGCGGCCACGAGGTCAACTTCATCTG

CGCCGACGATGCCCACGGTACACCGATCATGCTGAAAGCTCAGCAGCTTGGT

ATCACCCCGGAGCAGATGATTGGCGAAATGAGTCAGGAGCATCAGACTGATT

TCGCAGGCTTTAACATCAGCTATGACAACTATCACTCGACGCACAGCGAAGA

GAACCGCCAGTTGTCAGAACTTATCTACTCTCGCCTGAAAGAAAACGGTTTTA

TTAAAAACCCCAGCATCTCTCAGCTGGCATACGATCCGGAAAAAGGCATGTT

CCTGCCGGACCGTTTTGTGAAAGGCACCTGCCCGAAATGTAAATCCCCGGAT

CAATACGGCGATAACTGCGAAGTCTGCGGCGCGACCTACAGCCCGACTGAAC

TGATCGAGCCGAAATCGGTGGTTTCTGGCGCTACGCCGGCAGTAATGCGTGA

TTCTGAACACTTCTTCTTTGATCTGCCCTCTTTCAGCGAAATGTTGCAGGCATG

GACCCGCAGCGGTGCGTTGCAGGAGCAGGTGGCAAATAAAATGCAGGAGTG

GTTTGAATCTGGCCTGCAACAGTGGGATATCTCCCGCGACGCCCCTTACTTCG

GTTTTGAAATTCCGAACGCGCCGGGCAAATATTTCTACGTCTGGCTGGACGCA

CCGATTGGNTACATGGGTTCTTTCAAGAATCTGTGCGACAAGCGCGGCGACA

GCGTAAGCTTCGATGANACTGGAAGAAAGANTNCACCGCCGAGCTGTACCAC

TTCATCGGTAAAGATATGTTTACN

T123/S187 deletion variant

NAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGC

51

GGCCTGGTGCCGCGCGGCAGCCATATGACTCAAGTCGCGAAGAAAATTCTGG

TGACGTGCGCACTGCCGTACGCTAACGGCTCAATCCACCTCGGCCACATGCT

GGAGCACATCCAGGCTGATGTCTGGGTCCGTTACCAGCGAATGCGCGGCCAC

GAGGTCAACTTCATCTGCGCCGACGATGCCCACGGTACACCGATCATGCTGA

AAGCTCAGCAGCTTGGTATCACCCCGGAGCAGATGATTGGCGAAATGAGTCA

GGAGCATCAGACTGATTTCGCAGGCTTTAACATCAGCTATGACAACTATCACT

CGACGCACAGCGAAGAGAACCGCCAGTTGTCAGAACTTATCTACTCTCGCCT

GAAAGAAAACGGTTTTATTAAAAACCCC___ATCTCTCAGCTGTACGATCCGG

AAAAAGGCATGTTCCTGCCGGACCGTTTTGTGAAAGGCACCTGCCCGAAATG

TAAATCCCCGGATCAATACGGCGATAACTGCGAAGTCTGCGGCGCGACCTAC

AGCCCGACTGAACTGATCGAGCCGAAATCGGTGGTTTCTGGCGCTACGCCGG

TAATGCGTGAT___GAACACTTCTTCTTTGATCTGCCCTCTTTCAGCGAAATGT

TGCAGGCATGGACCCGCAGCGGTGCGTTGCAGGAGCAGGTGGCAAATAAAA

TGCAGGAGTGGTTTGAATCTGGCCTGCAACAGTGGGATATCTCCCGCGACGC

CCCTTACTTCGGTTTTGAAATTCCGAACGCGCCGGGCAAATATTTCTACGTCT

GGCTGGACGCACCGATTGGNTACATGGGTTCTTTCAAGAATCTGTGCGACAA

GCGCGGCGACAGCGTAAGCTTCGATGANACTGGAAGAAAGANTNCACCGCC

GAGCTGTACCACTTCATCGGTAAAGATATGTTTACTTCNN

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Yiyuan Xia

Citizenship: P. R. China Birth: Apr. 6th 1989

E-mail: xiayy0406@hotmail.com Telephone: +86-17712600578

EDUCATION

08/2012-05/2015: Master of Science in Chemistry, Wake Forest University

09/2008-06/2012: Bachelor of Science in chemistry, minor in Finance, Nanjing

University

SKILLS

Bilingual in Chinese and English Oral and poster presentations

Bacterial DNA/RNA primer design, subcloning and site-directed mutagenesis;

RNA primer design and in vitro transcription; protein expression and purification

Radiation safety and radioactive assay (35S and 32P) operation

Mentoring undergraduates for their researches

Preparing and supervising undergraduate lab course

Hazard category of product and waste

Nano-materials synthesis methods, and electrode modification with nano-materials

Analyzing experimental data from AFM, XRD, TEM, SEM, NMR, IR, UV-VIS,

CD and ICP

Software application including MS Office, Origin, Sigmaplot, ChemBioDraw ApE,

SwissPdb, and Endnote

RESEARCH EXPERIENCE

05/2014-12/2014: Functional effect of alterations to Escherichia coli methionyl-

tRNA synthetase β-linker length Advisor: Dr. Rebecca Alexander

01/2013-07/2014: Experimental analysis of Borrelia. burgdorferi tRNAMet

discrimination Advisor: Dr. Rebecca Alexander

07/2011-06/2012: A Nonenzymatic Electrochemistry Senser Based on Noble Metal

Nano-particles/Graphene Modified Electrode Advisor: Dr. Xudong Jia

02/2011-07/2011: A Novel Biosensor Based on Gold Nanoflowers/Hemoglobin/

Carbon Nanotubes Modified Electrode Advisor: Dr. Xudong Jia

OTHER EXPERIENCE

2013-2014 Regulatory Compliance Analyst Intern in INMAR

2011-2012 Secretary of 2008 Grade Student Party Branch in School of Chemistry

and Chemical Engineering, Nanjing University

2010-2011 Executive Vice President of Student Union in School of Chemistry

and Chemical Engineering, Nanjing University

2008-2010 Leader of the Committee of Grade 2008 in School of Chemistry and

Chemical Engineering, Nanjing University

2009-2010 Caption of Basketball Team in School of Chemistry and Chemical

Engineering, Nanjing University

2006-2008 President of Mudu Senior High School in Jiangsu Province

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SCHOLARSHIP & AWARD

2012-2014 General Chemistry & Analytical Chemistry Teaching Assistant Award

in Chemistry Department, Wake Forest University

2011 Outstanding Student Cadre in Nanjing University; People's Scholarship

2010 Scholarship of Singapore-Suzhou Industrial Park; Outstanding Student in

Nanjing University; People's Scholarship for Special Skills of Social Work & Arts

and Sports

2009 Scholarship of Shanghai Hersbit Chemical Company; People's Scholarship

for Special Skills of Social Work & Arts and Sports

2008 Final of Host Competition in Nanjing University