COEVOLUTION OF RIBOSOMES AND THE TRANSLATIONAL APPARATUS: THE STRUCTURE

148 Pages·2015·3.72 MB·English

COEVOLUTION OF RIBOSOMES AND THE TRANSLATIONAL
APPARATUS: THE STRUCTURE AND FUNCTION OF EUKARYOTIC
RIBOSOMAL PROTEIN uS7 FROM YEAST, Saccharomyces cerevisiae.

ARNAB GHOSH

Bachelor of Science in Microbiology
University of Calcutta
June 2003

Master of Science in Biotechnology
Utkal University
July 2005

Submitted in partial fulfillment of requirement for the degree
DOCTOR OF PHILOSOPHY IN REGULATORY BIOLOGY
at
CLEVELAND STATE UNIVERSITY
June 2015
We hereby approve this dissertation for

Arnab Ghosh

Candidate for the Doctor of Philosophy in Regulatory Biology degree for the

Department of Biological, Geological and Environmental Sciences

and the CLEVELAND STATE UNIVERSITY

College of Graduate Studies

_____________________________________________________________
Dissertation Chairperson, Dr. Anton A. Komar
_____________________________________________
Department & Date

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Dissertation Committee Member, Dr. Barsanjit Mazumder
_____________________________________________
Department & Date

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Dissertation Committee Member, Dr. G. Valentin Börner
_____________________________________________
Department & Date

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Dissertation Committee Member, Dr. Andrea N. Ladd
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Department & Date

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Dissertation Committee Member, Dr. Girish C. Shukla
_____________________________________________
Department & Date

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Dissertation Committee Member, Dr. Derek Taylor
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Department & Date

Student‟s Date of Defense: February 26, 2015
Dedicated to those I am most connected to: mom and dad and
mou, caffeine and sugar, and earth: three by DNA, two by
biochemistry, and one by biology. All with pleasure and love.
ACKNOWLEDGEMENTS

Firstly, I would like to express my heartiest thankfulness and gratitude
to Dr. Anton A. Komar for being my graduate advisor, giving me the
opportunity to work and his continued guidance. I would like to thank him for
his encouragement and patience, even when things were not going great. He
taught me many things, which I believe, made me a better student.
I thank Dr. G. Valentine Bӧrner, Dr. Barsanjit Mazumder and Dr. Andrea
N. Ladd for being a part of my advisory committee and also for their valuable
time, suggestions and criticisms, which significantly improved my thesis work;
Dr. Girish C. Shukla and Dr. Derek Taylor for agreeing to be a part of my
dissertation defense committee and all the faculty member of the department
and graduate program for their help as needed. I would like to express my
sincere gratitude and very special thanks to Dr. Ralph Gibson for his
numerous help and guidance whenever I needed them as a teaching
assistant.
I take this opportunity to acknowledge the help and support I received
from my lab mates Amber Bentley, Sujata Jha, Rita Rozmarynowycz, and
others during the years. Also thanks to new members of the lab, Supriya,
Amra and Puja. I would like to thank all the students, post docs and research
assistants (both past and present) from the department for their help during
the years with special mention to Neeraj, Sujan, Abhijit, Darshana, Jagjit,
Ranjodh, Rima, Unnati, Rohini, Jey, Jasvinder, Sanaa, William, Imaan and
Kavleen. I really appreciate that many of you allowed me to borrow things
many times from your lab for my thesis work. I would also like to thank the
TA‟s of the department for their help on many occasions especially Kaloyan,
Nicole, Abhilash, Anthony, Matt and Rabbani.
I would like to thank Dr. Jeffrey Dean for accepting my application for
admission into the graduate program; Dr. Crystal M. Weyman for numerous
help and guidance whenever I needed them the most; Dr. Barsanjit Mazumder
and Dr. Girish Shukla for numerous suggestions and going out of their way to
help me in many occasions; Dr. Donald Lindmark and Dr. Harry Vankeulen for
their help when I was working as a teaching assistant under their supervision.
I would also like to thank all the staff members of Department of
Biological, Geological and Environmental Sciences and Cleveland State
University (especially Michelle Zinner, Monica Warner and Carolee Pichler) for
their help and support. I thank Cleveland State University, College of Sciences
and Health Professions, Department of Biological, Geological and
Environmental Sciences, Cellular and Molecular Medicine Program and
Center for Gene Regulation in Health and Disease for numerous funding and
facilities, which were essential for the success of my work.
I would like to thank my parents and sister for their love and
encouragement. They always made me believe that tomorrow will be a better
day. I thank all my teachers (from primary school through college and
university) who taught me things for the first time and re-taught me things
which I didn‟t understand. They made me realize the difference between
knowing things and understanding them.
I acknowledge the help and support from my amazing friends. I have
shared excitement, fun, laugh, sadness, grief and many more emotions with
you all. You were always there whenever I needed you. It‟s still a great
pleasure to have most of you around. I appreciate the opportunity of working
with my wonderful colleagues in India. They helped me a lot to quickly adapt to
the new environment of industrial application, which was out of my comfort
zone. They trained me to embrace and finally love this new work culture, to
apply theoretical concepts into practical applications.
With this, I would like to thank you all once again including those,
whose name could not be mentioned here specifically due to limited space, for
your help and support leading up to this moment!
COEVOLUTION OF RIBOSOMES AND THE TRANSLATIONAL
APPARATUS: THE STRUCTURE AND FUNCTION OF EUKARYOTIC
RIBOSOMAL PROTEIN uS7 FROM YEAST, Saccharomyces cerevisiae.

ARNAB GHOSH

ABSTRACT
High-resolution structures of yeast ribosomes revealed that eukaryotic
and bacterial ribosomes share a common ribonucleoprotein core with majority
of the changes (addition of rRNA expansion segments and proteins) occurring
on the outer shell of the ribosome. The 80S yeast ribosome contains 79
proteins, of which 46 are eukaryote-specific and 34 proteins (15 and 19 in the
small and large subunit respectively) are universally conserved. Despite
general similarity in sequence and structure, many conserved ribosomal
proteins have evolved eukaryote-specific extensions, whose functional
significance is largely unknown and/or just beginning to emerge. It has been
hypothesized that eukaryote-specific extensions of the conserved ribosomal
proteins have evolved to accommodate eukaryote-specific features of the
eukaryotic translational apparatus. Yeast, uS7 protein (known as rpS5 in
yeast) from the small (40S) ribosomal subunit has evolved an N-terminal
extension of ~60 amino acid residues in length. The eukaryote-specific N-
terminal extension of rpS5 also forms contacts with N-terminal extension of
uS9 (known as rpS16 in yeast).
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To understand the evolutionary complexity of the eukaryotic (yeast)
ribosome and the function of yeast ribosomal protein rpS5, we have obtained
and characterized yeast strains in which the wild-type yeast rpS5 was
replaced by its truncated (from the N-terminal end) and/or mutant variants.
Mutations of rpS5 led to the disruption of the eukaryotic-specific rpS5-rpS16
interaction and impair translation initiation, cell growth and induction of GCN4
mRNA translation in a manner suggesting incomplete assembly of 48S
preinitiation complexes (PICs) at upstream AUG codons in GCN4 mRNA. We
have hypothesized that rpS5-rpS16 interaction modulates the correct
placement of the eIF2•GTP•Met-tRNAMet ternary complex (TC) in the P-site of
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the 40S ribosomal subunit and eIF5-stimulated GTP-hydrolysis/Pi release, in a
manner involving an altered location of the rpS16 C‐terminal tail in the 40S
decoding center. The rpS5-rpS16 interaction was also suggested to influence
the placement of eIF1, TC and eIF5 following start codon recognition.
Our study provided the first experimental evidence supporting the
functional significance of eukaryote-specific extensions and protein-protein
interactions within the ribosome that are absent in prokaryotes but represent a
defining feature of eukaryotic (yeast) ribosome.
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TABLE OF CONTENTS
Page
Abstract………………………………………………………………….. vii
List of Tables…………………………………………………………..... xiii
List of Figures…………………………………………………………... xiv
List of Abbreviations……………………………………………………. xvii

CHAPTER I
Introduction……………………………………………………………… 1
1.1 Ribosomes……………………………………………………… 1
1.2 Evolution of translation apparatus…………………………… 2
1.2.1 Evolution of ribosomes………………………………... 2
1.2.2 Evolution of translation process & factors…………... 9
1.3 Eukaryotic translation initiation……………………………… 15
1.3.1 Initiation events: what happens when?...................... 15
1.3.2 Eukaryotic initiation factors: Unanswered
questions………………………………………………………. 22
1.4 Translation reinitiation………………………………………... 29
1.5 Ribosomal proteins…………………………………………… 32
1.5.1 Ribosomal proteins: Importance……………………... 32
1.5.2 Eukaryotic ribosomal proteins: conserved yet
unique!................................................................................ 33
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Page
1.5.3 Eukaryote-specific extensions of the small subunit
ribosomal proteins…………………………………………..... 36
1.6 Eukaryotic ribosomal protein S5 (uS7)……………………... 41
CHAPTER II
RpS5 N-terminus is important for ensuring efficient initiation
events downstream of 48S complex assembly……………………… 46
2.1 Abstract………………………………………………………… 46
2.2 Introduction……………………………………………………. 48
2.3 Materials and methods……………………………………….. 50
2.3.1 Yeast strains, plasmids and constructs……………... 50
2.3.2 40S Biogenesis assay………………………………… 50
2.3.3 Re-initiation assays…………………………………… 51
2.4 Results…………………………………………………………. 53
2.4.1 Ribosome biogenesis is not impaired in the RPS5
mutants………………………………………………………… 53
2.4.2 Reinitiation is compromised in rps5-Δ1-46 mutant ... 54
2.4.3 rps5-Δ1-46 mutant exhibits leaky scanning
phenotype……………………………………………………... 59
2.5 Discussion……………………………………………………... 69

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