Exam for 5MO019: Gene regulation and expression technology
 Four hour written examination.
 For marking purposes the exams are coded – i.e. you do not write your name on the
answer sheet – only the code assigned to you.
 Questions are in English, but answers may be written in English or Swedish (or a
combination of both)
 Your written answers must be clear, both in form (i.e. legibility) and in content
(meaning), for us to be able to award marks.
Grading:
Grading system is used:
5 – pass with distinction (80% or higher)
4 – (65% to 79%)
3 – (50% to 64%)
Permissible Exam Aids 2013
 English-Swedish dictionary or Translation pen
Examples of previous exam questions:
Some of these (or similar style questions) will be on your exam
Victoria Shingler:
Victoria Shingler:
Victoria Shingler:
Victoria Shingler:
Victoria Shingler:
Jörgen Johansson:
Jörgen Johansson:
Matthew Francis:
Matthew Francis:
Tracy Nissan:
Victoria Shingler:
Yuri Schwartz:
Ruth Palmer:
Tracy Nissan:
Martin Gullberg:
Per Stenberg:
Bacteria Transcriptional Regulation I (RNAP, promoter etc)
Bacteria Transcriptional Regulation II (transcriptional regulators)
External stimuli and gene regulation: Two-component systems
Signal sensing, regulatory logic, and synthetic regulatory devices
Global regulation and the “omics”
Transcriptional termination, attenuation and riboswitches
Regulation of mRNA stability and function
Bacterial protein export systems
Regulation of extra-cytoplasmic components in bacteria
Bacterial translation and control of protein synthesis
Protein expression systems: bacterial
Eukaryotic transcriptional control
Manipulating gene expression via RNA interference (RNAi)
The eukaryotic ribosome and regulation of protein synthesis
Protein expression systems: eukaryotic
Transcriptomics and RNA sequencing
1
Victoria Shingler:
Bacteria Transcriptional Regulation I and II
RNA polymerase, sigma-factor, promoters paradigms of bacterial transcriptional
regulators, and DNA bending
(you will find many of the answers to this series of questions in discussion group 1 /PSC1)
Q1.
A) Explain why many positive bacterial transcriptional activators are dual function proteins
that can activate transcription from some promoters and act as repressors at other promoters.
B) Describe the basic modes of action when acting as an activator and when acting as a
repressor in terms of the important DNA motifs for RNA polymerase binding to bacterial
promoters. (Xp)
--------------------------------------------------------------------------------------------Q2. Compare and contrast bacterial transcriptional activator and repressor proteins with
respect to:
a) Their mode of action on RNA polymerase function at a promoter, and
b) How their activity is controlled.
Aid your answer with illustrations and examples. (Xp)
-----------------------------------------------------------------------------------------------------Q3
A) Briefly describe, with the aid of illustrations(s), the DNA features of a typical bacterial promoter
sequence that are important for binding of RNA polymerase. Indicate which subunit or sub-region of a
subunit of RNA polymerase is involved in binding of each region of the promoter DNA.
B) Explain why many bacterial transcriptional regulators are dual function proteins that can activate
transcription from some promoters and act as repressors at other promoters. (Xp)
Expected answer (in note form) would include:
A) A figure something like one of the three below...
A typical promoter recognized by 70-RNA polymerase
T T G A C A
T A T A A T
= consensus
69 79 61 59 54 54
77 76 60 61 56 82
% occurrence
spacer 16 -19 bp
17 bp spacer
43% occurrence
-60 UP element
Bound by the
-subunits of
RNA polymerase
-35
-10
Additional TGn
recognised in extended
-10 promoters
(recently renamed the
TGGT -15 element)
+1
Discriminator
GGG
Bound by the -factors
Should minimally include the information that.....
That the -subunits and sub-region of the -factor interact to promote binding to promoters:
-subunits with UP-element DNA; -35 recognised by 4 domain; -10 recognised by 2
domain; extended -10 by the 3 domain (in really good answers also that: discriminator is
recognised by -region 1.2) (I would not expect the consensus sequences...)
A) Here I would expect an answer that explains how binding-site (operator) location differs at
different promoters and how this affects the outcome...
2
-----------------------------------------------------------------------------------------------------Q4.
A) Describe the subunit structure of Escherichia coli holoenzyme RNA polymerase and the
role of the sigma () factor.
B) Contrast the properties of the holoenzyme using the house-hold (housekeeping) 70 factor
with that of the holoenzyme using the alternative 54-factor.
C) What repercussions (consequences) does the 54-factor have on how transcription from 54
promoters is regulated? (Xp)
-----------------------------------------------------------------------------------------------------Q5.
A) What is the “specificity subunit” of bacterial RNA polymerase and how is it physically
displayed on RNA polymerase to aid its function?
B) What are the primary steps in transcriptional initiation and what are the major parameters
that affect their impact on promoter output? (Xp)
-----------------------------------------------------------------------------------------------------Q6.
A) Briefly describe, with the aid of an illustration(s), the structural features of a typical
bacterial promoter that are important for RNA polymerase binding and the subunits (or subregions of subunits) of RNA polymerase that are involved in binding these DNA regions.
B) Describe, with the aid of an illustration(s), the steps in transcriptional initiation. Be sure to
include in your answer the major parameters that affect these steps to alter promoter output?
-----------------------------------------------------------------------------------------------------Q7.
A) Describe the subunit structure of Escherichia coli holoenzyme RNA polymerase and their
involvement in interactions with the DNA at a bacterial promoter.
B) Describe why the -subunit is also known as the “specify factor” and how it is physically
displayed in the holoenzyme to aid its function.
C) What is an anti--factor? With aid of an illustration, describe one of the ways an anti-factor can work. (Xp)
-----------------------------------------------------------------------------------------------------Q8. Explain three different mechanisms by which a bacteria promoter can be made codependent on the activities of two different regulators. Aid your explanations with
Illustrations in each case. (Xp)
-----------------------------------------------------------------------------------------------------Q9. In the following situations, a) to c) below, state whether the changes made would increase
or decrease the activity of a bacterial promoter. Explain why in each case.
a) Introduction of an intrinsic bend within the spacer region between the –35 and –10 motifs
of a typical σ70-dependent promoter.
b) Introduction of an intrinsic bend so that it curves an AT-rich DNA upstream region
(between –40 and –60) towards the RNA polymerase
c) Introduction of the same intrinsic bend as in (b) above, except that there is an extra 5 bp
insert between the –35 and –40 regions.
-----------------------------------------------------------------------------------------------------Q10.
Within a bacterial promoter region, a regulatory protein may sometimes have a DNA binding
site that is distant (30-140 bases upstream) to the RNA polymerase binding site. DNA
3
segments of this size have limited flexibility. The stiffness of the DNA within this region may
prevent the regulatory protein and RNA polymerase from interacting with each other, which
is required for transcription to occur.
Bacteria have evolved two solutions (ways) to help the RNA polymerase to interact with the
regulatory protein bound upstream. What are they? Give a short explanation of how these
solutions allow the RNA polymerase to interact with a regulatory protein bound at a distance
from the RNA polymerase binding site.
Intrinsic DNA bends (caused by polyA or polyT tracts) are found in the sequence giving it more
flexibility without the need for additional proteins to bend the DNA. The sequence between both
protein-binding sites may be already curved in the appropriate direction or may show an increased
flexibility. This then allows the DNA to have a more dynamic structure and the two proteins can
come into contact more easily.
Additional protein factors are required to bind and to bend the DNA (e.g. IHF, HU etc) between
the two protein-binding sites. The binding of these proteins to DNA provokes a conformational
change (bend) inf the DNA that has a precise directionality and can line up non-adjacent binding
sites, thus, allowing otherwise unlikely or infrequent protein-protein interactions to occur
Q11.
The plaG gene encodes an enzyme for production of a bio-plastic. However, when expressed
in E. coli, the PlaS protein causes the cells to burst open. Therefore, for production of this
protein you would like to put it under a regulatory system so that it only becomes expressed
during the stationary phase of growth.
Design and describe two different hypothetical promoters for preferential gene expression in
the stationary phase of growth in E. coli. Motivate your design. (Xp)
Q12.
Pseudomonas putida is naturally white. However, when a synthetic (geneG) is expressed it
produces a green pigment, while expression of a different synthetic (geneB) results in
production of a blue pigment.
In addition to the two synthetic genes, you have at your disposal:
I] a dual functional regulatory protein - Mol1 - that can bind to its DNA operator site only
when activated by arabinose, and
II] two promoters:
promoter 1: has a good -35 and -10 box and Mol1 operator site that completely over laps
the -10 box
promoter 2: lacks a -35 box but has a Mol1 operator site located at -60 that will allow
appropriate interaction of a regulator with the -subunit of RNA polymerase to efficiently
recruit RNA polymerase.
Design a regulatory circuit for production of the green pigment in the absence of arabinose,
but a blue pigment in the presence of arabinose in the growth media.
------------------------------------------------------------------------------------------------------
4
Victoria Shingler: External stimuli and gene regulation: Two-component systems
Including Chemotaxis and Quorum sensing
Q1. Dephosphorylation is as important as phosphorylation in determining the biological
success of bacterial regulatory His-Asp phosphotransfer systems (so-called “two component”
or HAP systems). True or false?
Discuss this statement and motivate your answer considering the duration of responses
needed for appropriate regulation of different physiological processes. (Xp)
-----------------------------------------------------------------------------------------------------Q2. “Two component systems” that involve His to Asp phosphotransfer are one of the
primary forms of communication used to control prokaryotic transcription in response to
environmental stimuli.
With the aid of illustrations,
A) describe the basic features of the proteins involved in these regulatory systems, and
B) describe the kinds of proteins and reactions that are important for the flow of information
from the external environment to control the response to a signal. (Xp)
-----------------------------------------------------------------------------------------------------Q3. Flagella mediated taxis is one of the best understood behavioural responses in E. coli.
A) Briefly describe the proteins involved in the sensing of attractants/repellents, and signal
transduction that controls the phosphorylation status of CheY (that interacts with the flagella
motor) and CheB (the methyl esterase).
B) What advantage is conferred (given) by coupling of the receptors to the histidine kinase in
this system, rather than having sensing directly built into the histidine kinase? (X p)
-----------------------------------------------------------------------------------------------------Q4.
A) Why is flagella driven motility important for bacteria?
B) Briefly describe, with the aid of an illustration, the proteins and signal transduction
mechanisms that control the rotation of the flagella in response to
attractants/repellents.
Expected answer would include:
A) Bacteria need to be motile to be able to find the most energetically favourable environment
and/or interaction partners (for symbiotic-, mutualistic-, or pathogenic interactions).
B) See Figure – chemo- and metabolism-dependent
receptors have a common signalling domain that
interacts with histidine kinase CheA (via CheW) to
control downstream events via phosphorylation of
the response regulators (CheY and CheB). The
active form of CheB (CheB-P) removes methyl
groups from MCPs to reset sensitivity (adaption),
while the active form of CheY
(CheY-P) interacts with the flagella motor (via
FliM) to change the rotation direction of the flagella.
Signalling results in prolonged swimming through
5
Repellent/Attractant
CheA
CheW
MCPs
Or Aer receptors
Methyl Esterase
CheZ phosphatase
(Activity to counteract methylation
by CheR; MCPs only)
PCheBP
(active)
Controls ½ life of the active form
PCheYP
(active)
Interacts with FliM
(motor switch protein
of the flagellum) to
change rotation
direction
gradients in the direction of favourable
environments (or away from noxious ones)
-----------------------------------------------------------------------------------------------------5.
A) With the aid of an illustration, briefly describe the general structure of receptors for
bacterial chemotaxis responses and metabolism-dependent taxis responses.
B) What is the key conceptual difference behind these two taxis responses?
6
Victoria Shingler: Signal sensing, regulatory logic, and synthetic regulatory devices
(new lecture for 2010)
Q1) In bacterial regulatory circuits, a few simple regulatory devices can be combined to
achieve sophisticated regulatory outcomes. Describe how a simple feed-forward loop
involving two activators that both act on a common promoter can distinguish the duration of a
signal i.e. distinguish between a short irrelevant signal (noise) and a signal of long duration
that the bacterium needs to respond to.
---------------------------------------------------------------------------------------------------------------Q2) In bacterial regulatory circuits, a few simple regulatory devices can be combined to
achieve sophisticated regulatory outcomes. Describe how
A) a simple positive feedback loop leads to increased levels of a gene product only so long as
a signal is present.
B) a simple negative feedback loop can be used to provide a constant level of transcription of
a gene.
---------------------------------------------------------------------------------------------------------------Q3) Bacterial sensor-regulators are transcriptional repressors or activators that can directly
bind and respond to the presence of certain chemicals or metabolites.
A) Explain how these types of regulatory circuits can be employed in biosensor applications.
B) Explain how you might manipulate a bacterial sensor-regulator to respond to a new
compound.
---------------------------------------------------------------------------------------------------------------Q4) In bacterial regulatory circuits, simple positive and negative feed-back loops result in
different outcomes in terms of protein production. What are these outcomes? - In each case
motivate your answer.
---------------------------------------------------------------------------------------------------------------Q5) Describe a bacteria flip-flop regulatory device.
----------------------------------------------------------------------------------------------------------------
7
Victoria Shingler: Global regulation and the “omics”
Q1.
(new lecture for 2011)
A) What is bacterial global regulation?
B) What kind of molecules can mediate this level of regulation?
Expected answer would include (note form):
Definition given:
A) Fundamental concept 1: Global regulation can be mediated via different kinds of factors,
however, whatever the factor-type, if it results in altered activity of multiple promoters it is
classified as a “global regulator”.
# The group of promoters (cistrons/operons) that the given factor controls constitutes a
regulons.
# For a signal, e.g. salt, heat etc, a group of factors that respond to that signal are called a
stimulon.
(potential bonus points if they bring up direct versus indirect cascade regulation...).
B) proteins, small regulatory RNAs, and small regulatory molecules. (minimalistic answer),
Full point if they give an example of each kind and briefly describe how they work
---------------------------------------------------------------------------------------------------------------Q2) Choose one bacterial global regulator that you have heard about and describe how it can mediate
different effects at different bacterial promoters.
A. There can be many correct answers to this (as there are many different global regulators...)
The ones most extensively discussed were sigma-factors (particularly RpoS σS), ppGpp, CRP and
IHF
----------------------------------------------------------------------------------------------------------------
Q3. Briefly describe how cascade regulation can occur in bacteria.
(Hint, think about the examples of ppGpp/DksA and small regulatory RNA DrsA)
---------------------------------------------------------------------------------------------------------------Q4. Expression of RpoS is highly regulated – true or false – Motivate your answer.
----------------------------------------------------------------------------------------------------------------
8
Q5) Briefly describe
A) transcriptomics,
B) proteomics and
C) metabolimics.
Be sure to include in your answer the basic methodology used in each case and the kind of
information that can be gained from each of these “omics”
Expected answer would include:
A) Transcriptomics - the systematic
Microarrays – a snap shot of transcription
identification of all the RNA
Culture A
Culture B
transcripts in a organism/tissue at any
given moment, including mRNAs and
regulatory RNAs. It gives a “snapshot” view of the RNA present at the
DNA Microarray
(DNA Chip)
moment of extraction i.e. events that
are occurring on the transcriptional
level. Basic methodology: as in Fig
opposite: i.e. differentially labelled test
RNA (more usually
cDNA) are hybridized to a DNA chip. Lazer scanning and imaging is used to
determine which genes are differentially expressed. Usually used to identify
differentially transcribed genes - thus regulation on the transcriptional level.
e.g. 1 Same strain grown under different conditions, or
e.g. 2 Wild-type versus a mutant derivative
Prepare RNA probe (often copied to cDNA)
label with Fluorescent Dye
Combine Equal amounts
Hybridize to microarray
Scan, image and quantify
Expressed in B but not A
Expressed in A but not B
Each spot contains
defined DNA
representing a small
portions of the genome
B) Proteomics - The systematic analysis
of proteins for their identity, quantity
and function. As with transcriptomics,
it provides a “snap shot”, in this case of
the proteins at the moment of
extraction. Basic methodology: as in
Fig opposite i.e. Separation by 2D
PAGE, identification through protein
extraction followed by either peptide
mass fingerprint or peptide sequence.
Usually used to identify differentially
expressed proteins and regulation on
the translational level.
SDS-PAGE (mw)
SDS-PAGE (mw)
Quantification
Isoelectric focusing (pI)
2D gel
electrophoresis
Sample
Analysis
Identification
Excise a spot and digest with a protease
(trypsin, pepsin etc)
Peptide mixture from one spot
MALDI-TOF-MS
Peptide mass fingerprint
ESI-MS/MS
Peptide sequence
Protein sequence
General flow for metabolmic analysis
High performance liquid
chromatography (HPLC)
Separation
Gas chromatographymass spectrometry (GC-MS)
Mass spectroscopy (MS)
Identification/
Quantification
C) Metabolomics - the systematic study
of the unique chemical fingerprints that
specific cellular processes leave
behind. Specifically, the study of
small-molecule metabolite profiles. As
in A) and B) it provides a “snap shot”,
at the moment of extraction. Basic
identification by MS or NMR. Usually
used to identify changes in metabolite
levels under certain condition.
General flow for proteomics analysis
Nuclear magnetic resonance
(NMR)
---------------------------------------------------------------------------------------------------------------9
Q6)
A) What is genomics?
B) Describe how bacterial genomes are sequenced today.
C) Describe the kind of information that having the genome sequence of an organism can
provide you with.
---------------------------------------------------------------------------------------------------------------Q7.
A) What is transcriptomics?
B) Describe how you could use transcriptomics to determine which transcripts are altered in a
mutant bacterial strain that lacks the regulatory protein X.
---------------------------------------------------------------------------------------------------------------Q8.
A) What is proteomics?
B) Describe how you could use proteomics to determine alterations in bacterial gene
expression in response to two different growth conditions.
----------------------------------------------------------------------------------------------------------------Q9.
A) What is metabolmics?
B) Describe how you could use metabolmics to determine if CRP is likely to be in its active
DNA binding form or not.
----------------------------------------------------------------------------------------------------------------
10
Jörgen Johansson: Transcriptional termination, attenuation and riboswitches
(new lecture for 2011)
Q1. What is fundamental difference between attenuation regulation and riboswitch regulation?
A. Attenuation regulation sense the level of metabolites indirectly (differences in transcription and
translation), whereas riboswitch regulation sense the amount of metabolites directly, through direct
binding to the RNA-structure (aptamer) causing a regulatory outcome.
-----------------------------------------------------------------------------------------------------Q2. How can analogs of metabolites that normally bind specific riboswitches, function as possible
novel antibiotics?
-----------------------------------------------------------------------------------------------------Q3. How does the pyrBI attenuation mechanism take place?
-----------------------------------------------------------------------------------------------------Q4. What are the main differences between rho-dependent and -independent transcriptional
termination?
11
Jörgen Johansson: Regulation of mRNA stability and function
Q1. In E. coli the enzyme RNaseE is part of a multiprotein complex, “the Degradosome”,
which is implicated in mRNA degradation. What other three types of enzymatically active
proteins are found in the complex? (Xp).
-----------------------------------------------------------------------------------------------------Q2. The stability of mRNA and its processing in bacteria is determined by several parameters.
Circle the correct answer for the three most important parameters:
A) RNases, transcription efficiency, translation efficiency
B) Transcription efficiency, mRNA structure, RNAses
C) mRNA structure, transcription efficiency, translation efficiency
D) RNases, translational efficiency, mRNA structure
E) All of the above
-----------------------------------------------------------------------------------------------------Q3. What are the main parameters determining mRNA stability? (Xp)
-----------------------------------------------------------------------------------------------------Q4. What are the two main types of RNAses, and how do they act?. illustrations are
encouraged (Xp)
-----------------------------------------------------------------------------------------------------Q5. Describe how a small regulatory RNA such as DsrA, can both stimulate and inhibit
translation from different bacterial mRNAs. Illustrations are encouraged (Xp).
-----------------------------------------------------------------------------------------------------Q6. Describe one method to determine the mRNA half-life of a specific transcript in the
bacteria. Illustrations are encouraged (4p).
-----------------------------------------------------------------------------------------------------A (in note form), Figure also expected
Add rifampicin (block function of RNAP)
Harvest bacteria at different time-points after addition of rifampicin
Extract RNA
Run Northern, probe against transcript of interest
Plot amount of transcript against time
Calculate the time when half the amount of the transcript has been degraded = T1/2
-----------------------------------------------------------------------------------------------------Q7. An ribonuclease in Bacillus subtilis has been identified that show a novel type of function.
What is this type of specific nucleolytic activity called and what is the name of the enzyme?
-----------------------------------------------------------------------------------------------------
12
Matthew Francis: Bacterial protein export systems
Q1. Bacteria have evolved multiple pathways for protein transport into, and beyond, the
bacterial envelope. Often these pathways can be connected by common conserved features.
Connect the following pairs of matching statements by drawing a connecting line between the
two. (3p)
Statement 1
Statement 2
Gram-positive bacteria
Chaperone/usher pathway
T3SS
Sortases for protein anchoring
Type 1 pilus biogenesis
DNA conjugation
T4SS
Solute influx and efflux systems
T2SS
Flagella mediated motility
T1SS
Secretion of folded proteins
Statement 1
Statement 2
Gram-positive bacteria
Chaperone/usher pathway
T3SS
Sortases for protein anchoring
Type 1 pilus biogenesis
DNA conjugation
T4SS
Solute influx and efflux systems
T2SS
Flagella mediated motility
T1SS
Secretion of folded proteins
A
-----------------------------------------------------------------------------------------------------------------
13
Q2. Answer whether the following statements are true (T) or false (F)
a) Bacteria evolved protein export and secretion systems to overcome the physical
barriers imposed by the cytoplasmic and outer membranes.
b) Gram-positive bacteria possess dedicated systems for protein secretion through their
outer membrane.
c) SRP-dependent protein export to the periplasm is a feature common to both Gram
positive and Gram negative bacteria.
d) With the appropriate recognition sequences, periplasmic protein intermediates can be
secreted across the outer membrane via a T2SS, T5SS or a chaperone/usher (CU)
pathway.
e) T3SS and T4SS have the capacity to transport proteins directly from the bacterial
cytoplasm into an infected eukaryotic cell.
f) Requiring only two components to mediate protein secretion, the CU pathway is the
most simple secretion system known.
-----------------------------------------------------------------------------------------------------------------
14
Q3. With respect to bacterial protein export pathways, answer the following multiple choice
questions (½ point each; only one answer is correct per question)
a) Which of the following is NOT true about the SRP-dependent export pathway?
1) common to both Gram positive and Gram negative bacteria
2) requires energy through the hydrolysis of ATP
3) responsible for the export of integral inner membrane proteins
4) a direct interaction between SRP and the Sec-translocon in the inner membrane
mediates substrate export
5) results in an altered form of the exported protein
6) SRP is a complex composed of both a protein and a RNA molecule
b) Which of the following is NOT true about the Tat-dependent export pathway?
1) fully folded proteins are exported into the periplasm
2) a general cytoplasmic chaperone targets substrates to the Tat-translocon in the inner
membrane
3) is conserved even in distantly related bacteria
4) folding and activity of substrates may require cytoplasmic co-factors
5) substrates usually contain a pair of arginine residues at the N-terminus
6) may contribute to the process of two-step protein secretion in Gram negative bacteria
c) Which of the following is NOT true about the Sec-dependent export pathway?
1) requires the SecB chaperone for substrate folding
2) is associated with Gram positive bacteria
3) is energised by SecA-dependent hydrolysis of ATP
4) the SecYEG translocon forms a transport channel in the inner membrane
5) substrates are altered by the activity of a signal peptidase during export
6) important export signals are located at the substrate N-terminus
d) Which of the following is NOT true about protein export in Gram positive bacteria?
1) special mechanisms for compartmentalization of exported proteins is due to the
absence of an outer membrane
2) exported proteins may possess cell wall anchor (CWA) sequences at their C-terminus
3) exported proteins lacking CWA sequences are freely secreted to the environment
4) sortases are unique to these bacteria
5) sortases cleave CWA sequences and then covalently link these altered proteins to the
inner membrane
6) type I secretion contributes to protein export in Gram positive bacteria
-----------------------------------------------------------------------------------------------------------------
15
Q4. With respect to bacterial protein secretion pathways, choose the ONE alternative that
BEST completes the statement. (½ point each)
a) Type I protein secretion systems:
1) are unique to Gram negative bacteria
2) are related to nutrient influx and waste efflux systems
3) secrete substrates in two steps
4) are independent of ATP hydrolysis
5) secrete substrates with a N-terminal secretion signal
6) all of the above
b) Type II protein secretion systems:
1) secrete substrates in a one-step mechanism
2) are related to type 1 pilus assembly machines
3) recognize unfolded periplasmic intermediates
4) directly deliver toxic substrates into the cytosol of target eukaryotic cells
5) represent a terminal branch of the Sec-dependent and Tat-dependent export pathways
6) all of the above
c) Type III protein secretion systems:
1) secrete at least two protein classes – the translocators and the effectors
2) secrete substrates that have no periplasmic intermediate
3) are responsive to contact with eukaryotic cells
4) are dependent on a dedicated cytoplasmic chaperone for the controlled secretion of
each substrate
5) can be used as a tool for heterologous antigen delivery in vaccine development
6) are all of the above
d) Type IV protein secretion systems:
1) Function in cell adhesion, twitching motility and DNA uptake
2) are universally found among all Gram negative bacteria
3) recognise substrates by their N-terminus
4) are able to translocate protein and protein-DNA complexes into eukaryotic cells
5) are a descendent of the bacterial flagella secretion organelle
6) all of the above
e) Type V protein secretion systems:
1) are highly promiscuous, each capable of secreting large numbers of different
substrates
2) are primarily responsible for the secretion of integral outer membrane proteins
3) are a solution to the problem of secreting highly toxic proteins
4) establish a pore in the outer membrane through which the remainder of the substrate
transits to gain access to the outside environment
5) secrete protein unchanged from the translated form
6) all of the above
f) Chaperone-usher secretion systems:
1) require a periplasmic chaperone and an outer membrane usher for the assembly of
multi-subunit fibres on the bacterial surface
2) are dependent on the Sec-transocon
16
3) are found only in Gram negative bacteria
4) often contribute to adhesion or immune evasion strategies
5) rely on varying affinity of chaperone-substrate interactions to establish ordered
secretion of substrates
6) are all of the above
Q5. Answer whether the following statements are true (T) or false (F). Each correct answer
is worth 0.5p (Total: 4p).
a) Complex protein secretion systems with specialized functions have often evolved from
ancestral systems involved in routine housekeeping tasks.
b) Despite notable differences in cell wall structures of Gram positive and Gram negative
bacteria, systems of protein export into or across the inner membrane remain
universal.
c) All bacterial protein export and secretion systems depend upon ATP hydrolysis for
substrate transport.
d) Substrates of the T1SS contain a Sec-dependent N-terminal secretion signal.
e) Type IV pili structures are assembled via a chaperone/usher secretion system.
f) All T2SS substrates fold only after they have gained access into the periplasm via the
Sec or Tat export systems.
g) T4SS can transport cargo into both eukaryotic cells and bacterial cells.
h) The Bam complex is required for outer membrane insertion of the -barrel domain
associated with substrates of T5SS.
-----------------------------------------------------------------------------------------------------------------
17
Q6. Bacteria possess multiple pathways for protein transport into, and beyond, the bacterial
envelope. Concerning some of these systems, a series of short statements are given below.
Draw lines to connect the best matching statements on the left to those on the right. (0.5p for
each correct answer)
Statement 1
Statement 2
A system dedicated to the injection of proteins into
eukaryotic cells
Chaperone affinity for substrate dictates a secretion
hierarchy that is critical for assembly
Inner membrane located ATP binding cassette proteins
are integral components
Individual substrates require cognate chaperones
Adhesive organelles for tight binding to eukaryotic
cells
A secretion signal for substrate recognition exists in the
tertiary structure
Gram-positive bacteria lack an outer membrane
A chaperone, such as SecB, might be required to
maintain substrates in an unfolded state competent for
Sec-independent secretion
Substrates enter the secretion system as folded
periplasmic intermediates
These substrates either remain attached at the outer
membrane or are released free from the bacterial
surface
Substrates are recognised on the basis of a C-terminus
enriched in positively charged amino acids
Additional factors ensure proper insertion of these
proteins into the inner membrane
SRP-dependent
translocon
Sortases are required for covalently linking proteins to
the cell wall
protein
export
utilises
the
Sec-
Also a system for the transfer of DNA and proteins into
bacterial cells
Restricted to the secretion of a single substrate
----------------------------------------------------------------------------------------------------------------Q7. Bacteria possess multiple pathways for protein transport into, and beyond, the bacterial
envelope. Briefly describe three (3) biotechnological applications that can benefit from a
detailed knowledge of one (or more) of these systems.



Protein purification – link protein with a secretion signal to mediate secretion to the
extracellular environment
Vaccine development (antigen delivery) – link protein with a secretion signal to
deliver antigen to the cell cytosol for MHCI or MHCII presentation (can dictate
whether you achieve an antibody response or a cell-mediated response)
Secretion systems as targets for novel antibiotic development (examples given in
lecture were inhibition of T3SS or CU secretion system)
----------------------------------------------------------------------------------------------------------------Q8. Gram-negative and Gram-positive bacteria display fundamental differences in the
anatomy of their bacterial envelope.
a) Briefly describe these differences.
(1p)
18
b) Briefly describe how this anatomical difference impacts on the choice of protein export
pathways utilised by the two broad bacterial groups for protein transport into or across the
inner membrane.
(1p)
c) Briefly describe how this anatomical difference impacts on the choice of protein secretion
pathways utilised by the two broad bacterial groups for protein transport into the
extracellular space.
(3p)
19
Matthew Francis: Regulation of extra-cytoplasmic components in bacteria
Q1. Industrial scale protein purification strategies are a compromise between the goals of high
purity, high yield and low cost. How might you utilize knowledge of the bacterial stress
response to modify your chosen purification strategy, in order to improve the quality and
quantity of your product?
A (in note form)
Stress response monitoring:
recombinant protein production induces cellular stress
-a stressed cell performs poorly – low protein yield
-use transcriptomic or proteomic methods to develop a genetic blueprint of the stress response
-transcriptional fusions of key gene promoters to monitor in ‘real-time’ culture health during
protein production
-Use of chaperones and folding factors: co-express with recombinant protein will help
recombinant protein to reach native, biologically active, confirmation
Control of proteolysis:
protein degradation – low protein yield
reduce the risk of proteolysis at the:
protein level (modify the protein sequence by mutagenesis or fusion technologies; engineer it
to be secreted outside)
cellular level (protease deficient strains)
culturing conditions (physiochemical alterations, protease
----------------------------------------------------------------------------------------------------------------Q2. Biogenesis of the bacterial envelope requires the coordinated synthesis and assembly of
several components, such as outer membrane proteins, lipopolysaccharide, phospholipids,
lipoproteins and peptidoglycan. Errors in the assembly of one or more of these components
generate extracytoplasmic stress that is poorly tolerated by bacteria.
State the name and regulatory control mechanisms of the two major regulatory systems
located in the bacterial envelope that maintain quality control during envelope biogenesis.
----------------------------------------------------------------------------------------------------------------Q3. Incorrectly folded proteins are damaging to bacteria. Bacteria therefore possess quality
control mechanisms that assist and oversee protein folding. Briefly describe four functionally
distinct protein categories within the periplasmic quality control toolkit that function to watch
over biogenesis of the bacterial envelope. (4p)
-----------------------------------------------------------------------------------------------------------------
20
Q4. With respect to the extracytoplasmic compartment in bacteria, which of the following
statements is false? (½ point each – 3 points in total; only one correct answer per statement)
A) Bacterial extracytoplasmic components.......
1) form a compartment that contributes to cell shape and protects cytoplasmic contents
from the environment
2) includes phospholipids, lipopolysaccharide, peptidoglycan, lipoproteins and outer
membrane proteins
3) are likely to be essential for bacterial viability because deleting their relevant gene(s)
is often not possible
4) are produced in the periplasm during extracytoplasmic stress
5) undergo final assembly in the absence of ATP
6) can function cooperatively to ensure their correct localization and activity
B) Bacterial phospholipids.......
1) consist of two fatty acid chains and a glycerol head group that might also be modified
by the addition of a phosphoryl group
2) routinely aggregate to form lipid bilayers in aqueous solution
3) modify their fatty acid chain length to regulate membrane viscosity during bacterial
growth in low temperature
4) contribute to proton motive force generation through the creation of a charge
differential across the cytoplasmic membrane
5) are required for stable insertion of proteins in the outer membrane
6) form bilayers naturally permeable to the transport of nutrients into and out of the cell
C) Lipopolysaccharides.......
1) are a core component of the RpoE and CpxR~P regulon in both non-pathogenic and
pathogenic bacteria
2) can contain a hypervariable repeating sugar unit that extends outward from the
bacterial surface
3) require ABC transporters to assist with delivery to the outer membrane
4) consist of an invariant lipid A membrane anchor essential for viability in all bacteria
5) at the bacterial surface provide protection against attack from various antimicrobial
agents
6) are endotoxigenic
D) Bacterial lipoproteins.......
1) are integral membrane proteins that need not penetrate completely through the lipid
bilayer
2) may constitute ~10% of the proteins in a typical bacterial genome reflecting the
multiple functions they perform
3) are lipid linked at an invariant cysteine residue near their N-terminus
4) are exported to the periplasm as unfolded substrates
5) with an aspartate residue at their N-terminus are retained at the cytoplasmic membrane
6) form connecting bridges between the peptidoglycan layer and the inner and outer
membranes
F) Bacterial porins.......
1) are a major protein component of the outer membrane
2) generally function as trimers
21
3)
4)
5)
7)
cause bacterial osmotic lysis when they are not gated (closed)
are water-filled
are involved in small solute transport
penetrate completely through the outer membrane
G) Quality control in the bacterial envelope.......
1) requires specialist regulatory pathways responsive to protein misfolding in the
periplasm
2) is dependent upon protein folding and degradation factors that function in the
periplasm
3) can affect the efficiency of protein secretion into the extracellular environment
4) requires protein export across the inner membrane
5) is affected by phase variation
6) is controlled by regulatory pathways that are feedback inhibited
-----------------------------------------------------------------------------------------------------------------
22
Q5. With respect to extracytoplasmic stress (ECS) sensing in bacteria, which of the following
statements is false? (0.5 point each – 3 points in total; only one correct answer per statement)
A) Bacterial extracytoplasmic stress.......
1)
affects protein folding in the periplasm
2)
may disrupt outer membrane integrity
3)
can reduce ATP energy levels in the cytoplasm
4)
can cause cell death if relief mechanisms are not activated
5)
is induced by outer membrane protein overexpression
6)
is commonly referred to as the heat shock response, but in fact, can involve multiple
physio-chemical stresses
B) The Cpx pathway.......
1)
is a good example of a simple two-component sensor kinase/response regulator
signalling mechanism
2)
consists of the membrane-localized CpxA and the cytoplasmic-localized CpxR
3)
when activated, can amplify other ECS responsive pathways
4)
is both auto-amplified by CpxP and feed-back inhibited by CpxR
5)
is regulated by the dual kinase and phosphatase activities of CpxA
6)
activity is dependent on ATP hydrolysis
C) PPIases.......
1)
are often jointly regulated by the RpoE and Cpx pathways in response to ECS
2)
target proteins with multiple proline residues
3)
are not restricted to a function in the periplasm
4)
enhance cis/trans isomerisation of peptidyl-prolyl bond rotation to influence protein
folding
5)
are inactive in the absence of ECS
6)
are required for proper OMP assembly
D) Members of the Dsb protein family.......
1)
can be either integral inner membrane proteins or periplasmic proteins
2)
target proteins as they emerge from the SecB- and Tat-dependent export pathways
3)
establish disulphide bond formation in proteins containing two cysteine residues
4)
exchange disulphide bonds between different pairs of cysteine residues
5)
participate in redox reactions and electron transfer
6)
perform protein folding functions in the periplasm regardless of the bacterial growth
phase or growth conditions
E) The DegP protein.......
1)
is responsible for activation of the RpoE pathway through regulated proteolysis of the
membrane-localised anti-RpoE factor (RseA)
2)
binds substrates through C-terminal PDZ domains
3)
PDZ domains block access to the enzymatic site of DegP
4)
functions as a protease degrading misfolded proteins, but can also refold proteins with
chaperone activity
5)
has an important function during bacterial growth at high temperature
6)
characterizes a family of related proteins all with at least one PDZ domain
23
F) Predicting ECS responsive regulons.......
1)
may uncover novel virulence factors required for the survival of bacterial pathogens in
the infected host
2)
will benefit our understanding of how bacterial pathogens cope with stress
3)
first requires a good understanding of the promoter architecture of known responsive
genes
4)
might enable the creation of individual biosensors to monitor real-time stress levels in
a bacterial culture grown to overproduce a commercially valuable recombinant protein
5)
is most useful for the study of sigma factors with known promoter binding sites
6)
could identify novel protein folding or assembly factors
----------------------------------------------------------------------------------------------------------------Q6. With respect to the bacterial extracytoplasmic compartment, which of the following
statements is false? Read them carefully! (½ point each – 3 points in total; only one correct
answer per statement)
A) Bacterial extracytoplasmic components.......
1.
form a compartment that contributes to cell shape and protects cytoplasmic contents
from the external environment
2.
includes phospholipids, lipopolysaccharide, peptidoglycan, lipoproteins and outer
membrane proteins
3.
are quite often essential for bacterial viability
4.
are produced in the periplasm during extracytoplasmic stress
5.
undergo final assembly in the absence of ATP
6.
can function cooperatively to ensure their correct localization and activity
B) Bacterial phospholipids.......
1.
consist of two fatty acid chains and a glycerol head group that might also be modified
by the addition of a phosphoryl group
2.
routinely aggregate to form symmetrical lipid bilayers in aqueous solution
3.
modify their fatty acid chain length to regulate membrane viscosity as a bacterial ploy to
adapt to certain environmental stresses
4.
contribute to proton motive force generation by maintaining a charge differential across
the cytoplasmic membrane
5.
actively pump out protons into the periplasm to establish a proton motive force
6.
function as lipochaperones for the folding of proteins in the membrane
C) Bacterial lipopolysaccharides.......
1.
are a core component of the RpoE and CpxR~P regulon in both non-pathogenic and
pathogenic Gram-negative bacteria
2.
can contain a hypervariable and immunodominant repeating sugar unit that extends
outward from the bacterial surface
3.
require a type I secretion system (T1SS) to assist with their delivery to the outer
membrane
4.
consist of an invariant lipid A membrane anchor essential for viability in almost all
Gram-negative bacteria
5.
at the bacterial surface provide protection against attack from various antimicrobial
agents
6.
are necessary for the correct folding of integral outer membrane proteins
24
D) Bacterial lipoproteins.......
1.
with an aspartate residue at their N-terminus, lipoproteins will be exported by the Lol
complex to the outer membrane
2.
are peripheral membrane proteins that need not penetrate completely through the
membraneous lipid bilayer
3.
are a common component of the bacterial envelope reflecting the multiple and diverse
functions they may perform
4.
are lipid modified at an invariant cysteine residue near their N-terminus
5.
are exported as unfolded substrates to the periplasmic face of the inner membrane by the
Sec-translocon
6.
form connecting bridges between the peptidoglycan layer and the inner and outer
membranes
F) Bacterial porins.......
1.
are a major protein component of the outer membrane
2.
are inserted into the outer membrane as monomers, but generally function as trimers
3.
are closed (gated) when not in use to prevent the bacteria from undergoing osmotic lysis
4.
are water-filled
5.
are involved in small solute transport
6.
form an open conduit that spans the entire outer membrane
G) Quality control in the bacterial envelope.......
1.
is controlled via parallel regulatory pathways located in the inner membrane that are
responsive to misfolded proteins present in the periplasm
2.
is dependent upon protein folding and degradation factors that function in the periplasm
3.
can affect the efficiency of protein secretion into the extracellular environment
4.
operates in an ATP vacuum
5.
functions independently of protein export systems such as the Sec-translocon
6.
is controlled by regulatory pathways that are feedback inhibited
-----------------------------------------------------------------------------------------------------------------
25
Q7. With respect to extracytoplasmic stress (ECS) sensing in bacteria, which of the following
statements is false? (½ point each – 3 points in total; only one correct answer per statement)
A) Bacterial extracytoplasmic stress.......
1.
induces protein unfolding in the periplasm
2.
may disrupt outer membrane integrity
3.
can reduce ATP energy levels in the cytoplasm due to disruption of the proton motive
force
4.
can cause cell death if relief mechanisms are not activated
5.
is induced by outer membrane protein over-expression
6.
is overcome by elevated levels of cytoplasmic chaperones and proteases induced by an
activated RpoH (32) sigma factor
B) The Cpx pathway.......
1.
is a good example of a simple two-component sensor kinase/response regulator twocomponent regulatory system
2.
consists of the membrane-localized CpxA sensor kinase and the cytoplasmic-localized
CpxR response regulator
3.
when activated, can amplify other ECS responsive pathways
4.
is auto-amplified by a positive feedback loop involving CpxP
5.
relies on the phosphorylation of CpxR for activation
6.
phosphorylated CpxR can act as both a repressor and an activator of gene transcription
C) PPIases.......
1.
are often jointly regulated by the RpoE and Cpx pathways in response to ECS
2.
commonly target proteins with multiple proline residues
3.
are not restricted to a function in the periplasm
4.
enhance cis/trans isomerisation of peptidyl-prolyl bond rotation to influence protein
folding
5.
are transported across the inner membrane via a flip-flop mechanism involving ATP
binding cassette proteins
6.
are required for proper OMP assembly
D) Members of the Dsb protein family.......
1.
can be either integral inner membrane proteins or soluble periplasmic proteins
2.
target proteins as they emerge from the Tat-dependent export pathway
3.
establish disulphide bond formation in proteins containing at least two cysteine residues
4.
can exchange disulphide bonds between different pairs of cysteine residues within a
protein
5.
participate in redox reactions and electron transfer
6.
perform protein folding functions in the periplasm regardless of the bacterial growth
phase or growth conditions
E) The DegP protein.......
1.
is responsible for activation of the RpoE pathway through regulated proteolysis of the
membrane-localised anti-RpoE factor (RseA)
2.
binds to substrates through its C-terminal PDZ domains
3.
contains two PDZ domains that act as ‘gatekeepers’, blocking access of potential
substrates to the enzymatic site of DegP
4.
functions as a protease degrading terminally misfolded proteins in the periplasm
26
5.
6.
functions as a chaperone refolding proteins in the periplasm
was originally identified as a high temperature regulated protein
F) Using bioinformatics approaches to predict whole-genome ECS responsive
regulons.......
1.
in bacterial pathogens may uncover novel virulence factors required for survival in an
infected host
2.
will benefit our understanding of how bacterial pathogens cope with stress
3.
first requires a good understanding of the promoter architecture of known responsive
genes
4.
might enable the creation of individual biosensors to monitor real-time stress levels in a
bacterial culture grown to overproduce a commercially valuable recombinant protein
5.
will identify all extracytoplasmic components that function in the bacterial envelope
6.
could identify novel protein folding or assembly factors
--------------------------------------------------------------------------------------------------------------Q8. In what ways might the bacterial envelope influence fundamental processes of the
bacterial cell macromolecular machinery?
A: (in note form)
1. Protects the macromolecular machinery from the harmful external environment
2. Acts as a permeability barrier, controlling the flow of essential ions
3. Is an assembly platform for integral membrane proteins necessary for:
 Solute influx and efflux
 Protein export/secretion
 Electron transport chain
 Signal transduction (the nerve centre of the cell)
 Cell division
4. Establishes and maintains a proton motive force
27
Tracy Nissan: Bacterial translation and control of protein synthesis (new 2011)
Q2. During the elongation process on the ribosome the tRNA moves to three physically different sites
on the ribosome. If we say that tRNA is bound at a P/A-site, how is it bound to the translating 70S
ribosome? Describe in words and draw a figure. (1p)
-----------------------------------------------------------------------------------------------------Q3. During translation of bacterial mRNA, which reactions are catalyzed by the elongation factors EFTu and EF-G?
-----------------------------------------------------------------------------------------------------Q4. During bacterial translation, the charged tRNA (aa-tRNA) is transported to the ribosome as a
“ternary complex” consisting of aa-tRNA, EF-Tu, and GTP. This complex binds with the anticodon to
the codon in the A-site and the aminoacyl end of the tRNA to a place on the 50S subunit, which is
distinct to the A-site of the 50S. Following the hydrolysis of GTP on the ribosome the aa-tRNA is
accommodated by a movement of its 3’-end to the A-site on the 50S ribosomal subunit. What is it that
induces this movement of the 3’ end of the tRNA; i.e. the accommodation step?
-----------------------------------------------------------------------------------------------------Q5. What is a modified base? What is their purpose? Give an example of a modified base and where
it comes from
-----------------------------------------------------------------------------------------------------Q6. What is a reoccurring principle of bacterial translation regulation? Give two examples and explain
how they operate to control translation.
-----------------------------------------------------------------------------------------------------Q7. How can bacteria with less than 61 tRNA be able to read all possible sense codons (i.e. those for
amino acids)? Describe the reasoning why this would occur.
A. tRNA anti-codons can wobble. The base pairing of the interactions are strong for the first two
nucleotides for the codon, but promiscuous on the last nucleotide. This is enhanced by the use of
modified bases, which can further base pair to more nucleotides.
-----------------------------------------------------------------------------------------------------Q8. Describe the steps in which amino acids become attached to tRNA. Where does this occur? How
is this process made more accurate? What steps does this increased accuracy involve and why?
-----------------------------------------------------------------------------------------------------Q9. Why can bacterial mRNA carry the information for many genes on it? Explain and justify your
answer.
------------------------------------------------------------------------------------------------------
28
Q10. What is the RNA World Hypothesis? What are two examples that we can observe in translation?
A. The RNA world hypothesis proposes that self-replicating RNA molecules were precursors to
current life, which is based on DNA, RNA and proteins. Evidence supporting this hypothesis is that
many essential molecules of translation are composed of RNA such as tRNA and Ribosomes; The
ribosome is a catalytic RNA or ribozyme.
-----------------------------------------------------------------------------------------------------Q11. What is an example of a G protein that plays a role in translation? How do they work, and what
intrinsic property allows them to function.
-----------------------------------------------------------------------------------------------------Q12. Which of the following pairs of codons might you expect to be read by the same tRNA
as a result of wobble? Justify you choice.
(a)
CUU and UUU
(b)
GAU and GAA
(c)
CAC and CAU
(d)
AAU and AGU
-----------------------------------------------------------------------------------------------------Q13. Figure A below shows the stage in translation when an incoming aminoacyl-tRNA has
bound to the A-site on the ribosome. Using the components shown in figure A as a guide,
show on Figure B and C what happens in the next two stages to complete the addition of the
new amino acid to the growing polypeptide chain.
Answer as in Fig. below
29
Victoria Shingler: Protein expression systems: bacterial
Q1.
A) Define a fusion protein and a fusion tag.
B) explain why His-tagging of proteins is a commonly used strategy for purifying proteins.
Answer
Fusion proteins are created by a translational fusion of the coding sequence for the protein of
interest to a gene encoding a highly expressed protein partner. In other words, two genes are
fused into one transcript instead of two transcripts.
Fusion proteins are chosen because they are well expressed, highly soluble, and have
characteristics that aid purification. All of these characteristics may be conferred to your
protein of interest aiding its over-expression and purification. For His tags, specifically add
information on Ni (or other metal affinity purification).
----------------------------------------------------------------------------------------------------Q2. Controllable transcriptional promoters are a necessary component of protein overexpression vectors for both bacterial and eukaryotic systems.
The T7 RNA polymerase promoter is a very popular choice for expression vectors used in E.
coli.
A) Explain why the T7 promoter is a popular choice
B) Explain how protein expression from a T7 promoter is controlled such as systems.
-----------------------------------------------------------------------------------------------------Q3. From a bacterium that grows with a doubling time of 1 week, you have finally managed
to clone a gene that encodes a protein that degrades plastic. The biotechnical company that
you work for is very interested in this protein. They have given you the task of obtaining a
large quantity of this protein in a pure and active state. Briefly describe the experimental
design (i.e. give the necessary steps) that you would use to get a pure and active wild-type
protein in less than 1 week using a bacterial expression system. Explain why you made these
choices. ( p)
A (in note form)
There are several options here that can be right - so long as the student justifies their answer and
gets most of the steps. The anticipated answer is use an E. coli expression system using a His-tag
or some other type of single step purification.
Clone the gene encoding your plastic protein into an expression vector so that it is:
1-Place the target gene under the control of an inducible promoter (Should preferably say what
type of promoter e.g. T7). The promoter allows you to control when the protein is over expressed
in case it is toxic to the cell in large amounts.
2-Fused the gene (in-frame) to a DNA sequences encoding a His polypeptide (6 or more
consecutive histidine codons) so that the protein will be tagged and can be purified easily (there
are other tags that can be used). The His-tag allows easy one step purification from a bacterial cell
lysate.
3-Also incorporate the DNA sequence encoding a protease cleavage site located such that the
fused amino acid of the His-tag can be cleaved away when treated with the protease. This will
allow purification of your native protein by removing the His-tag.
30
Once the cloning is done then:
4-You must choose an appropriate E. coli strain for your protein expression system. The
expression vector was also chosen so that the work may be done in E. coli.
5-You will use affinity chromotography to purify the protein based on what type of fusion system
was chosen for easy purification. With the His-tag, the nickel agarose can be used.
6-Once purified, cleave off the fused amino acid sequences with the protease and pass the protein
back through the nickel agarose to purify your protein away from the cleaved product.
------------------------------------------------------------------------------------------------------
Q4.
A) Define a fusion tag and explain why this strategy is often chosen for protein expression
systems.
B) Give four reasons why Escherichia coli is usually the organism of choice when choosing a
protein expression system?
C) What are two main disadvantages of bacteria expression systems?
-----------------------------------------------------------------------------------------------------Q5. With the aid of an illustration, describe the important features of a bacterial expression
plasmid to be used for high level expression of a protein. Be sure to also describe in words
what each of the features you illustrate is for. (X p)
------------------------------------------------------------------------------------------------------
31
Yuri Schwartz: Eukaryotic transcriptional control (new lecture 2012)
Q1. Specific DNA sequences within eukaryotic chromosomes are involved in controlling
gene expression. Describe how the following three DNA elements operate and their influence
on the transcriptional process.
i) enhancer elements
ii) insulator elements, and
iii) promoters
----------------------------------------------------------------------------------------------------------------------
Q2. In yeast TBP protein can be part of general transcription factor TFIID or function
independently of TAFs but in concert with SAGA complex. Is there a preference for TATAbox containing and TATA-less promoters for either of these complexes? Explain why.
A in short form:
Promoters with TATA box tend not to bind TFIID but have TBP binding in concert with
SAGA. In this case SAGA is recruited by co-activator and helps to remodel chromatin to
evict nucleosome from TATA-box and TSS. TATA-less promoters have an element weakly
resembling TATA-box (TATA-like element). They preferentially associate with TFIID. In
this case some of the TAFs are co-recruited by co-activators and this stabilizes the binding of
TBP to otherwise low affinity DNA binding sequence. In addition TAFs compete with +1
nucleosomes, which helps to keep TSS open.
----------------------------------------------------------------------------------------------------------------------
Q3. Briefly outline the concept of a eukaryotic transcription factory.
----------------------------------------------------------------------------------------------------------------------
Q4. Which part of the histone molecule gets most extensively post-translationally modified?
And what kinds of modifications are known to occur?
----------------------------------------------------------------------------------------------------------------------
32
Ruth Palmer: Manipulating gene expression via RNA interference (new lecture 2012)
Q1. What is RNA interference? Describe the mechanism involved.
Answer:
RNAi is a PTGS mechanism which leads to the degradation of the targeted mRNA.
Description of the mechanism should include:
- dsRNA
- processing by Dicer (nuclease) to form siRNAs (21-25nt)
- RISC complex (large complex, triggers mRNA degradation in response to siRNA;
contains helicase, nuclease etc), requirement for ATP
- homologous mRNA target recognition
- target cleavage
- dsRNA can destroy an excess of mRNA – (pointing to an enzymatic mechanism)
- dsRNA needs to be directed towards an exon of a gene to by effective
- need homology between dsRNA and the target gene/mRNA
-----------------------------------------------------------------------------------------------------Q2. Compare and contrast siRNA and miRNA and their effects
-----------------------------------------------------------------------------------------------------Q3. You have just performed a microarray experiment and found several genes with unknown
function. One of the genes (gene X) encodes a protein which has recent been connected to a
human disease. You would like to examine the function of gene X by employing PTGS.
A) What is meant by PTGS?
B) How would you go about characterising the function of gene X in mammalian cells
experimentally? (4p)
-----------------------------------------------------------------------------------------------------Q4: Genetic approaches are powerful tools to analyse the importance of different gene
products involved in the control of gene expression.
A) Describe what is meant by the following terms forward genetics and reverse genetics.
B) Describe how RNAi (RNA interference) can be used as a tool in forward genetics by
outlining the experimental procedure that you would used for ONE of the following model
systems: C. elegans, Drosophila, OR mammalian cells. (p)
-----------------------------------------------------------------------------------------------------Q5: Genetic approaches are powerful tools to analyse the importance of different gene
products involved in the control of gene expression.
A) Describe what is meant by the following terms forward genetics and reverse genetics.
B) Describe how RNAi (RNA interference) can be used as an experimental tool in reverse
genetics (p)
------------------------------------------------------------------------------------------------------
33
Tracy Nissan: The eukaryotic ribosome and regulation of protein synthesis (new 2011)
Q1. Describe the principal difference of cap-dependent initiation of translation in eukaryotes
and initiation of translation in bacteria? (p)
------------------------------------------------------------------------------------------------------
Q2. Why is phosphorylation of initiation factor eIF-2 inhibitory for initiation of eukaryotic
translation? (p)
Answer
GTP/ GDP exchange factor eIF2B regenerate eIF2; GTP from eIF2; GDP. Ternary complex
eIF2;GTP;met-met-tRNA Met/I, required for translational initiation can only be formed from
eIF2; GTP. In the cell, the concentration of eIF2; GDP is higher than eIF2B. If eIF2; GDP is
phosphorylated the exchange reaction is inhibited as phosphorylated eIF2; GDP has a high
affinity for eIF2B and eIF2B will be titrated out. Thereby no ternary complex eIF2;GTP;metmet-tRNA Met/i can be formed.
-----------------------------------------------------------------------------------------------------Q3. Mention two eukaryotic proteins in which the efficiency of initiation of translation is
affected by their phosporylation status. In each case, describe the molecular mechanism by
which phosphorylation of the initiation factors leads to inhibitory and/or stimulatory effects
on translation. (p)
-----------------------------------------------------------------------------------------------------Q4. What is a polysome?
-----------------------------------------------------------------------------------------------------Q5. What are two regions on a eukaryotic mRNA that can regulate translation? What is the
advantage(s) of each of them? Which is the major one in terms of regulation of translation in
eukaryotes?
-----------------------------------------------------------------------------------------------------Q6. What are two reasons that mRNA decay is important in gene expression?
-----------------------------------------------------------------------------------------------------Q7. What are two advantages of microRNA as a method of translational regulation to the
cell?
34
Martin Gullberg: Protein expression systems: eukaryotic (new lecture for 2011)
Q1. In general, protein expression in eukaryotic systems is less efficient than in bacterial
systems.
A) Why might you still want to express a human protein using a eukaryotic based system?
B) Taking the above considerations in mind, what organism would you choose for your
purposes? Motivate you choice.
-----------------------------------------------------------------------------------------------------Q2. When expressing a protein in a eukaryotic based system, why is getting the protein
secreted in an active form a priority?
------------------------------------------------------------------------------------------------------
Q3. What are the potential differences between proteins expressed in the cytosol of E. coli,
fungi, insect cells, and mammalian cells?
A: in note form
- N-terminal modifications e.g. fMet in bacteria, other types of modifications in
eukaryotic cells. fMet serves as a chemoattractant for phagocytes (a type of innate
immune cell that is present in all metazoans (animals). Other types of modifications
can control the active state and/or influence protein folding.
- In eukaryotic cells, serine or threonine may become phosphorylated (very rare in
bacteria).
- There are also other types of modifications (e.g. methylations) that may differ.
------------------------------------------------------------------------------------------------------------Q4. What are the differences between proteins secreted to the external media from E. coli,
fungi, insect cells, and mammalian cells?
A: in note form
- In all eukaryotic (but not bacterial) cells, proteins passing through the secretory
pathway become glycosylated. This is likely to affect the half-life of the proteins if
subsequently injected into a patient.
-
The specific types of glycosylation differ between fungi, insect cells, and mammalian
cells. This may be amended by transplanting the glycosylation machinery from
mammals into other eukaryotic expression hosts (deleted of their native machinery).
------------------------------------------------------------------------------------------------------------Q5. Discuss what properties of the methylotrophic yeast Pichia pastoris make it useful as a
eukaryotic expression system?
-----------------------------------------------------------------------------------------------------Q6. Why is baculovirus a useful eukaryotic expression system? Motivate your answer.
35
Per Stenberg: Transcriptomics and RNA sequencing (new lecture 2013)
Q1. What are the major advantages and disadvantages with microarray (transcriptomic
analysis) versus RNA-sequence analysis when doing genome wide expression analysis?
Advantages
Disadvantages
Microarray
Cheap, fast, easy analysis
Compressed dynamic range,
only measure predetermined
transcripts
RNA-seq
Potentially better dynamic
range, can measure
unannotated transcripts
Still more expensive,
complicated analysis, very
large data files
-------------------------------------------------------------------------------------------------------Q2. You have access to data with numbers of RNA-seq reads covering each position in the
human genome. You have data obtained from a healthy individual and from an individual
with a disease. Your task is to discover genes that are differentially expressed in the two
individuals.
Explain how you would calculate gene expression values for each gene using the above data
as well as annotation information on all genes in the human genome.
-------------------------------------------------------------------------------------------------------Q3. What can we miss and/or potentially discover when using a reference genome (together
with gene annotation) as compared to when using de novo assembly of RNA-seq reads?
-------------------------------------------------------------------------------------------------------Q4. Explain what technical and biological replicates are and when we need to use them.
-------------------------------------------------------------------------------------------------------Q5. What is the advantage of using more biological replicates in an RNA-seq experiment that
aims to detect genes that are differentially expressed in a mutant as compared to a wild type
sample?
-------------------------------------------------------------------------------------------------------Q6. Why do we need to normalize transcriptomics data sets before comparing them? List at
least two ways how this can be done.
36