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Species-Specific Differences in Recogniton of tRNA(Pro)
by Prolyl-tRNA Synthetases

A THESIS
SUBMITTED TO TH E FACULTY OF TH E GRADUATE S C H O O L
O F TH E UNIVERSITY OF MINNESOTA
BY

Brian William Burke

IN PARTIAL FULFILLMENT OF TH E REQUIREMENTS
FOR THE DEGREE O F
DOCTOR OF PHILOSOPHY

Karin Musier-Forsyth, A dvisor
May, 2001

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UMI Number: 3008671


Copyright 2001 by
Burke, Brian William
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© Brian W. Burke 2001

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UNIVERSITY O F MINNESOTA


This is to certify that I have exam ined this copy of a doctoral thesis by

Brian W illiam Burke

and have found that it is complete and satisfactory in all respects,
and th a t any and all revisions required by the final
exam ining com m ittee have been made.

Karin Musier-Forsyth
Name of Faculty Adviser

Signature o f Faculty Adviser

May 22, 2001
Date

GRADUATE SCHOOL

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Preface
The following works in this thesis have been previously published.
Chapter 2:
Catherine Stehlin, Brian Burke, Fan Yang, Hongjian Liu, Kiyotaka Shiba and Karin
Musier-Forsyth (1998) Species-Specific Differences in the Operational RNA Code for
Aminoacylation o f tR N A Pro. Biochem istry 37, 8605-8613.
©1998 American Chem ical Society
Chapter 3:

Brian Burke, Fan Yang, Fei Chen, Catherine Stehlin, Barden Chan and Karin MusierForsyth (2000) Evolutionary Co-Adaptation of the M otif 2-Acceptor Stem Interaction in
the Class II Prolyl-tRNA Synthetase System. B iochem istry 39, 15540-15547.
©2000 American Chem ical Society
Chapter 4:
Brian Burke, Richard S. Lipman, Kiyotaka Shiba, Karin Musier-Forsyth and Ya-Ming Hou
(2001) Divergent Adaptation of tRNA Recognition by Methanococcus jannaschii ProlyltRNA Synthetase. J. Biol. Chem. 27, in press.
©2001 The American Society fo r Biochemistry and M olecular Biology

i

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B n s Brake
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ThnvzsityafMm iicaon

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I a m * m in j id j e q a n t p n a u a a n t o m e th e fa Z k n asg m e m e r i p t ( a f w i a d i I m s a t
an th e r i n ia y d s n t t H s o ,w iA tb e B c d e x tn ^ c q g A n th e ie g iix n e d c o p y r ig h t c m d B t Sue
w iH a j x a rtw flx th e t a m gMi f l t t t t t o p c r m w k i n a o n ly Her flic m g a c a a f la w d e

Buxke; Bnaz,IkpmK&,Sk&«nlS^ Shiba. Kyoaka.Masscx-Eoayih, Karin, and Hmoc.Y*^£ng (2001) D Ivexgeig Adaptation o f tRXA Recognaioa by ife jkanooocem i
ftw n a td d i Prclyl-tRXA Syrffhrtmr. Io to n o i o fB io lo g ic a l C h em istry, in. pan a.

le a n t* reached by phone a t612-624-0510 and by FAX a t612-626-7541 or by e-na £Lat
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Acknowledgments
First o f all, I would like to thank m y advisor, Professor Karin Musier-Forsyth, for
her years o f support, assistance, and advice. She is an excellent exam ple of what an
advisor should be. She has pushed me to grow as a scientist and researcher with
encouragement all along. I am grateful that she gave me the opportunity to travel and
experience another culture with my several trips to Grenoble, som ething most graduate
students do not get the chance to do.
I would also like to thank my other committee members, Prof. Marian Stankovich,
Prof. Greg Connell, Prof. Edgar Arriaga, Prof. George Barany and Prof. Hal Swafford, for
their participation in my graduate career.

I am also grateful to many members of the KMF lab fo r making w ork much more
fun than would be expected. I thank them fo r their helpful discussions and materials
they have given me throughout the years. Dr. Catherine Stehlin taught me most of the
procedures I have carried out in lab and about the “blah-blah”. I am glad she was so
patient with a new student with no biological lab experience. Dr. Barden Chan, Dr. Tim
Stello, and Dr. Penny J. Beuning will always bring back fond memories. I can’t imagine
a better set of graduate students to learn from. They have done so m uch for me and I
hope I carried m yself as a mentor and example to the younger students as well as they
did for me. Barden, otherwise know as the Eatus alotus, was always a wealth of
information fo r me as well as a good source o f amusement. I thank Stello for not only
putting up with me in lab, but also as a roommate as well. Penny (that is HRH PJB) has
been a great labm ate as well as a good friend. I am thankful for all the oligos she has
had to make me since I don’t know how to use the synthesizer! I also w a n t to thank
Minh Hong fo r being such a good Bob. Special thanks also to Abbey Rosen for
iv

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preparation of oligos. I m ust also thank several technicians who have helped me on
projects throughout the years, Hongjian Liu, Fan Yang, Fei-Fei Chen, and Carmen
Silvers. I have also been fortunate to have som e great experiences with undergraduate
students who did a great deal o f w ork fo r me. Thank you Melissa Sue Walker, Jaime
Vaeth, and Cuong Pham. It was fun getting to know you.
I thank the basketball group fo r many years of fun and at least som e exercise. I
would also like to thank the other non-KM F people who have helped keep me sane,
including Tamara Kale (TK), Carrie Buss, and Dr. Michelle Douskey. Thanks T K fo r
always being a good friend and confidant and always being right next door. I thank
Carrie fo r being my friend, even if she does hide from me. Special thanks to my friend
and roomy Michelle fo r helping me through hard tim es and just being a good person. I

will miss you all.
I would like to thank my parents, Harlan and Sherry Burke, fo r all they have given
me. They are great parents and have always encouraged and believed in me. Thank
you so much. I would also like to specially thank my brothers, Keith and Dwight.
Finally, Valerie Frydrychowski has been my best friend and supporter. She is a
great person and I admire her fo r many reasons. She has given me encouragement and
love and has always believed in me. Thanks fo r being in my life.

v

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Table of Contents
Preface
Acknowledgments
Table of Contents
List of Tables and Figures
List of Abbreviations
Abstract

i
iv
vi
ix
xii
xv

1. Introduction
1.1. The Aminoacylation Reaction

1.2 Classes o f Aminoacyl-tRNA Synthetases
1.3 tRNA Structure
1.4 tRNA Recognition
1.5 Class II Aminoacyl-tRNA Synthetases
1.6 Prolyl-tRNA Synthetase
1.6.1 Prokaryotic-Like ProRS
1.6.2 Eukaryotic-Like ProRS
1.7 Conclusions
1.8 References

1
1
1
4
6
10
14
15
16
19
20

2. Species-Specific Differences in the Operational RNA Code for
Aminoacylation of tRNAPro
2.1 Introduction
2.2 Materials and Methods
2.2.1 Enzyme Purification and Activity Assays
2.2.2 RNA Preparation
2.2.3 Sequence Analysis
2.3 Results

2.3.1 Acceptor Stem and Anticodon Mutations
2.3.2 Cross-Species Aminoacylation by Human ProRS
2.3.3 Sequence Analyses
2.4 Discussion
2.4.1 Changes in tRNApro Recognition through Evolution
2.4.2 Class II Aminoacyl-tRNA Synthetase Evolution and Relationship
to Acceptor Stem Changes
2.5 Acknowledgments
2.6 References
3. Evolutionary Co-Adaptation of the Motif 2-Acceptor Stem Interaction
in the Class II Prolyl-tRNA Synthetase System
3.1 Introduction
3.2 Materials and Methods
3.2.1 Enzyme Purification and Site-Directed Mutagenesis o f ProRS
3.2.2 Activity Assays
3.2.3 RNA Preparation and Mutagenesis
3.2.4 Cross-Linking Procedure
vi

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31
31
34
34
34
34
35
36
38

41
45
45
48
51
51

57
57
60
60
60
61
61


3.3 R esults
3.3.1 M otif 2 Loop Mutations
3.3.2 E. c o li ProRS Cross-Linking Studies
3.3.3 A tom ic Group Mutagenesis
3.3.4 Cross-Species Aminoacylation by E. c o li ProRS
3.4 D iscussion
3.5 Acknow ledgm ents
3.6 R eferences
4. Divergent A daptation of tRNA Recognition by M ethanococcus
ja n n asch ii P rolyl-tR N A Synthetase
4.1 Introduction
4.2 M aterials and Methods
4.2.1 Preparation of M. jannaschiitR N A p'°
4.2.2 Preparation of Recombinant M. Jannaschii ProRS

4.2.3 Am inoacylation with Proline
4.2.4 Sequence Analysis
4.3 R esults and Discussion
4.3.1 M. jan n a sch ii ProRS is a “Eukaryotic-Like” Enzyme
4.3.2 Recognition of tRNA by M. jannaschii ProRS
4.3.3 Co-Adaptation of Synthetase Motifs with Changes o f tRNA
S pecificity Determinants
4.4 A cknow ledgm ents
4.5 R eferences

62
62
64
65
68
69
73
73

78
78
81
81
81
82
82
82
82
83
87

91
91

5. C haracterization of a Critical tRNAPro-Prolyl-tRNA Synthetase Interaction
via an 8-O xopu rine Cross-Linking Strategy and Am inoacylation Analysis
95
5.1 Introduction
95
5.2 C ross-Linking Strategy
96
5.3 Am inoacylation Analysis
97
5.4 M aterials and Methods
98
5.4.1 Enzym e Purification and Site-Directed M utagenesis of ProRS
98
5.4.2 A ctivity Assays
98
5.4.3 R N A Preparation
99
5.4.4 C ross-Linking Procedure
99
5.5 R esults and Discussion
100
5.5.1 M otif 2 Loop Mutations
100
5.5.2 E. c o li ProRS Cross-Linking Studies
101
5.5.3 Suppression Analysis
105

5.6 C onclusions
106
5.7 A cknow ledgm ents
107
5.8 R eferences
1 07
6. Further E fforts Towards the Elucidation of tR N A Pro Recognition
6.1 Introduction
6.2 Investigation o f the Motif 2 Loopof M. jannaschii ProRS
6.2.1 M . jan n a sch ii ProCysRS
6.2.2 M aterials and Methods
v ii

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112
112
112
112
113


6.3

6.4

6.5
6.6
6.7


6.2.2.1 Enzyme Purification and Site-Directed Mutagenesis
o f ProRS
6.2.2.2 Preparation of M. jannaschii tR N A Pro
6.2.2.3 Aminoacylation with Proline
6.2.3 Results and Discussion
E. coli ProRS Recognition of the tRNAPro Backbone
6.3.1 The Role o f Backbone Phosphate Oxygens in Nucleic Acids as
Investigated by Phosphorothioate Substitutions
6.3.2 Materials and Methods
6.3.2.1 RNA Preparation
6.3.2.2 Enzyme Preparation
6.3.2.3 Aminoacylation with Proline
6.3.3 Results and Discussion
Crystallization o f a Prokaryotic-Like ProRS
6.4.1 The Structure of ProRS
6.4.2 M aterials and Methods
6.4.2.1 RNA Preparation
6.4.2.2 Enzyme Purification
6.4.2.3 Crystallization of E. coli ProRS
6.4.3 Results and Discussion
Conclusions
Acknowledgm ents
References

v iii

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113
113


113
113
115

115
117
117
118
118
119

121
121
123
123
123
123
124
126
127
128


List of Tables and Figures
C hapter 1
Scheme 1.1. The aminoacylation reaction

2


Table 1.1. Two classes of am inoacyl-tRNA synthetases

3

Figure 1.1. Secondary and tertiary structure o f tRNA

5

Figure 1.2. Ribbon diagram of T. thermophilus ProRS

18

C hapter 2
Figure 2.1. Sequence of human tR N APr0 and variant transcripts

36

Table 2.1. The effect of single and multiple nucleotide changes on
aminoacylation of human tRNAPr0 transcripts by human ProRS

39

Figure 2.2. Chimeric E. coWhuman tRNAPro variants tested as substrates
for human ProRS

40

Figure 2.3. Alignment of tw enty prolyl-tRNA synthetases

42


Figure 2.4. Phylogenetic tree o f tw enty prolyl-tRNA synthetase sequences

43

C hapter 3
Figure 3.1. Sequence alignment comparing the motif 2 loop o f E. coli and
human ProRS

59

Table 3.1. The effect of single am ino acid changes in the m otif 2 loop o f
E. coli and human ProRS on aminoacylation of their cognate tR N A transcripts

63

Figure 3.2. Design and results o f cross-linking experiments using E. coli
ProRS variants

64

Figure 3.3. Structures of purine bases substituted at positions 73 and 72
of a semi-synthetic E. coli tRNAPro construct

66

Figure 3.4. Chimeric human/E. c o litR N APro variants tested as substrates
for E. coli ProRS

67


Figure 3.5. Proposed hydrogen bonding interaction between R144 and G72

72

ix

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Chapter 4
Figure 4.1. Schematic diagram o f the architecture of the tw o groups of ProRS

80

Figure 4.2. Sequence and cloverleaf structure of M. ja n n a s c h iitRNAPro

84

Figure 4.3. Comparison of a representative set of initial rates of
aminoacylation by M. ja n n a sch ii ProRS

85

Figure 4.4. The sequence and cloverleaf structures o f E. coli, human,
and M. jannaschii tRNAPro

87

Figure 4.5. Alignm ent o f the motif 2 region o f representative ProRS

sequences

89

Figure 4.6. Alignment o f the anticodon-binding region of representative
ProRS sequences

90

Chapter 5
Table 5.1. Effect of single amino acid changes at position 144 in the motif
2 loop of E. coli ProRS on aminoacylation of E. coli tRNAPro transcripts

100

Scheme 5.1. Proposed mechanism of 8-oxoG activation and cross-linking

101

Figure 5.1. E. coli ProRS cross-linking with 8-oxo-tRNAPr° variants

102

Figure 5.2. 8-oxoG72 tR N A Pro cross-linking with E. coli ProRS motif
2 loop variants

103

Figure 5.3. Cross-linking is inhibited by substrates and a substrate analog


105

Table 5.2. Effect of m otif 2 loop mutations of E. coli ProRS on aminoacylation
of tRNAPr0 acceptor stem mutants

106

Chapter 6
Table 6.1. The effect o f single amino acid changes in the m otif 2 loop of
M. jannaschii ProRS on aminoacylation of cognate tRNAPro transcripts

114

Figure 6.1. Comparison of phosphate versus phosphorothioate backbone
in nucleic acid

116

Figure 6.2. Results of phosphorothioate substitutions in E. c o litRNAPro

119

Figure 6.3. HPLC separation of the substituted A73 16-m er oligonucleotide
into its phosphorothioate components

120

x

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Figure 6.4. Crystals o f E. co li ProRS

124

xi

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List of Abbreviations
A
Hi
p.M
'P
2AP
8-oxoA
8-oxoG
aaRS
A
A, Ala
Af, A. fulgidus
ATP
BMH
C
C, Cys
Cam, C. albican
Ce, C. elegans
Ci

Co2+
Ct, C. trachomatis
CTP
dA
dG
D
D, Asp
Dm, D. meianogaster
DNA
DTT
eW T
E, Glu
Ec, E. coli
EDITH
EDTA
F, Phe
FPLC
G
G, Gly
G. lamblia
GMP
GTP
hr
hWT
H, His
HEPES

Angstrom
M icroliter
M icrom olar

Pseudouridine
2-aminopurine
7,8-dihydro-8-oxoadenine
7,8-dihydro-8-oxoguanine
Am inoacyl-tRNA synthetase
Adenosine
Alanine
Archaeoglobus fulgidus
Adenosine triphosphate
1,6-bismaleimidohexane
Cytosine
Cysteine
Candida albican (mitochondrial)
Caenorhabditis elegans
Curie
Cobalt
Chlamydia trachomatis
Cytosine triphosphate
Deoxyadenosine
Deoxyguanosine
Dihydrouridine
Aspartic acid
Drosophila m eianogaster
Deoxyribonucleic acid
Dithiothreitol
E. coli wild-type
Glutam ic acid
Escherichia coli
3-ethoxy-1,2,4-dithiazoline-5-one
Ethylenediaminetetraacetic acid

Phenylalanine
Fast Protein Liquid Chromatography
Guanosine
Glycine
Giardia lamblia
Guanosine monophosphate
Guanosine triphosphate
Hours
Human wild-type
Histidine
/V-(2-hydroxyethyl)-1 -piperazine-A/-2-ethane-sulfonic acid
x ii

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Hi, H. influenzae
HIV
Hp, H. pylori
HPLC
Hs, H. sapiens
I, lie
kDa
K, Lys
L, Leu
LPSA
mCi
ml
min
mM

mRNA
M, Met
M
Mg, M. genitalium
Mg2+, MgCI2
Mj, M. jannaschii
Mp, M. pneumoniae
MPD
Mt, M. tuberculosis
MTF
Mtt, M. thermoautotrophicum
nM
N
N, Asn
Ni2+
Ni-NTA
Ng, N. gonorrhoeae
NMR
P, Pro
PAGE
PCR
PEG
PPf
Q, Gin
R, Arg
RNA
s
S, Ser
Sc, S. cerevisiae
SDS

Scm, S. cerevisiae
Sec
Sp, S. pyogenes
Sy,

Haemophilus influenzae
Human Immunodeficiency Virus
Helicobacter pylori
High Performance Liquid Chromatography
Hom o sapiens
Isoleucine
Kilodalton
Lysine
Leucine
5’-0-[A/-(L-prolyl)-sulfamoyl]adenosine
Millicurie
Milliliter
Minutes
M iilimolar
Messenger ribonucleic acid
Methionine
M olar
Mycoplasma genitalium
Magnesium, Magnesium chloride
Methanococcus jannaschii
Mycoplasma pneumoniae
(±)-2-methyl-2,4-pentanediol
Mycobacterium tuberculosis
Methionyl-tRNA formyltransferase
Methanobacterium thermoautotrophicum

Nanomolar
Any nucleotide
Asparagine
Nickel
Nickel-Nitrilotriacetic acid
Neisseria gonorrhoeae
Nuclear Magnetic Resonance
Proline
Polyacrylamide Gel Electrophoresis
Polymerase Chain Reaction
Polyethyleneglycol
Pyrophosphate
Glutamine
Arginine
Ribonucleic acid
Seconds
Serine
Saccharomyces cerevisiae
Sodium dodecyl sulfate
Saccharomyces cerevisiae (mitochondrial)
Selenocysteine
Streptococcus pyogenes
Synechocystis spirochete PCC6803
x iii

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tRNA
T, Thr

T. therm ophilus
Tris
U
UTP
V, Val
W .T rp
WT, w t
Y, Tyr
X
Zm, Z. m oBilrs

Transfer ribonucleic acid
Threonine
Thermus thermophilus
Tris (hydroxymethyl) am inom ethane
Uracil
Uridine triphosphate
Valine
Tryptophan
W ild-type
Tyrosine
Any am ino acid
Zym om onas mobilts

x iv

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Abstract

Am inoacyl-tRNA synthetases (aaRSs) are a fam ily o f enzymes essential in the
process o f translation o f the genetic code. Class II prolyl-tR NA synthetase (ProRS) is
responsible fo r the specific attachment o f the amino acid proline onto tRNAPro. ProRSs
can be divided into two groupings based upon sequence analysis, a “prokaryotic-like”
and a “eukaryotic-like" group. We have explored recognition o f tRNAPro by three
ProRSs, representative of each of the three Domains o f life: Escherichia coli, human,
and Methanococcus Jannaschii.
Site-directed mutagenesis revealed that the eukaryotic-like ProRSs recognize
mainly the anticodon of their cognate tRNAs. This is in contrast to E. coli ProRS, which
strongly recognizes both the anticodon and acceptor stem of E. c o litRNAPro. Using
atomic group m utagenesis, we determined the critical functional groups in the acceptor
stem of E. coli tR N A Pro are the N7 of A73 and the 6-keto oxygen and N7 of G72.
The class ll-specific motif 2 loop interacts with the acceptor stem of cognate
tRNAs. Sequence analysis reveals evolutionary divergence in the motif 2 region of
ProRSs. We set out to determine if elements in the m otif 2 loop o f these ProRSs are in
close contact with th e ir cognate tRNAs. Site-directed m utagenesis of the motif 2 loop of
human ProRS has not supported direct interaction with tR N A Pro in a base-specific
manner. To date, a m oderate effect on aminoacylation by M. jannaschii ProRS has
been observed for a T135A mutation. In contrast, m utagenesis o f the motif 2 loop of E.
coli ProRS has revealed an arginine residue (R144) th a t is critical for efficient
aminoacylation. Based upon these results, we propose that R144 recognizes the m ajor
groove functional groups of G72 in E co li tRNAPro. The proxim ity of the acceptor stem of
E. coli tRNAPro and R144 of E. coli ProRS was verified by two methods of cross-linking,
xv

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including a novel method involving 8-oxopurine nucleotides not previously reported for
RNA-protein cross-linking.

Taken together, our results have revealed species-specific differences in tRNAPro
recognition by ProRSs. Specifically, the different modes of acceptor stem recognition
between the two groupings o f ProRS illustrate how synthetases and tRNAs have co­
adapted through evolution.

xvi

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1. Introduction
Deoxyribonucleic acid o r DNA encodes the necessary information fo r cells to
carry out all life functions. Ribonucleic acid o r RNA is chemically sim ilar to DNA except
that it contains a hydroxyl group at the 2'-position of the ribose sugar. RNA participates
in many critical functions in the cell and in doing so, is almost always complexed to
proteins. Compared to DNA-protein interactions, less is known about RNA-protein
interactions. Transfer RNA (tRNA) participates in protein synthesis by taking amino
acids to the ribosome to be added to the growing polypeptide chain. Amino acids are
covalently attached to tRNAs by aminoacyl-tRNA synthetases (aaRSs). The interactions
between tRNAs and aaRSs are highly specific and make excellent model systems for
learning more about RNA-protein interactions. This chapter will review what is known
about the structures of tRNA and aaRSs as well as tRNA recogniton, with specific
emphasis on the proline system.
1.1 The Aminoacylation Reaction
The fidelity of protein translation depends upon accurate recognition between
aaRSs and their cognate tRNA molecules. The aaRSs catalyze the esterification of
amino acids to their corresponding tRNAs via a two step reaction (Scheme 1.1) (7). In
the first reaction, the amino acid is ATP-activated to form an enzyme-bound aminoacyladenylate intermediate. In the following step, the amino acid is covalently attached tothe
3'-end of the tRNA molecule, forming an aminoacyl-tRNA or “charged” tRNA that can
then proceed to the ribosome to participate in protein synthesis.

1.2 Classes of Aminoacyl-tRNA Synthetases

1

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Although aaRSs carry out a common function, this family o f enzym es is quite
diverse in sequence, quartenary structure and size. The twenty aaRSs are divided into

1

nh

ATP

*

O

H

2

PPi
i
-P -O -i

^ -


i.

H

O

„

V

H

?

OH

proline

l

OH

aminoacyl-adenylate

2
nh

/

2


NH2
tRNA-CCA-OH

*

tR N A -C C -p — O - i

C N> ^ o- r o w
H
°r"T
OH

>

N

AMP

'

„

N

N

^

i.


O

OH

OH

H

aminoacyl-adenylate

aminoacyl-tRNA

Scheme 1.1. The am inoacylation reaction as illustrated with the am ino acid
proline.
two classes with ten members each based on characteristic structural dom ains as
determined from prim ary sequence alignments (Table 1.1) (2 , 3). The two classes are
also divided into subgroups based mainly upon anticodon binding dom ains (4, 5). Class
I aaRSs are usually m onom eric, aminoacylate the 2'-OH of A76 of th e ir cognate tRNAs
and contain the characteristic peptide sequences HIGH (His-lle-Gly-His) and KMSKS
(Lys-Met-Ser-Lys-Ser), which form a Rossmann nucleotide binding fold active site (6).
The Rossmann nucleotide-binding fold consists of a five-stranded parallel (3-sheet
situated between two pairs o f a-helices, but is not unique to aaRSs as it has been
observed in other proteins. The function of the Rossmann fold is prim arily to bind ATP

2

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but not the tRNA as class I enzymes have a separate domain w hich interacts with the
m inor groove of the tRNA acceptor stem.
Class II enzymes are generally larger than class I enzymes and form dimers or
tetramers. Class II synthetases attach amino acids to the 3'-OH o f A76 (except for
phenylalanyl-tRNA synthetase (PheRS)). The class II enzymes contain an antiparallel 13-

Table 1.1 Two Classes of Aminoacyl-tRNA Synthetases
Class 1
Rossmann fold
HIGH and KMSKS motifs
aminoacylate at 2'-OH
Subclass la
cysteine
isoleucine
leucine
valine
arginine
methionine

Quartenary
Structure

oc
cc
oc
a
oc

Class II
antiparallel (3-sheet

Motif 1, 2, and 3
aminoacylate a t 3'-OH
Subclass lla
serine
proline
threonine
histidine
glycine
alanine

oc2
Subclass lb
glutamic acid
glutamine
lysine3
Subclass Ic
tryptophan
tyrosine

cc
a
cc
CC,

Quartenary
Structure

OC,

oc,

OC,

cc2, ( cc(3)2
cc,cc4

Subclass lib
aspartic acid
asparagine
lysine

cc,
oc,
cc,

Subclass lie
phenylalanine13

( a p ).

CC2

aAlthough most LysRS are class II, class I LysRS has been found in some
archaebacteria and a small num ber of eubacteria. bPheRS am inoacylates at the
2'-OH.

sheet catalytic domain, which is larger than the Rossmann fold o f class I enzymes.
Unlike the Rossmann fold, the class II (3 -sheet catalytic domain binds the tRNA CCA-3'end and contains three structural motifs known as motifs 1, 2, and 3. M otif 1 is involved
3

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