Regulation of Gene Expression in Bacteria Flashcards
What is most regulation of gene expression in bacteria?
transcriptional regulation
What is constitutive transcription?
transcription that is continuously occurring
some genes are needed to continuously perform routine tasks - they are constitutively transcribed
When is transcription of genes regulated?
when the genes are needed for responses to environmental conditions and not required ALL the time
What two things does transcription regulation control?
the initiation and amount of transcription
What is negative control of transcription?
when a repressor protein binds to a regulatory DNA sequence
What does negative control of transcription cause?
the repressor binding prevents transcription of a gene or gene cluster
Describe positive control of transcription
this occurs when an activator protein binds to a regulatory DNA sequence
What does positive control of transcription result in?
initiation of transcription of a gene or gene cluster
What function do repressor proteins have?
they exert negative control of transcription by binding to regulatory sequences like operators and blocking transcription initiation
What is an operator?
a regulatory sequence that occurs on DNA after the promoter region
activated repressors bind to operators
What binds to operators?
activated repressor proteins
What are the 2 active sites on repressor proteins?
DNA-binding domain
allosteric domain
describe the DNA-binding domain of a repressor protein
one of the active sites on a repressor protein that locates and binds to the operator DNA sequence
describe the allosteric domain of a repressor protein
one of the active sites
it binds a molecule or protein which causes a conformational change in the DNA-binding domain
How does positive control of transcription occur?
when activator proteins bind to regulatory sequences of DNA called activator binding sites
What do activator proteins bind to? AKA What does CAP bind to?
activator binding sites (regulatory DNA sequences)
CAP binds to CAP sites when it is already bound to cAMP
What does activator protein binding facilitate?
RNA polymerase to bind at promoter and initiate transcription
What domains does an activator protein have?
DNA-binding domain
allosteric domain
How many modes of action are there for positive control of transcription?
2
Describe the mode of action for positive control of transcription that includes the allosteric binding site of repressor proteins
the DNA-binding domain of an activator protein is inactive until
an allosteric effector compound (ligand) binds to the allosteric domain of the repressor protein and induces a conformational change in the DNA-binding domain of the repressor protein which prevents it from binding to the DNA
Describe the alternative mode of positive control of transcription
certain activator proteins have a DNA-binding domain that is converted to inactive conformation by the binding of an inhibitor to the allosteric domain
Describe an operon
A cluster of genes undergoing coordinated transcriptional regulation by a shared regulatory region
What genomes are operons common in?
bacterial genomes
What is the purpose of operons?
to allow coordinated expression of genes required for the same biochemical process
What is the function of the lactose (lac) operon in E. coli?
it produces two polypeptides required for lactose metabolism
What carbon source do bacteria prefer? Which will they use if this isn’t present?
glucose is preferred carbon source
will use lactose if no glucose is present
What does it mean for a system to be inducible?
it is not on all of the time, it will be induced
Is the lac operon system on all the time or is it induced?
it is induced only when lactose is available
Describe lactose
it is a disaccharide consisting of glucose and galactose and joined by Beta-galactoside linkages
What are the 2 components of lactose? what are they linked by?
glucose and galactose are linked by beta-galactoside linkage
What kind of medium can bacteria with the lac+ phenotype grow on?
media containing lactose as the only sugar
How do bacteria with the lac+ phenotype grow on media with lactose as the only sugar?
they produce:
a permease channel
the beta-galactosidase enzyme
What is the purpose of the permease channel?
it allows lactose to enter the cell
What gene codes for the permease enzyme channel?
lacY
What is the purpose of the beta-galactosidase enzyme?
it breaks down the beta-galactoside linkage in the lactose
What gene codes for beta galactosidase?
lacZ
what happens to the glucose in lactose?
when lactose is broken down, the glucose enters glycolysis
What function does galactose have in lactose when lactose is breaking down?
it is processed to produce glucose
What does the breakdown of lactose produce a small amount of? what is its function?
A small amount of allolactose
allolactose acts as an inducer
What does allolactose do?
act as an inducer
T or F: bacteria with a lac- phenotype are unable to utilize lactose
true
What does lac operon consist of?
a multipart regulatory region and three structural genes
What are the 3 structural genes of lac operon?
lacZ
lacY
lacA
What 3 things does the regulatory region of the lac operon contain?
a promoter that binds RNAP
an operator that binds the lac repressor protein
a CAP binding site that binds CAP-cAMP
What does the operator overlap with? What does this cause?
the binding location for RNAP
this causes the blocking of RNAP binding cause the repressor protein binds to the regulatory region
Which structural gene of lac operon encodes for beta-galactosidase?
lacZ
Which structural gene of lac operon encodes for permease?
lacY
Which structural gene of lac operon encodes for transacetylase?
lacA
How are the 3 structural genes transcribed?
as a single (polycistronic) mRNA
What does translation of the polycistronic mRNA produce?
three distinct polypeptides
T or F: lacA, lacZ, lacY are transcribed individually
False! they are transcribed as a single polycistronic mRNA
T or F: the lac operon consists of lacZ, lacA, lacY, and lacI
false! lacI is not part of the lac operon
What is the function of lacI? where is it located in relation to the lac operon?
lacI codes for the lac repressor protein and is constitutively expressed
it is located next to the lac operon
Is the expression of the lacI gene constitutive or inducible?
constitutive
T or F: the lac operon is transcriptionally active when there is no lactose available or when glucose is available
false!! it is transcriptionally silent
Why is the lac operon transcriptionally silent when there’s no lactose available or if glucose is available?
if no beta-galactosidase is produced by lacZ, there is no allolactose and the lac repressor protein can bind to lacO and prevent transcription
What is an example of negative control of transcription?
the lac operon being transcriptionally silent in the absence of lactose or presence of glucose
When is transcription of the lac operon induced?
when lactose is available to the cell and glucose is not
Describe how transcription of the lac operon is induced
when lactose is available, beta-galactosidase is produced which degrades the lactose into glucose, galactose, and allolactose
When allolactose is produced, it binds to the lac repressor which forms the inducer-repressor complex and causes a conformational change in the repressor protein that prevents it from binding to the operator
What forms when allolactose binds to the lac repressor? What does this do?
the inducer-repressor complex forms and causes a conformational change in the DNA-binding domain of the repressor and prevents it from binding to the operator
this allows transcription of the lac operon
When does basal transcription occur?
When both glucose and lactose are present
How much polycistronic mRNAs are transcribed when basal transcription occurs?
a small number
T or F: basal transcription is sufficient to generate enough copies of the lac operon mRNA for metabolism of lactose
false! basal transcription only produces a small number of polycistronic mRNAs
Where does positive control of the lac operon occur?
at the CAP binding region of the lac promoter
What are the two domains of the CAP activator protein?
DNA-binding domain that binds to the CAP binding region of the promoter
allosteric domain that binds cAMP
What does the DNA-binding domain of the CAP activator do?
bind to the CAP binding region of the lac promoter
What does the allosteric domain of the CAP activator protein do?
binds cAMP
What restrictions are placed on CAP binding to the CAP binding region of the lac promoter?
cAMP must be bound to the allosteric domain
What function does DNA-bound CAP have?
it interacts with RNAP and increases its ability to bind to the lac promoter
How does glucose prevent activation of lac operon?
by modulating levels of cAMP
What does cAMP stand for?
cyclic adenosine monophosphate
How is cAMP synthesized?
from ATP by adenylate cyclase
T or F: during glycolysis, adenylate cyclase is limited in quantity
true
What is a result of adenylate cyclase being limited during glycolysis?
almost no cAMP is produced = almost no CAP-cAMP can form = inefficient lac gene transcription
How does the production of cAMP affect lac gene transcription?
cAMP production is limited by the small quantity of adenylate cyclase during glycolysis (which makes cAMP from ATP)
cAMP binds to CAP to allow it to bind to the lac promoter region
if there’s not much cAMP to bind to CAP, the lac gene transcription is not very efficient
What happens to cAMP levels when glucose is absent?
they are high
How does the absence of glucose affect transcription of the lac gene?
no glucose = high cAMP
CAP-cAMP complex forms efficiently and can bind to the CAP binding domain
CAP-cAMP binds to the lac promoter to interact with RNAP and increase transcription
List the steps of lac gene transcription when glucose is absent
- cAMP levels rise because adenylyl cyclase is more available
- CAP-cAMP complex forms and binds to the CAP site of the lac promoter
- allolactose forms as B galatosidase breaks down lactose
- repressor protein binds allolactose = conformational change = released from operator
- RNAP can bind and transcribe
When does no lac operon transcription occur?
when glucose is present and there’s no lactose
or
when neither glucose or lactose are present
T or F: the lac repressor protein binds irreversibly to the operator sequences and cannot be released from lacO
false! it binds reversibly and can occasionally be released
How were each of the structural genes of the lac operon discovered?
by genetic analysis of the lac operon mutants (Jacob, Monod, Lwoff, et al.)
Aside from identifying each of the structural genes and the regulatory region of the of the lac operon, what else did the work of Jacob, Monod, Lwoff, etc. do?
created a foundation for the description of transcriptional regulation at the DNA sequence level
What are two mutations of the lacI gene?
lacI-
lacI^S
What is the result of the lacI- mutation?
the repressor protein product is unable to bind to the operator
What is the result of the lacI^S mutation?
the repressor protein is a ‘super-repressor’ and is unable to bind the inducer (allolactose) which means it blocks all transcription
What is a mutation of the lacZ gene?
lacZ-
What is the result of the lacZ- mutation?
no functional beta-galactosidase is produced = lactose cannot be broken down
What mutations does the lacY gene have?
lacY-
What is the result of the lacY- mutation?
no functional permease protein is produced = lactose cannot be transported across the membrane
What mutations does the lacA gene have?
no transacetylase is produced
What mutations can the lacO gene have?
lacO^C
What is the result of the lacO^C mutation?
the repressor protein cannot bind to the operator = continuous transcription
What mutations can the lacP gene have?
lacP-
What is the result of the lacP- mutation?
RNAP cannot be bound or it is bound weakly to the promoter
How were lac- mutants synthesized?
by treating E. coli with mutagens and running complementation tests
What two complementation groups did complementation analysis classify lac- mutations into?
bacteria with mutations in the lacZ gene
bacteria with mutations in the lacY gene
What organisms was complementation analysis conducted in?
partial diploids produced by conjugation between F’ (lac) and F- bacteria
How can the genotypes of a partial diploid bacteria be written?
I+ P+ O+ Z+ Y- / I+ P+ O+ Z- Y+
What is one copy of the operon of a partial diploid unable to produce? What about the other?
one copy is unable to produce a functional permease (lacY-)
the other is unable to produce functional beta-galactosidase (lacZ-)
How do partial diploids have complementation even with mutations?
in combination, the mutations of each copy of the operon complement because the WT allele of each gene is dominant to the mutant
What phenotype would I+P+O+Z+Y+ produce?
wild type (lac+)
What phenotype would I+P+O+Z-Y+ produce?
no functional beta-galactosidase = lac-
What phenotype would I+P+O+Z+Y- produce?
No permease = lac-
What phenotype would I+P+O+Z+Y-/ I+P+O+Z-Y+ produce?
wild type (lac+)
Where constitutive mutants located? Why?
to the lacO and lacI regions
because these mutations affect the production of the lacI regulatory protein or its DNA-binding site (lacO)
Are lac operator mutations exclusively cis- or trans-acting? what does this mean?
cis-acting
means they influence transcription of genes on the SAME chromosome
How do lacO^C mutants affect downstream structural genes?
OC mutants have altered operator sequences which repressor proteins cannot bind to = structural genes downstream are constitutively expressed because there’s no negative control of transcription
In experiments with partial diploids, what resulted from an OC mutation being adjacent to Z+ and an O+ next to a Z-?
constitutive expression of Z+
In experiments with partial diploids, what resulted from an OC mutation being adjacent to Z- and an O+ next to a Z+?
normal expression of Z+
In experiments with haploid cells, what resulted from a I- P+ O+ Z+ Y+ genotype?
the Z and Y genes are constitutively expressed
In experiments with partial diploids, what resulted from I+ P+ O+ Z- Y+ / I- P+ O+ Z+ Y-
normal expression of both Z and Y genes
Is the regulatory protein produced by lac+ cis- or trans-acting? what does this mean?
trans-acting which means it influences transcription of genes on different chromosomes
How is the lac+ produced regulatory protein trans-acting?
it is able to interact with both operators in a partial diploid (both chromosomes) because it can diffuse through the cytoplasm
Describe cis-acting
influence on transcription of genes on the same chromosome
Describe trans-acting
influence on transcription of genes on different chromosomes
How can a lacI- gene lead to constitutive transcription?
lacI- produces a mutant form of the repressor protein that cannot bind to the operator = transcription cannot be repressed
In partial diploids, what happens if both lacI- and lacI+ are present?
the gene will function normally and the repressor protein will function normally
Is lacI- dominant or recessive to lacI+?
lacI- = recessive
lacI+ = dominant
Describe the lacI^S mutation
results in a non-inducible operon where the Z and Y genes are not expressed
alters the allosteric domain so that allolactose cannot bind to it
is the lacI^S mutation dominant or recessive to the lacI+ allele?
IS = dominant
I+ = recessive
How does mutations of lacP affect transcription of lacZ and lacY?
most mutations of lacP reduce or eliminate the transcription of Z and Y
What phenotype would result from I-P+O+Z+Y+?
the lacI- mutation will cause constitutive transcription
What phenotype would result from I+ P+ OC Z+ Y+?
constitutive transcription because of the mutant lacOC
What phenotype would result from IS P+ O+ Z+ Y+?
IS mutation makes transcription not inducible
What phenotype would result from I+P-O+Z+Y+?
lacP- mutation prevents transcription
What is a corepressor?
a molecule that binds to the allosteric site of a repressor protein and helps it do its job of repressing gene transcription
What is an inducer?
a molecule that binds to the allosteric site of a protein and stimulates gene expression
ex. inducer binds to the activator protein to stimulate gene expression
ex. inducer binds to the allosteric site of repressor protein and causes conformational change in DNA binding site which prevents repression of transcription
