Lecture 8 Flashcards
Gene Regulation
List four reasons why cells do not express genes at the maximal level.
- Protein production takes up energy and resources
- Space in the cell is limited
- Some proteins might have contradictory functions
- Other proteins can be detrimental when produced abundantly
True or False: In general, control at the DNA level is the most drastic and least reversible, whereas control at the protein level is the most rapid and most reversible.
True
What is a major site of
regulatory control in bacterial cells?
Transcription initiation
What help cells sense
internal or external changes and alter gene
expression in response?
Regulatory proteins
List four ways regulatory proteins affect gene expression.
- bind DNA, usually upstream of the coding
sequence within the promoter region - Can act as repressors or activators, to
respectively prevent or promote RNA
polymerase binding to promoter - DNA-binding domain interacts with major
groove of DNA - Often form dimers
Regulatory proteins usually interact
with DNA sites containing ____________.
Inverted repeats
What interacts with the inverted
repeats, whereby each monomer
binds one repeats?
Dimers
_________ determines affinity of
the regulatory proteins.
DNA sequence
What are ligands?
molecules that can modulate activity of the regulatory proteins
True or False: Ligands allow the cell to modulate gene expression based on the changes in ligand availability in the cell.
True
Ligands that stimulate/induce gene expression are called ________.
Inducers
________ bind to regulatory
sequences in the DNA and
prevent transcription of target
genes.
Repressors
How can a ligand induce gene expression?
dissociate the repressor to
activate gene expression
What is a Co-repressor?
ligand that
promotes repressor binding
to repress expression
__________ bind to regulatory
sequences in the DNA and
stimulate transcription of
target genes
Activators
True or False: Activators usually bind an inducer.
True
Absence of the inducer
results in ___________.
repression
_____________ sense the external environment
Two-component signal transduction
systems
Where is the sensor kinase located?
Cell membrane
What does the sensor kinase do and how is it activated?
- Binds to environmental signal
- Activates itself via phosphorylation
Where is the response regulator located?
Cytoplasm
What does the response regulator do and how is it activated?
- Bind DNA to regulate gene expression
(often for several genes) - Activated through phosphorylation by
sensor kinase
True or False: Phosphorylation is a covalent modification (NOT a ligand!!)
True
What two scientists proposed the revolutionary idea that genes could be regulated in 1961?
Jacques Monod and François Jacob
What is Lactose?
a disaccharide sugar comprised of GLUCOSE and GALACTOSE that can be utilized as a carbon and energy source
What are the two components of lactose metabolism?
Lactose permease (LacY) & β-galactosidase (LacZ)
What function does lactose permease (LacY) serve?
Imports lactose into the cell
What molecule cleaves lactose into the monosaccharides glucose and galactose?
β-galactosidase (LacZ)
In E. coli, the genes that encode LacY and LacZ (and LacA) form a __________.
lacZYA operon
What regulates the expression of lacZYA genes?
a single promoter (PlacZYA) and a
series of operator sequences (lacO)
What is encoded by lacI gene upstream and is expressed
from an independent constitutive (always ON) promoter PlacI?
Lactose repressor (LacI)
What is Lactose limited?
*LacI tetramer has high
affinity and binds to the operator region
* represses lac transcription
* prevents open complex formation by
RNA polymerase
Lactose Operon: LacI
What is Lactose abundant?
*basal level of LacZ produces allolactose inducer
which binds to LacI and decreases affinity for the operator region
* activates lac transcription
* permits open complex formation by RNA polymerase
Lactose Operon: LacI
Maximum expression of the lac
operon also requires the presence of ____________ and ______________.
cyclic AMP (cAMP), a dimeric
cAMP receptor protein (CRP)
Where does the cAMP-CRP complex bind?
60 nt units upstream of the lac promoter
How does the cAMP-CRP complex maximize expression of the lac operon?
interacts with RNA pol to increase
the rate of transcription initiation
What produces cAMP from ATP during glucose
starvation by sensing the phosphorylation status of EIIA components of the Glucose PTS?
Adenylate cyclase (AC)
True or False: When glucose levels are high AC is inhibited, and cAMP levels are decreased. This means that the lac operon will not be expressed.
True
True or False: When glucose levels are low AC becomes active, and cAMP levels are increased. Therefore, the lac operon will be expressed.
True
____________ occurs when an operon enabling the catabolism of one nutrient is repressed by the presence of a more favorable nutrient (commonly glucose)
Catabolite repression
__________ is a biphasic curve of a culture growing on two carbon sources.
Diauxic growth
____________ occurs when uptake of a
more favorable carbon source (e.g.
glucose) inhibits uptake of a less favorable
carbon source (e.g. lactose).
Inducer exclusion
In E. coli glucose transport by the _________ excludes lactose (lac inducer) uptake.
PTS
(If lactose cannot enter the cell, the lacZYA
operon cannot be induced, hence inducer
exclusion)
True or False: When glucose levels are high so are the levels of EIIIA. Therefore, lacY is inhibited.
True
True or False: When glucose levels are low, the levels of EIIIA- P remain high. This is when lacY will be active.
True
lac is activated when:
- Lactose ↑ - LacI binds operator loosely (activation)
- Glucose ↓ - cAMP ↑
- Glucose ↓ - EIIA-P ↑ - LacY active
True or False: Some proteins have dual regulatory function and can serve as both activators
(positive control) and repressors (negative control)
True
What is a major regulator of arabinose utilization?
AraC
How does AraC regulate arabinose utilization?
- Regulates araBAD operon encoding proteins for arabinose conversion into
xylulose-P, an intermediate in a biosynthetic pathway - can activate or repress ara genes depending on the availability of arabinose
What happens when AraC acts as a repressor?
When arabinose is absent, the N-terminal arm binds its on C-terminal DNA binding domain.
What happens when AraC acts as an activator?
When arabinose is attached, the N-terminal arm binds to the dimerization domain of the other monomer
AraC–like regulators are collective members of the
AraC/XylS family of
transcriptional regulators
Genes encoding biosynthetic (anabolic) enzymes typically are regulated by inactive repressors in
the apo conformation (aporepressor)
Aporepressor complexed with corepressor is referred to as ____________.
Holorepressor
What binds the operator sequence to inhibit transcription?
Holorepressor
What codes for enzymes involved in the biosynthesis of the amino acid tryptophan?
trp operon
When internal trp levels exceed cellular needs, the excess tryptophan (the corepressor) will bind to______________.
an inactive aporepressor, TrpR
True or False: TrpR holorepressor then binds to an operator DNA sequence upstream of the trp operon to inhibit transcription initiation
True
_____________ is a regulatory mechanism in which translation of a leader peptide affects transcription of an operon’s downstream structural genes
Attenuation
The attenuator region of the trp operon has how many tryptophan codons?
2
True or False: The attenuator region of the trp operon is capable of making stem loop structures.
True
The formation of _________ leads to the termination of transcription
attenuator stem loop
When tryptophan is abundant:
▪ Availability of Trp tRNA allows for ribosome to translate through Trp codons and STOP
▪ Attenuator stem-loop 3:4 forms and dissociates RNA pol (transcription
attenuation)
When tryptophan is limited:
▪ Ribosome stalls at the Trp codons
▪ Anti-attenuator stem-loop 2:3 forms allowing for RNA Pol to proceed with transcription and expression of Trp biosynthetic genes (less energetically 3:4 stem cannot form)
What are two things that genes can be formed into?
Operons and Regulons
_________ are controlled by the same DNA control sequence.
Operons
__________ are controlled by the same control factor.
Regulons
What can regulons be controlled by?
activators, repressors, and alternative sigma factors (differ from regular sigma factors like σ70)
Many Gram-negative bacteria like E. coli have an _________ that protect the bacteria from STARVATION and other stress of STATIONARY PHASE GROWTH.
alternative sigma
factor (RpoS, or σS or σ38)
When nutrients are abundant and cells are rapidly dividing, σS is
rapidly degraded by proteolysis via ________.
ClpXP
When cells enter __________ degradation of σS stops.
stationary phase growth
What does σS bind to to initiate transcription?
RNA polymerase
What can bind to sigma factors and inhibit their activity?
Anti-sigma factors
What is an anti-Sigma
factor that inhibits FliA (σ28)
during early phase of
flagellum assembly?
FlgM
Anti-sigma factors can
themselves be neutralized
by _______________.
anti-anti-sigma factors
According to this hypothesis, RNA stored both genetic information and catalyzed the chemical reactions in primitive cells. Only later in evolutionary time did DNA take
over as the genetic material and proteins become the major catalyst and structural component of cells
RNA World Hypothesis
Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition
RNAs can regulate gene expression in several ways:
- Transcriptional attenuation (trp operon)
- Riboswitch RNA elements
- Untranslated regulatory RNAs
Regulation by RNA
________ are RNA sequences that control gene expression by folding into three-dimensional structures that bind specific
metabolites to sense their abundance in the cell
Riboswitches
___________ usually found in the 5’ untranslated region (5’UTR) of an mRNA molecule, upstream of a coding sequence (CDS).
cis elements
Assume secondary structures that switch in response to ________ or ____________.
metabolite binding or change in conditions (i.e. temperature)
Riboswitches control gene regulation at what level?
transcription or translation
Untranslated regulatory RNAs are
grouped into two general categories:
Trans-acting small RNAs (sRNAs) and cis-acting small RNAs (asRNAs)
Characteristics of trans-acting small RNAs (sRNAs) include:
- Usually intergenic-encoded
▪ Have wide regulons
▪ Utilize short imperfect complementarity
regions for regulation
Characteristics of cis-acting small RNAs (asRNAs) include:
▪ encoded on nontemplate strands of mRNA-encoding genes
▪ Have narrow regulons (usually regulate the gene they overlap)
▪ Utilize longer perfect complementarity
regions for regulation
Idling ribosomes synthesize the
signal molecule guanosine
pentaphosphate (ppGpp), which
then ___________of rRNA
genes and other genes.
decreases the affinity of RNA pol to promoters
The result, called the __________ is lower transcription of genes required for rapid
growth, and generation of fewer
ribosomes.
stringent respone
_____________ is a second
messenger used by E. coli cells to transition between a motile, single-cell (planktonic) state and an adhesive multicellular (biofilm)
state
Cyclic di-GMP (c-di-GMP)
What synthesizes cyclic di-GMP (c-di-GMP) during the stationary phase?
diguanylate cyclases (DGCs)
What degrades cyclic di-GMP (c-di-GMP) during the exponential phase?
phosphodiesterases (PDEs)
The transition is achieved by the balance
between the _________ of c-di-GMP.
synthesis and degradation
Low c-di-GMP favors the
planktonic state (stationary)
High c-di-GMP favors
Biofilm formation
__________ is a pathway for sensing an increase in cell density; means of
cell-to-cell communication.
Quorum sensing
In what bacteria was quorum sensing first discovered?
Vibrio fischeri
__________ is a membrane-permeable second messenger.
Autoinducer
Characteristics of autoinducers include:
- concentration of autoinducer rise as the cell density grows
- allows for coordinated response when population density increases
__________ in Gram-positive (+) bacteria involves the cell density-dependent establishment of competency
Natural transformation
________ cells that can import free DNA fragments and integrate them into their genomes by recombination.
Competent
Transformation competence is induced by a threshold concentration
of an autoinducer called _____________.
competence stimulation peptide (CSP)
CSP activates a phosphorylation cascade that ultimately activates
ComX which is:
an alternative sigma factor used specifically to transcribe genes that establish competency
