Post on 12-Jul-2020
FUNCTIONAL ANALYSIS OF THE RQC2 HOMOLOG OF HALOFERAX VOLCANII
By
QINYIN LING
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2016
© 2016 Qinyin Ling
To my family and girlfriend
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ACKNOWLEDGMENTS
I thank my professor, Dr. Maupin-Furlow, for giving me the project and guiding
me through it, and my committee member Dr. Romeo. I thank all the members of Dr.
Maupin’s lab, Dantuluri Swathi, Xian Fu, Shiyun Cao, Sungmin Hwang and Lana
McMillan, also former members Dr. Hepowit, Dr. Martin and Rui Liu for providing me
suggestions for this project.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 7
LIST OF FIGURES .......................................................................................................... 8
LIST OF ABBREVIATIONS ............................................................................................. 9
ABSTRACT ................................................................................................................... 10
CHAPTER
1 INTRODUCTION .................................................................................................... 12
Ribosome ................................................................................................................ 12
Ribosome Stalling ................................................................................................... 13 Ribosome Rescue System ...................................................................................... 14 Ribosome Quality Control Complex Subunit 2 ........................................................ 16 Haloferax volcanii – A Model Archaeon .................................................................. 17
Hypothesis .............................................................................................................. 19
Specific Aims .......................................................................................................... 19
2 METHODS .............................................................................................................. 23
Media ...................................................................................................................... 23 Strains and Growth Conditions ............................................................................... 23 DNA Extraction and Cloning ................................................................................... 24
Generation of mutant strains ................................................................................... 24 Phenotypic Analysis of Δrqc2 Mutant Strains ......................................................... 25
Protein Purification .................................................................................................. 26 Immunoblotting Analysis ......................................................................................... 27 Ribosome Stalling Reporter .................................................................................... 28
3 RESULTS ............................................................................................................... 34
Bioinformatic Analyses of Rqc2 .............................................................................. 34 Generation of C-terminal Tagged Rqc2 Expression Strains ................................... 35 Generation of rqc2 Deletion Strain .......................................................................... 36
Deletion of rqc2 Reduced Cell Growth .................................................................... 37 Deletion of rqc2 Increased Sensitivity to Translation Inhibitor Blasticidin S ............ 37
Generation of Ribosome Stalling Reporters ............................................................ 37
4 DISCUSSION ......................................................................................................... 46
6
LIST OF REFERENCES ............................................................................................... 48
BIOGRAPHICAL SKETCH ............................................................................................ 55
7
LIST OF TABLES
Table page 2-1 Strains ................................................................................................................ 30
2-2 Plasmids ............................................................................................................. 31
2-3 Primers ............................................................................................................... 32
3-1 Ribosome rescue factors of bacteria, eukaryotes and Hfx. volcanii .................... 39
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LIST OF FIGURES
Figure page 1-1 Schematic representation of the tmRNA mediated trans-translation pathway
for ribosome rescue in bacteria .......................................................................... 21
1-2 Model of ribosome rescue system in eukaryotes ................................................ 21
1-3 Rqc2 mediated C-terminal alanine and threonine (CAT) tail formation ............... 22
2-1 pyrE2 based pop-in/pop-out gene knockout system ........................................... 33
2-2 Ribosome stalling reporter plasmid encoding nonstop GFP fusion proteins containing lysine residues encoded by rare codons followed by mCherry .......... 33
3-1 Domain architectures of Rqc2 proteins in Saccharomyces cerevisiae and Haloferax volcanii ............................................................................................... 40
3-2 HvRqc2-StrepII expression in trans .................................................................... 41
3-3 HvRqc2-StrepII expression from the Rqc2-StrepII integrant strain ..................... 42
3-4 Protein bands at 120 kDa (HvRqc2-StrepII-His) and 60 kDa from the HvRqc2-StrepII-His integrant strain .................................................................... 43
3-5 Identification of Δrqc2 mutant by PCR ................................................................ 43
3-6 Schematic of Southern blotting analysis of the Δrqc2 mutant strain ................... 44
3-7 The Δrqc2 mutant strain grows slower than the H26 parent strain under standard aerobic growth condition ...................................................................... 45
3-8 The Δrqc2 strain is more sensitive to the treatment with the translation elongation inhibitor blasticidin S compared to H26 parent strain ........................ 45
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LIST OF ABBREVIATIONS
CAT
NFACT
PCR
RQC
C-terminal alanine and threonine
Protein family including NEMF, FbpA, Caliban, Tae2
Polymerase chain reaction
Ribosome-associated quality control
Rqc2 Ribosome quality control complex subunit 2
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science
FUNCTIONAL ANALYSIS OF THE RQC2 HOMOLOG OF HALOFERAX VOLCANII
By
Qinyin Ling
August 2016
Chair: Julie A. Maupin-Furlow Major: Microbiology and Cell Science
The ribosome is one of the oldest macromolecular assemblies of extant life,
playing a major role in the process of translation during protein biosynthesis, which is
essential for all living cells. The failure of translation arising from various factors can
lead to the stalling of the ribosome, which reduces the capacity for protein synthesis and
disrupts cellular fitness. While ribosome rescue systems have been studied in bacteria
and eukaryotes, the mechanism how archaea handle a stalled ribosome remains largely
unknown. To further understand the ribosome rescue system of archaea, the
HVO_2883 (HvRqc2) protein was investigated for its putative role as a ribosome rescue
factor in the model archaeon, Haloferax volcanii (Hfx. volcanii). Comparative genomics
revealed that HVO_2883 is homologous to Rqc2, a component of the eukaryotic
ribosome-associated quality control (RQC) complex which recognizes peptidyl tRNA on
the stalled ribosome to trigger the ribosome rescue pathway and to mediate mRNA-
independent C-terminal alanine and threonine extensions (CAT tails) to the stalled
nascent polypeptide chains. The presence of the catalytic N terminal NFACT (Protein
family including NEMF, FbpA, Caliban, Tae2; Burroughs and Aravind, 2014) domain
suggests that HvRqc2 may have similar functions during ribosome stalling. An Δrqc2
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mutant strain was generated for phenotype analysis. The growth of the Δrqc2 mutant
strain was found to be 8.2% slower than the H26 parent strain, suggesting that HvRqc2
is important for the growth of Hfx. volcanii. In addition, the Δrqc2 mutant strain was
identified with increased growth sensitivity to the translation elongation inhibitor
blasticidin S than the H26 parent strain, strongly indicating that HvRqc2 is involved in
the process of translation during protein synthesis. An HvRqc2-StrepII-His integrant
strain was generated to analyze HvRqc2 associated protein partners. A protein with
molecular mass of 60 kDa was co-purified with HvRqc2 by StrepII-His tandem affinity
chromatography. Ribosome stalling reporters were generated to induce translation
stalling in Hfx. volcanii to determine whether HvRqc2 can mediate CAT tail modification
of the stalled nascent chains. Future work in characterizing the function of HvRqc2 will
provide an insight in understanding ribosome rescue system in the domain archaea.
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CHAPTER 1 INTRODUCTION
Ribosome
Ribosomes were first discovered by George Palade in 1954 (Palade, 1955). As a
complex molecular machine, the ribosome is a major site for protein biosynthesis, which
is essential to life. The synthesis of protein is guided by genetic information, which is
stored by the sequences of deoxyribonucleic acid (DNA) in all organisms (Watson and
Crick, 1953). During protein synthesis, genetic information in DNA is first copied to
messenger ribonucleic acids (mRNAs) by transcription, followed by the process of
translation assisted by different elements like translation initiation factors, elongation
factors, termination factors, which takes place on the ribosome. The ribosome is
responsible for the translation process, decoding mRNAs and joining amino acids
together in the order specified by mRNAs to form polypeptide chains, which can fold
into proteins and perform multiple functions.
The distribution of ribosomes can be classified as free ribosomes in the cytosol in
bacteria, archaea and eukaryotes, and membrane-bond ribosomes which are found in
eukaryotes and Hfx. volcanii, a species of archaea (Ring and Eichler, 2004). Ribosomes
consist of ribosomal proteins as well as ribosomal RNAs and these molecules are
arranged into a large subunit and a small subunit. According to sedimentation
coefficients measured in Svedberg units, bacterial and archaeal ribosomes are
designated as 70S ribosomes made of 50S (large) subunits and 30S (small) subunits,
while the eukaryotic 80S ribosome is larger and more complex consisting of a larger
60S subunit and a smaller 40S subunit. Despite the difference in size and distribution,
ribosomes from all three domains of life share a similar core structure and function. All
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the ribosomes have three binding sites for transfer RNAs (tRNAs), the A site for binding
aminoacyl tRNAs, the P site for binding peptidyl tRNAs and the E site for binding free
tRNAs before exiting the ribosome (Ramakrishnan, 2002). The small subunit of the
ribosome binds to mRNA and the anticodon loop area of tRNA, contributing to the
mRNA decoding process by monitoring the base pairing between the codon and
anticodon and controlling the fidelity. The large subunit of the ribosome catalyzes the
formation of a peptide bond between an amino acid on A-site tRNA and the nascent
peptide chain at the P-site (Ramakrishnan, 2002). The ribosome is necessary to all
cellular life, and the quality control of every step during the translation process on the
ribosome needs to be highly effective.
Ribosome Stalling
Every biosynthetic pathway has its own intrinsic limits on overall fidelity, leading
to a low but tangible rate of biosynthetic errors. Environmental factors or changes may
also influence or even disrupt the process of cellular biosynthesis (Rodrigo-Brenni and
Hegde, 2012). Under various circumstances, the process of translation on the ribosome
can fail and result in the stalling of the translational machinery. Environmental reasons
include high temperature induced heat shock, nutrient limitation induced amino acid
starvation, as well as oxidative stress, which induce the pausing of translation and
stalling of the ribosome (Shalgi et al., 2013). Antibiotics, such as cycloheximide, have
been widely used to block the elongation step of eukaryotic translation, leading to the
stalling of the ribosome (Alamgir et al., 2010). Translation of aberrant mRNAs with
truncations, depurination, pseudoknots, and/or extensive stem loop structures can also
cause ribosome stalling (Mansouri et al., 2012; Tholstrup et al., 2012; Li et al., 2006).
Furthermore, mRNAs templates with specific sequences, like consecutive rare codons
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or lacking stop codons may stop the ribosome from elongating and cause stalling
(Letzring et al., 2010; Dimitrova et al., 2009). Translation of mRNA into polybasic protein
sequences, such as poly(A) encoding polylysines, can cause strong electrostatic
interaction with the negatively charged ribosomal exit tunnel and will also induce the
stalling of the ribosome (Ito-Harashima et al., 2007; Brandman et al., 2012; Lu and
Deutsch, 2008).
In general, ribosome stalling caused by all these factors will reduce the
availability of translation-competent ribosomes and retain the incomplete nascent
polypeptide chains associated with the large ribosomal subunits. If left uncontrolled,
these partially-synthesized or misfolded polypeptides have the potential to disrupt
cellular homeostasis, reduce fitness and cause cell death (Rodrigo-Brenni and Hegde,
2012). Therefore, every cell requires systems for rescuing the stalled ribosomes and
handling the aberrant products.
Ribosome Rescue System
To ensure accurate translation and maintain the protein synthesis capacity of the
cell, all organisms have evolved mechanisms to manage and recognize the stalled
ribosomes and initiate pathways for ribosome recycling, aberrant mRNA degradation
and nascent peptide degradation (Figure 1-3; Shao et al., 2015).
Bacteria mainly use a hybrid transfer-messenger RNA (tmRNA) mediated trans-
translation system for ribosome rescue (Figure 1-1; Keiler et al., 1996). The specialized
RNA molecule, tmRNA, has both tRNA- and mRNA-like properties. The tRNA-like
region together with a small protein B (SmpB) can mimic a tRNA molecule structurally,
which enables the aminoacylation of tmRNA with alanine for interacting with ribosome
(Karzai et al., 1999; Keiler, 2015). After accommodation, the tRNA-like domain is placed
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in the peptidyl transferase center and the nascent polypeptide chain is transferred to the
tmRNA complex (Weis et al., 2010). With the help of elongation factor G (EF-G), the
peptidyl-tmRNA complex is translocated to P site. The original faulty mRNA will be
removed from the stalled ribosome during the process and will be degraded to avoid re-
initiation of translation by the ribosome, thus preventing the translation of the faulty
mRNA and the synthesis of aberrant protein products in the future (Ramrath et al.,
2012). Upon the removal, the mRNA-like region of the tmRNA is placed at the decoding
center to resume the process of translation. The mRNA-like region contains an open
reading frame (ORF) with a ‘resume’ codon at the 5’ end which is translated into a
degradation SsrA tag (ANDENYALAA) which is attached to to the C-terminus of the
nascent peptide chain (Karzai et al., 1999). After reaching the stop codon of the ORF in
the mRNA-like region, the nascent polypeptide chain is terminated and released, and
the stalled ribosomes are rescued and recycled. Finally, with the help of different
adaptor proteins, the tagged nascent polypeptides are recognized and degraded by
different proteases, such as FtsH, ClpAP, ClpXP, Lon, HflB and Tsp (Keiler et al., 1996;
Choy et al., 2007; Gottesman et al., 1998; Herman et al., 1998; Flynn et al., 2001).
Eukaryotes use a fundamentally different system consisting of factors unrelated
to the bacterial tmRNA system for ribosome rescue (Figure 1-2). Firstly, eukaryotes use
the splitting factors Pelota, Hbs1 and ABCE1 for the disassociation of the stalled
ribosome and releasing the mRNA for degradation (Shoemaker et al., 2010; Shoemaker
et al., 2011; Pisareva et al., 2011). However, unlike the eukaryotic translation
termination factors eRF1 and eRF3, these rescue factors cannot catalyze the hydrolysis
of peptidyl-tRNA, so the aberrant nascent chain cannot be readily released to rescue
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the stalled ribosome (Pisareva et al., 2011). To release the nascent polypeptide chains
for degradation, eukaryotes have a ribosome-associated quality control (RQC) pathway.
How this pathway works has not been fully understood. According to the current model,
in yeast, the RQC complex consists of Rqc1, Rqc2, Ltn1, Cdc48, Npl4 and Ufd1. Rqc2
specifically binds to the peptidyl tRNA on the stalled ribosome, and the ubiquitin E3
ligase Ltn1 binds to Rqc2 on the stalled ribosome and triggers the ubiquitynation of the
nascent chains (Bengtson and Joazeiro, 2010; Brandman et al., 2012). With the
assistance of Rqc1, the Cdc48 complex, made up of an AAA-ATPase Cdc48 with its
cofactors Npl4 and Ufd1, will extract the ubiquitinated nascent chains from the stalled
ribosomes for proteasomal degradation so the ribosome can be rescued and recycled
(Defenouillère et al., 2013; Verma et al., 2013).
For archaea, the ribosome rescue system is still poorly understood. The bacterial
tmRNA system has not been identified in archaea, while some factors related to the
eukaryotic ribosome rescue system have been found, such as archaeal homologs of the
splitting factors Pelota and ABCE1 (Becker et al., 2012). Like eukaryotes, archaea have
a ubiquitin-like proteins and proteasomes for protein tagging and degradation
(Humbard, M.A. et al., 2010; Maupin-Furlow, 2013). However, with limited data it is hard
to determine the model for the archaeal ribosome rescue system.
Ribosome Quality Control Complex Subunit 2
Rqc2, the abbreviation for Ribosome quality control complex subunit 2, also
named as Tae2 (Translation associated element 2) in yeast, Caliban in Drosophila, and
NEMF (nuclear export mediator factor) in mammals, is a component of the eukaryotic
RQC complex (Burroughs and Aravind, 2014). Rqc2 belongs to a highly conserved
nucleic acid–binding protein family termed NFACT family which can be traced to the
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LUCA (last universal common ancestor) of life among three domains of life (Shao and
Grishin, 2000; Doherty et al., 1996; Aravind et al., 1999). Proteins of the NFACT family
contain two HhH (helix-hairpin-helix) domains, several coiled-coil domains, NFACT-N, -
R and -C domains. The N-terminal NFACT-N domain was found to recognize and bind
to peptidyl-tRNA using two conserved aspartate residues and a conserved arginine
residue (Burroughs and Aravind, 2014). The yeast Rqc2 consists of 1038 amino acids,
and the NFACT-N domain D9, D98, R99 residues of this Rqc2 were characterized to
play a role in binding RNA (Shen et al., 2015; Burroughs and Aravind, 2014).
Interestingly, Rqc2 also mediates the mRNA-independent C-terminal addition of
alanine and threonine extensions (CAT tails) to nascent chains on stalled ribosomes
(Shen et al., 2015). Rqc2 can recognize the A-site alanine and threonine tRNAs by the
anticodon and D loops, and stimulate the addition CAT tails onto the nascent chains
(Shen et al., 2015). The CAT tail modification of the nascent polypeptide chains results
in the formation of detergent-insoluble aggregates (Yonashiro et al., 2016; Choe et al.,
2016). However, the biological purpose of CAT tail formation is not fully understood.
Haloferax volcanii – A Model Archaeon
The group of single-celled microorganisms termed archaea are recognized as a
seperate domain of life for biological classification (Woese and Fox, 1977; Woese et al.,
1990). Initially classified as bacteria, archaea are found generally similar to bacteria in
size and shape and do not contain a cell nucleus, interior membranes or organelles.
However, some unique features separate archaea from the bacteria including the lack
of peptidoglycan in the cell wall and use of ether-linked phospholipids with isoprenoid
chains. More importantly, based on the results of genome sequencing, many archaeal
genes and enzymes of basic information processing systems are found to be largely
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different from bacterial ones but similar to eukaryotic ones, making some archaeal
machinery more related to the eukaryotic system, especially for the process of DNA
replication (MacNeill, 2001), transcription (Bell and Jackson, 2001) and translation
(Dennis, 1997).
Many archaea are extremophiles existing in some of the most extreme
environmental conditions on Earth. Some archaea survive temperatures as high as 122
ºC (Takei et al., 2008), pressures as high as 40 MPa (Marteinsson et al., 1999), and pH
as low as 0 (Schleper et al., 1995).
Hfx. volcanii is a halophilic archaeon found in different hypersaline environments
such as the Dead Sea (Mullakhanbhai and Larsen, 1975). To inhabit environments at
NaCl concentrations of 1.7-2.5 M (Mullakhanbhai and Larsen, 1975), Hfx. volcanii cells
have evolved mechanisms for tolerating intracellular cations (Mevarech et al., 2000) and
maintaining protein activity in high concentration of salt.
In addition to being halophilic, Hfx. volcanii is also mildly acidophilic and grows
optimally at the temperature of 42 ºC in aerobic conditions (Mullakhanbhai and
Larsen,1975) on various carbon resources, such as sugars, sugar alcohols or low
molecular weight acids (Javor, 1984). Hfx. volcanii can also grow anaerobically with
dimethyl sulfoxide (DMSO), trimethylamine oxide (TMAO) (Oren and Truper, 1990),
fumarate (Oren, 1991) or nitrate (Bickel-Sankotter and Ufer, 1995) as alternative
electron acceptors in the absence of oxygen.
Different from many extremophiles, Hfx. volcanii can be cultured in the laboratory
on simple defined media without much difficulty, making it possible for further
biochemical and genetic study. A variety of methods have been developed in Hfx.
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volcanii, including genome sequencing, targeted gene deletion and affinity protein
purification (Soppa, 2006; Allers and Mevarech, 2005), making it a key model organism
for the biological research of archaea. Hfx. volcanii has a relatively stable genome, and
the genome of Hfx. volcanii strain DS2 was sequenced in 2010. It consists of a 2.848
Mb main chromosome and three smaller chromosomes named pHV1 (85 Kb), pHV3
(438 Kb), pHV4 (636 Kb) and a pHV2 plasmid (6.4 Kb) (Hartman et al., 2010).
Hypothesis
Our hypothesis for this study is that the HvRqc2 (HVO_2883, Rqc2 homolog of
Hfx. volcanii) is involved in ribosome rescue pathways of Hfx. volcanii. We propose that
HvRqc2 plays important role with protein partners in the rescue of the stalled ribosomes
during the process of translation elongation and the degradation of the stalled nascent
peptide chains.
Specific Aims
To test the hypothesis above, a gene deletion strain of rqc2 will be generated. If
the deletion of rqc2 is not lethal, the phenotype of this mutant will be compared to H26
parent strain in several growth assays. A growth assay under standard growth condition
will be performed to determine the importance of HvRqc2 for cell growth, while growth
assays with treatment with different antibiotics inhibiting translation initiation, translation
elongation and transcription will be used to determine a possible function of HvRqc2
during protein synthesis. An integration strain, which expresses C-terminal StrepII-His
tagged HvRqc2 from the genome, will be constructed to analyze the associated protein
partners of HvRqc2 by StrepII-His tandem affinity chromatography and mass
spectrometry. Reporter plasmids containing optimized stalling sequences will be
generated to induce ribosome stalling in Hfx. volcanii to facilitate the association of
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HvRqc2 with its protein partners and determine whether HvRqc2 protein can mediate
CAT tail modification of the nascent polypeptide chains on the stalled ribosome.
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Figure 1-1. Schematic representation of the tmRNA mediated trans-translation pathway
for ribosome rescue in bacteria. Photo courtesy of Kenneth C. Keiler. Schematic pathway showing the mechanism of trans-translation. 2015. Source: Keiler, K.C. (2015). Mechanisms of ribosome rescue in bacteria. Nat. Rev. Microbiol. 13, 285-297.
Figure 1-2. Model of ribosome rescue system in eukaryotes. Photo courtesy of Tarek
Hilal and Christian M. Spahn. Scheme of the Ribosomal Quality Control. 2015. Source: Hilal, T. and Spahn, C.M. (2015). Ribosome rescue and protein quality control in concert. Mol. Cell 57, 389-90.
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Figure 1-3. Rqc2 mediated C-terminal alanine and threonine (CAT) tail formation. (A)
Recognition of the A-site tRNA via the anticodon loop and D loop by Rqc2. (B) Rqc2 mediated CAT tail elongation of nascent chains. Photo courtesy of Onn Brandman and Ramanujan S. Hegde. CAT-tail formation by Rqc2p. 2015. Source: Brandman, O. and Hegde, R.S. (2016). Ribosome-associated protein quality control. Nat. Struct. Mol. Biol. 23, 7-15.
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CHAPTER 2 METHODS
Media
Luria-Bertani (LB) medium contained 10 g NaCl, 10 g tryptone, and 5 g yeast
extract in deionized water brought to a final volume of 1 liter adjusted to pH 7. ATCC
974 liquid medium contained 125 g NaCl, 50 g MgCl2•6H2O, 5 g K2SO4, 0.134 g
CaCl2•2H2O, 5 g tryptone, and 5 g yeast extract in deionized water brought to a final
volume of 1 liter adjusted to pH 6.8. Solid agar media consisted of the same ingredients
mixed with 15 g agar to make agar plates. Casamino acids (Hv-Ca) solid agar medium
contained 33 ml CA solution (1.65 g casamino acids and 776 l 1 M KOH in deionized
water to a final volume of 33 ml), 200 ml 30% (vol/vol) salt water (240 g NaCl, 30 g
MgCl2•6H2O, 35 g MgSO4•7H2O, 7 g KCl, and 20 ml 1 M Tris-HCl pH 7.5 in the final
volume of 1 liter), and 100 ml deionized H2O mixed with 5 g agar to make agar plates,
and 3 mM CaCl2, 0.8 g•ml-1 thiamin, 0.01 g•ml-1 biotin were added to the sterilized
mixture upon cooling to 50 ºC.
Strains and Growth Conditions
The strains used in this study are listed in Table 1-1. Recombinant plasmids were
constructed using Escherichia coli (E.coli) strain TOP10. E. coli strain GM2163 was
used to purify plasmid DNAs for transformation into Hfx. volcanii. Strains of E. coli were
grown at the temperature of 37 ºC under aerobic conditions in LB medium. Strains of
Hfx. volcanii were grown at the temperature of 42 ºC aerobically in ATCC 974 or Hv-CA
medium. Cells were grown aerobically in liquid medium with rotary shaking at 200 rpm
(revolutions per minute) or on solid agar plates. Ampicillin (100 g•ml-1), novobiocin (0.2
g •ml-1), 5-flouroorotic acid (5-FOA, 50 g•ml-1), uracil (50 g•ml-1) and tryptophan (2.5
24
mM) were added as needed. Glycerol stocks for all strains were prepared by mixing
equal volume of stationary-phase cell culture with 40% (vol/vol) sterile glycerol in
cryogenic tubes. The glycerol stocks were maintained at -80 ºC.
DNA Extraction and Cloning
Genomic DNA of Hfx. volcanii was extracted from 8 ml cell cultures at optical
density at 600 nm (OD600) of 0.6 using the method of spooling (Dyall-Smith, 2009).
Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit from Qiagen (Hilden,
Germany). DNA was sequenced by Sanger sequencing (Interdisciplinary Center for
Biotechnology Research at University of Florida and GENEWIZ, South Plainfield, NJ).
Custom oligonucleotide primers were synthesized by Integrated DNA Technologies
(Coralville, IA).
The plasmids and primers used for this study are listed in Table 1-2 and Table 1-
3, respectively. DNA polymerases and restriction enzymes were from New England
Biolabs (Ipswich, MA). Phusion high-fidelity DNA polymerase was used for PCR
cloning. Taq DNA polymerase was used for colony PCR screening.
Generation of mutant strains
Hfx. volcanii mutant strains were generated by a pyrE2-based ‘pop-in/pop-out’
method (Figure 2-1; Bitan-Banin et al., 2003; Allers et al., 2004). Deletion and integrant
strains were identified by PCR using primer pairs specific to the genome immediately
flanking the genomic region on the plasmid used for the homologous gene
recombination in Hfx. volcanii. The deletion strain was further confirmed by Southern
blotting analysis using an optimized hybridization probe immediately 5’ to the targeted
gene. The probe was labeled by digoxigenin (DIG)-11-dUTP (Roche, Indianapolis, IN),
which was detected by polyclonal anti-DIG-alkaline phosphatase-linked antibody
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(Roche). The pattern of hybridization was visualized by chemiluminescence using
CSPD (Roche) with X-ray film (Hyperfilm; GE Healthcare Life Sciences, Pittsburgh, PA).
Phenotypic Analysis of Δrqc2 Mutant Strains
Strains were inoculated from -80 ºC glycerol stocks to ATCC 974 plates. The
ATCC 974 plates were incubated aerobically at 42 ºC for 5 days and kept at room
temperature (20 ºC) for 5 or 15 days. Strains were inoculated from ATCC 974 plates
into 4 ml ATCC 974 liquid medium in 13 x 100 mm culture tubes to serve as seed
cultures. Seed cultures were grown under standard aerobic growth conditions with
rotary shaking at 200 rpm at 42 ºC to early log phase (OD600 of 0.4). Seed cultures of
each strain were inoculated into 4 ml ATCC 974 medium in 13 x 100 mm culture tubes
to a final OD600 of 0.003 to serve as starter cultures. To obtain a standard growth curve
for each strain, starter cultures were grown aerobically at 42 ºC with rotary shaking at
200 rpm until cells reached an OD600 of 0.1, and then inoculated to 40 ml ATCC 974
medium in 250 ml Erlenmeyer flasks to a final OD600 of 0.003. Cultures were incubated
aerobically at 42 ºC with rotary shaking at 200 rpm. Experiments were performed in
biological triplicate and the growth of each strain was monitored at OD600 over a 50-hour
time period. To analyze drug sensitivity of Δrqc2 mutant strains, seed cultures were
inoculated to 4 ml ATCC 974 medium in 13 x 100 mm culture tubes. Cultures were
grown aerobically at 42 ºC with rotary shaking at 200 rpm until cells reached an OD600 of
0.4. Cells were treated with and without blasticidin S (200 g•ml-1) by incubation for 10
hours at 42 ºC with rotary shaking at 200 rpm. Serial dilutions (10-1 to 10-5) of the cell
cultures were performed. Each dilution was spotted in a row on ATCC 974 agar plates.
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The spotted ATCC 974 agar plates were grown aerobically at the temperature of 42 ºC
for 6 days.
Protein Purification
Strains were inoculated from ATCC 974 plates into 4 ml ATCC 974 liquid
medium in 13 x 100 mm culture tubes to serve as seed cultures. Seed cultures were
grown under standard aerobic growth conditions with rotary shaking at 200 rpm at 42 ºC
to early log phase (OD600 of 0.4). Seed cultures of each strain were subcultured into 4 1-
liter cultures in 2.8-liter Fernbach flasks to a final OD600 of 0.0003. Cells were grown
aerobically with rotary shaking at 200 rpm at 42 ºC to log phase (OD600 of 2) and
harvested by centrifugation (9,000 × g for 20 min at 20°C). Cell pellets were
resuspended at 5 ml per 1 g (wet weight) cells in Tris-salt buffer (2 M NaCl, 50 mM Tris-
HCl, pH 7.4) supplemented with 1 minitablet EDTA-free protease inhibitor cocktail
(Roche) per 40 ml buffer and lysed by French press 5 times (20,000 lb/in2). Cell debris
was removed by centrifugation (10,000 × g for 20 min at 4°C) and the cell lysate was
further clarified by filtration using 0.8-m and 0.45-m cellulose acetate filters (Thermo
Scientific). For StrepII affinity purification, clarified cell lysate (480 mg protein per 32 ml)
was applied to Strep-Tactin Superflow Plus Cartridge (1 ml; Qiagen). The Strep-Tactin
Cartridge was washed with 120 ml Tris-salt buffer to remove unbound proteins. StrepII-
tagged proteins were eluted in Tris-salt buffer supplemented with 2.5 mM d-
desthiobiotin. For StrepII-His tandem affinity purification, clarified cell lysate (480 mg
protein per 32 ml) was first applied to a HisTrap HP column (1 ml, GE Healthcare Life
Sciences). The HisTrap HP column was washed with 120 ml Tris-salt buffer
supplemented with 40 mM imidazole to remove unbound proteins, and proteins from the
27
HisTrap column were eluted in Tris-salt buffer supplemented with 500 mM imidazole.
Eluted fractions were then applied to the Strep-Tactin Cartridge for StrepII affinity
purification to further purify Strep-His tandem tagged proteins. Protein concentration
was determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockville,
IL) using bovine serum albumin (BSA) as the protein standard.
Immunoblotting Analysis
Immunoblotting analysis was performed as follows. Protein samples were boiled
for 10 min in SDS loading buffer (100 mM Tris-Cl, pH 6.8, 0.6 mg·mL−1 bromophenol
blue, 10% w/v glycerol, 2% w/v SDS and 2.5% w/v β-mercaptoethanol) and centrifuged
at 13,000 × g for 10 min to obtain the supernatant. Samples were separated by SDS-
PAGE (10% or 12%) and transferred to 0.5 m polyvinylidene difluoride (PVDF)
membranes (GE Healthcare Life Sciences) by electroblotting (150 Volts, 180 min).
StrepII affinity tagged proteins on PVDF membranes were analyzed by immunoblotting
using mouse anti-StrepII polyclonal antibody (Qiagen; 1:60,000 dilution) followed by
goat anti-mouse IgG (whole molecule)-alkaline phosphatase-linked antibody (Sigma-
Aldrich, St. Louis, MO; 1:60,000 dilution). Immunoreactive antigens were detected by
chemiluminescence using CDP-Star (Thermo Fisher Scientific) as the substrate for
alkaline phosphatase. smRSGFP were analyzed by immunoblotting using Living Colors
Aequorea victoria peptide rabbit immunoglobulin anti-GFP antibody (Clontech
Laboratories, Palo Alto, CA; 1:400 dilution) followed by Horseradish peroxidase-
conjugated anti-rabbit immunoglobulin (H+L) antibody raised in goats (Southern
Biotechnology, Birmingham, AL; 1:4,000 dilution). mCherry were analyzed by
immunoblotting using rabbit anti-mCherry polyclonal antibody (abcam, Cambridge, MA;
28
1:1000 dilution) followed by Horseradish peroxidase-conjugated anti-rabbit
immunoglobulin (H+L) antibody raised in goats (Southern Biotechnology; 1:4,000
dilution). Immunolabeled smRSGFP and mCherry were visualized by
chemiluminescence using ECL Prime Western Blotting Detection Reagent (GE
Healthcare Life Sciences). Chemiluminescence was monitored with X-ray film
(Hyperfilm; GE Healthcare Life Sciences). For loading control, an equivalent amount of
protein for each sample determined by BCA assay or OD600 of whole cells was
separated by SDS-PAGE (10% or 12%) and stained with Coomassie blue or SYPRO
Ruby (Thermo Fisher Scientific).
Ribosome Stalling Reporter
Ribosome stalling reporter plasmids were constructed by cloning genes encoding
two fluorescent proteins, smRSGFP (a soluble modified derivative of green fluorescent
protein [GFP] with red-shifted mutation; Reuter and Maupin-Furlow, 2004) and mCherry
fluorescent protein. Genes encoding smRSGFP and mCherry were amplified from
pJAM1020 and pFPV-mCherry plasmids respectively by PCR. The stop codon of
smRSGFP was replaced by tracts of consecutive rare codons encoding 4 lysine
residues, serving as the stalling signal (Figure 2-2). The DNA fragment was ligated with
T4 DNA ligase (New England Biolabs) to pJAM3136 plasmid. The fluorescent reporter
genes were positioned to be regulated by a tryptophan-inducible promoter PtnaA
promoter, which is tightly controlled by tryptophan (Large et al., 2009). A non-stalling
plasmid encoding smRSGFP and mCherry both with stop codons, was constructed as
the control for the ribosome stalling reporter. The ribosome stalling reporter plasmid and
control reporter plasmid were transformed into E. coli TOP10 cells, and the
29
transformants were screened by PCR for the presence of the inserted genes using the
primers which generated the inserts.
30
Table 2-1. Strains
Strain Description Source or reference
E. coli Top10
F– recA1 endA1 hsdR17(rK
– mK+)
supE44 thi-1 gyrA relA1
Invitrogen
GM2163 F– ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 rpsL136 dam13::Tn9 xylA5 mtl-1 thi-1 mcrB1 hsdR2
New England Biolabs
Hfx. volcanii
DS70 wild-type isolate DS2 cured of plasmid pHV2
Wendoloski et al., 2001
H26 DS70 ΔpyrE2 Allers et al., 2004 QL01 DS70 Δrqc2* This study
*rqc2, HVO_2883.
31
Table 2-2. Plasmids
Plasmid Description Source or reference
pJAM202c Ampr; Novr; Hfx. volcanii-E. coli shuttle plasmid, empty vector carrying P2rrna-His
Zhou et al., 2010
pJAM809 Ampr; Novr; pJAM202c carrying P2rrna-Hvo_1862-StrepII
Humbard et al., 2009
pTA131 pBluescript II carrying Pfdx-pyrE2 with MCS a
Allers et al., 2004
pJAM1020 Ampr; Novr; pJAM202c carrying P2rrnA-smRSGFP
Reuter and Maupin-Furlow, 2004
pJAM3136 Ampr; Novr; pJAM202c carrying PtnaAM3-StrepII
Hwang, unpublished
pJAM3250 Ampr; Novr; pJAM202c carrying P2rrnA-Rqc2-StrepII
This study
pJAM3251 Ampr; Novr; pTA131-based pre-knockout plasmid for rqc2
This study
pJAM3252 Ampr; Novr; pTA131-based knockout plasmid for rqc2
This study
pJAM3253 Ampr; Novr; pTA131-based Rqc2-StrepII, C-terminal fusion to StrepII
This study
pJAM3254 Ampr; Novr; pJAM202c carrying PtnaAM3-smRSGFP-STOP b
This study
pJAM3255 Ampr; Novr; pJAM202c carrying PtnaAM3-smRSGFP-K4-NSTOP c, d
This study
pJAM3256 Ampr; Novr; pJAM202c carrying PtnaAM3- smRSGFP-STOP-mCherry
This study
pJAM3257 Ampr; Novr; pJAM202c carrying PtnaAM3- smRSGFP-K5-NSTOP-mCherry
This study
pJAM3258 Ampr; Novr; pTA131-based Rqc2-StrepII-His, C-terminal fusion to StrepII and polyhistidine
This study
pJAM3259 Ampr; Novr; pJAM202c carrying P2rrnA-Rqc2
This study
pFPV-mCherry Ampr; pFPV vector carrying mCherry Addgene a MCS, multiple cloning site. b STOP, stop codon. c K4, sequence encoding 4 lysine residues using rare codons (AAA AAA AAA AAA). d NSTOP, no stop codon.
32
Table 2-3. Primers
Primer Pair Primer sequence (5’-3’) Description
Rqc2 NdeI FW Rqc2 KpnI RV
AGCATATGGATCCGAAGCGGGAACTCACG ATGGTACCCTCGTCGACGATATCGCTCCCGC
Rqc2-StrepII
Rqc2 500bp FW Rqc2 500bp RV
TCGGTACCACCCCGTCGTCGTGGAAGTATCCA TAAGCTTGGATCCGTCATGATGCCGCCGAAGATGCCCGC
rqc2 pre-knockout
Rqc2 INV FW Rqc2 INV RV
GGTAATCGGGCGTTAGACG GAACGAAAAGGGGTGTCGT
rqc2 knockout
Rqc2 700bp FW Rqc2 700bp RV Rqc2 21bp RV
TGAGGTACGTCTGGCTCCC GTCGGTCGGTCTCAGCGT ACGAAAAGGGGTGTCGTTTCCGCG
confirmation of rqc2 knockout
Rqc2 StrepII INT FW Rqc2 StrepII INT RV
CAGTTCGAAAAATGAACGTCGCCGCCTTCGGGT CGGGTGGCTCCAGGTACCCTCGTCGACGATATC Rqc2 C-terminal
fusion to StrepII
Rqc2 StrepII-His INT FW Rqc2 StrepII-His INT RV
CACCACTGAACGTCGCCGCCTTCG GTGGTGGTGGTGGGTACCTTTTTCGAACTG
Rqc2 C-terminal fusion to StrepII and polyhistidine
smRSGFP FW smRSGFP Stop RV smRSGFP NonStop RV
GCGGATCCTAGAAATAATTTTGTTTAA GAGCTCAGCTATTTTTTTCATTATTTGTATAGTTCATCCAT GAGCTCAGCTATTTTTTTTTTTTTTTGTATAGTTCATCCAT
smRSGFP-STOP smRSGFP-K4-NSTOP
mCherry S FW mCherry NS FW mCherry RV
ACAAATAATGAAAAAAATAGCGTGAGCAAGGGCGAGGAGGATAACATGGCC ATACAAAAAAAAAAAAAAATAGCGTGAGCAAGGGCGAGGAGGATAACATGGCC GTTATGCTAGTTATTGCTTACTTGTACAGCTCGTCCATGCCGCCGGTG
smRSGFP-STOP-mCherry smRSGFP-K4-NSTOP-mCherry
Rqc2 Comp FW Rqc2 Comp RV
AGCTTGCGGCCGCACTCGAGC CTCGTCGACGATATCGCTCCCGCC
Rqc2 complementation
33
Figure 2-1. pyrE2 based pop-in/pop-out gene knockout system. A plasmid carrying the
flanking regions of the gene to be deleted and pyrE2 marker is used for gene knockout in the ΔpyrE2 Hfx. volcanii H26 strain. Photo courtesy of Thorsten Allers, Hien-Ping Ngo, Moshe Mevarech and Robert G. Lloyd. Gene knockout system based on the pyrE2 gene. 2004. Source: Allers, T., Ngo, H.P., Mevarech, M., and Lloyd, R.G. (2004). Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes. Appl. Environ. Microbiol. 70, 943-953.
Figure 2-2. Ribosome stalling reporter plasmid encoding nonstop (lacking stop codons)
GFP fusion proteins containing lysine residues encoded by rare codons followed by mCherry.
34
CHAPTER 3 RESULTS
Bioinformatic Analyses of Rqc2
We identified the proteins which are hypothesized to be involved in ribosome
rescue pathways in the archaeon Hfx. volcanii by comparative genomic analysis (Table
2-1). Homologs of eukaryotic ubiquitin E3 ligase Ltn1 and Rqc1 were not identified in
Hfx. volcanii. However, homologs of eukaryotic ribosome rescue factors including
translation release factors RF1 and RF3, nonstop mRNA decay factor Ski7, stalled
ribosome rescue factor Pelota, ribosome recycling factor ABCE1, as well as RQC
components Rqc2 and the AAA ATPase Cdc48 were identified in Hfx. volcanii.
Proteasomes and ubiquitin-like SAMPs were also found in Hfx. volcanii. However, a
bacterial tmRNA-mediated ribosome rescue system was not identified in Hfx. volcanii.
Further analysis revealed that Hfx. volcanii (Hv) Rqc2 was related to yeast Rqc2
in protein domain architecture. The M domain of yeast Rqc2, which is likely to interact
with the eukaryotic ubiquitin E3 ligase Ltn1 N-terminal helix (Shao et al., 2015), was
missing in HvRqc2. However, the remaining protein domain architectures were similar.
Both yeast and archaeal Rqc2 proteins contained the catalytic N-terminal NFACT-N
domain, required for binding peptidyl tRNA on the stabled ribosome to trigger RQC
pathway, and mediate CAT tail synthesis of the stalled nascent polypeptide chains in
yeast (Figure 3-1). Given the homology to some of the eukaryotic ribosome rescue
factors in Hfx. volcanii, the results suggested that HvRqc2 may have similar functions
during ribosome stalling.
35
Generation of C-terminal Tagged Rqc2 Expression Strains
To further understand Rqc2 function in archaea, the HvRqc2 protein was
expressed in trans with a C-terminal StrepII tag and purified from Hfx. volcanii by StrepII
affinity purification. A single predominant protein band was detected in the purified
fractions by anti-StrepII immunoblotting from the strain expressing HvRqc2-StrepII in
trans compared to the empty vector control (Figure 3-2). The molecular mass of
HvRqc2-StrepII observed by SDS-PAGE (120 kDa) was larger than the theoretical
molecular mass (80 kDa) estimated by the deduced protein sequence. The theoretical
pI (isoelectric point) of HvRqc2-StrepII was calculated to be 4.36. Thus, the slower
migration of HvRqc2 may due to an altered SDS coating of the acidic polypeptide as
supported by previous study of other acidic proteins (Reed et al., 2013; Peck, 2006).
The protein samples purified by Strep-Tactin affinity chromatography were further
analyzed by staining with SYPRO Ruby to address the associated protein partners of
HvRqc2. However, we only identified a specific band for HvRqc2 from the strain
expressing HvRqc2-StrepII in trans compared to the empty vector control (Figure 3-2).
To maintain an appropriate ratio of HvRqc2 to its protein partners, an HvRqc2-
StrepII integrant strain was generated by a pyrE2-based ‘pop-in/pop-out’ method
(details in Methods). A StrepII affinity purification was performed and a single
predominant protein band was detected at 120 kDa by anti-StrepII immunoblotting,
which corresponded to the HvRqc2-StrepII integrant strain. However, the yield of protein
purified from the HvRqc2-StrepII integrant strain and H26 parent strain was low (protein
concentration was less than 5 g•ml-1). Multiple bands were detected for both the
HvRqc2-StrepII integrant strain and the H26 parent strain based on total protein stain
36
(Figure 3-3). HvRqc2 associated protein partners were not identified due to this high
background of non-specific proteins.
Thus, we generated a HvRqc2-StrepII-His integrant strain which had an added
polyhistidine tag to identify the associated protein partners of HvRqc2. The purity of
protein partners specifically associated with HvRqc2 was increased by a tandem affinity
tag purification approach using this HvRqc2-StrepII-His integrant strain. Two protein
bands, which corresponded to the HvRqc2-StrepII-His integrant strain, were detected by
SYPRO Ruby staining at 120 kDa (HvRqc2-StrepII-His) and 60 kDa (Figure 3-4).
Generation of rqc2 Deletion Strain
An rqc2 deletion strain was generated by pyrE2 pop-in/pop-out system for
homologous recombination (details in Methods). Deletion strains were identified by PCR
of genomic DNA using primers annealing specifically to 700 bp 5’ and 3’ of the rqc2
gene on the genome and outside the deletion plasmid. The PCR product generated from
the putative Δrqc2 mutant Hfx. volcanii QL01 was 1.4 kb, and carried only the flanking
regions of rqc2 gene (the open reading frame encoding the protein was deleted) (Figure
3-4). Compared to the putative Δrqc2 mutant strain, the PCR products generated from
the parental strain were 3.5 kb, which was consistent with amplification of the rqc2 gene
(2.1 kb) and its flanking regions (Figure 3-5).
To provide further evidence for the absence of the rqc2 gene from the genome of
the Hfx. volcanii QL01 strain, Southern blotting analysis was performed using a probe
immediately 5’ to the rqc2 gene. The genomic DNA from the putative Δrqc2 mutant
strain QL01 identified by PCR was digested by BanII restriction enzyme which was
predicted to cleave at 0.95 kb upstream and 0.54 kb downstream of the rqc2 gene
based on genome sequence. The presence of a band at 1.5 kb confirmed that strain
37
QL01 was an Δrqc2 mutant compared to the 3.6 kb band for the H26 parent strain
(Figure 3-6).
Deletion of rqc2 Reduced Cell Growth
While deletion of the rqc2 gene reduces the rate of protein biosynthesis in yeast
(Alamgir et al., 2010), the function of the rqc2 gene in archaea remains largely
unknown. Here we monitored growth of the Δrqc2 mutant compared to its parent H26A.
The growth of the Δrqc2 strain was found to be 8.2% slower than the H26 parent strain,
with a doubling time of 5.3 hours, compared to the doubling time of H26 parent strain,
which was 4.9 hours (Figure 3-7). These results suggest that rqc2 is an important gene
for the growth of Hfx. volcanii.
Deletion of rqc2 Increased Sensitivity to Translation Inhibitor Blasticidin S
To determine a possible role of Rqc2 during protein biosynthesis in Hfx. volcanii,
a chemical inhibitory compound was used to target a specific step of protein synthesis.
Blasticidin S, a compound that inhibits the process of translation elongation by binding
to the P site of 60S ribosomal subunit and prevents peptidyl-transferase reaction
(Svidritskiy et al., 2013), was used in the drug sensitivity analysis. Deletion of rqc2 was
found to confer increased sensitivity to blasticidin S compared to the H26 parent strain
(Figure 3-8), suggesting that HvRqc2 is involved in the process of translation during
protein synthesis.
Generation of Ribosome Stalling Reporters
Anti-GFP and anti-mCherry immunoblotting is ongoing to test whether the
ribosome stalling reporter can cause translation stalling in Hfx. volcanii. Protein bands of
25-kDa which correspond to the smRSGFP are expected to be detected by anti-GFP
immunoblotting for the strains carrying stalling reporters or non-stalling reporters, while
38
27-kDa protein bands corresponding mCherry are expected to be detected only for the
strains carrying non-stalling reporters by anti-mCherry immunoblotting.
39
Table 3-1. Ribosome rescue factors of bacteria, eukaryotes and Hfx. volcanii.
Ribosome rescue factors Bacteria Eukaryotes Hfx. volcanii
Translation release factors RF1 RF2 RF3 eRF1 eRF3 HvRF1 HvEF1α
Stalled ribosome rescue factors ArfA ArfB ePelota Hbs1 HvPelota HvEF1α
Non-stop mRNA decay factors SmpB-tmRNA-EF-tu Ski7 HvEF1α
Ribosome recycling factors RRF EF-G eABCE1 HvABCE1
RQC complex
Rqc1 ? eRqc2 HvRqc2 eCdc48 HvCdc48
Ubiquitylation/Ubiquitylation-like Ltn1 ?
Ubiquitin SAMPs
Nascent chain degradation Proteases Proteasome Proteasome
mRNA degradation RNase R Exosome Exosome Blank, no related factors. ?, homologs not predicted by bioinformatics.
40
Figure 3-1. Domain architectures of Rqc2 proteins in Saccharomyces cerevisiae and
Haloferax volcanii (upper and lower, respectively). CC, coiled-coil; HhH, helix-hairpin-helix.
41
A
B
Figure 3-2. HvRqc2-StrepII expression in trans. A) StrepII purified in trans expressed
HvRqc2-StrepII was detected by anti-StrepII immunoblotting B) SYPRO Ruby staining of purified fractions.
42
A
B
Figure 3-3. HvRqc2-StrepII expression from the Rqc2-StrepII integrant strain. A) StrepII
purified HvRqc2-StrepII from the Rqc2-StrepII integrant strain was detected by anti-StrepII immunoblotting. B) SYPRO Ruby staining of purified fractions.
43
Figure 3-4. Protein bands at 120 kDa (HvRqc2-StrepII-His) and 60 kDa from the
HvRqc2-StrepII-His integrant strain were detected by SYPRO Ruby staining of StrepII-His tandem purified fractions.
Figure 3-5. Identification of Δrqc2 mutant by PCR. A 1.4 kb PCR product generated
from the Δrqc2 mutant strain corresponds to gene flanking regions. A larger 3.5 kb PCR product generated from H26 parent strain corresponds to the gene plus gene flanking regions.
44
A
B
Figure 3-6. Schematic of Southern blotting analysis of the Δrqc2 mutant strain. A) A
probe immediately 5’ to the rqc2 gene was used to generate a 1.5 kb fragment from the Δrqc2 mutant strain corresponding to the flanking regions of rqc2, and a 3.6 kb fragment from H26 parent strain corresponding to the flanking regions and rqc2 gene. B) The Δrqc2 mutant was confirmed by the presence of 1.5 kb band compared to the 3.6 kb band for the H26 parent strain.
45
Figure 3-7. The Δrqc2 mutant strain grows slower than the H26 parent strain under
standard aerobic growth condition. OD600 was measured at time points from 15 to 51 hours to determine the growth rates. Error bars represent standard deviation. td, doubling time.
Figure 3-8. The Δrqc2 strain is more sensitive to the treatment with the translation
elongation inhibitor blasticidin S compared to H26 parent strain. The Δrqc2 mutant strain and H26 parent strain were serially diluted to 10-3 to 10-6 after 10 h treatment with and without blasticidin S and spotted on ATCC 974 solid medium.
46
CHAPTER 4 DISCUSSION
To overcome ribosome stalling during protein biosynthesis and maintain cellular
fitness, organisms have evolved mechanisms known as ribosome rescue (Defenouillère
et al., 2013). While ribosome rescue pathways have been investigated in bacteria and
eukaryotes, little work has been done in archaea. Proteins related to eukaryotic
ribosome rescue factors are found to be conserved in archaea, including translation
release factors RF1 and EF1α, splitting factor Pelota and ABCE1, as well as RQC
components Cdc48 and Rqc2. Here we provide evidence for understanding the function
of the Rqc2 homolog of Hfx. volcanii, which may provide an insight into understanding
ribosome rescue system in archaea.
Deletion of the rqc2 gene leads to a reduction in the rate of cell growth,
suggesting the importance of Rqc2 for the growth of Hfx. volcanii. The Δrqc2 mutant
strain also exhibited an increased sensitivity to blasticidin S, a translation elongation
inhibitor, strongly indicating that the function of Rqc2 is linked with protein biosynthesis.
Future experiments to be performed include using different inhibitory compounds
targeting specific steps of transcription and translation. Actinomycin D, which binds
strongly to duplex DNA and minimizes the synthesis of mRNA (Sobell, 1985), can be
used to investigate the role of Rqc2 for transcription. Puromycin, an analog of the
terminal aminoacyl-adenosine part of aminoacyl-tRNA which disrupts translation
process by causing premature peptide chain termination before chain elongation (Sanz
et al., 1993), can be used to investigate the role of Rqc2 for translation elongation. With
previous work showing that in eukaryotes the RQC complex rescues stalled ribosomes
during translation elongation (Yonashiro et al., 2016), it is possible that the function of
47
Rqc2 is involved in translation elongation. To assign the ribosome-associated functions
of Rqc2, it is important to analyze the Rqc2 protein partners. The Rqc2-StrepII-His
integrant strain was generated to facilitate this analysis by enabling the stoichiometry of
Rqc2 to be maintained in the cell and allowing the Rqc2 associated partners to be
purified by tandem affinity purification. A protein with molecular mass of 60 kDa with
HvRqc2 were detected in the purified fractions. Future work will include identification of
the co-purified protein at 60 kDa by mass spectrometry analysis.
The experiments to confirm that the ribosome stalling reporter leads to translation
stalling are still ongoing. Expression of only one fluorescent protein (smRSGFP) is
expected for stalling reporter compared to expression of both fluorescent proteins for
non-stalling control reporter. To determine whether Rqc2 mediates CAT tail modification
of the nascent polypeptide chains on the stalled ribosome, future work may include size
comparison of smRSGFP expressed from the Δrqc2 mutant strain/H26 parent strain
carrying the stalling reporter and amino acid analysis of the modified smRSGFP.
48
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BIOGRAPHICAL SKETCH
Qinyin Ling grew up in Tongling, Anhui, China. He graduated from Tongling NO.1
High School in 2009. He attended Nanjing Agricultural University from 2009 to 2013 as
an undergraduate student, majored in biological sciences. Upon completion of his
undergraduate degree, he went on to graduate school at the University of Florida, and
he was expected to graduate with his Master of Science in microbiology and cell
science.