Lecture 31 + DLA 23 Flashcards
What are the purpose of repressor proteins in bacteria?
bind to an operator region and prevent RNA
polymerase from initiating transcription
What are the purpose of activator proteins in bacteria?
bind to or near an operator region and allow RNA
polymerase to initiate transcription
What is the purpose of the operator region in bacteria?
The operator is a sequence of DNA which is a binding site for specific proteins that help to regulate gene expression
When lactose is present E.Coli produces three protein products…. which are
lacZ
LacY
LacA
What does lacZ do?
it is a beta-galactosidase
digests lactose into glucose and galactose
What does LacY do?
it is a permease
allows for activate transport of lactose across the cell membrane
What does lacA do?
it is a transacetylase
it transfers an acetyl group from acetyl coA to beta-galactosides (acetylegalactose)
What does the lacI gene do?
encodes a repressor protein that shuts the system down when lactose is not present
how does the Lac operon get turned on?
this is an inducible system, thus lactose must be present
when lactose is not present the repressor is bound to the operator, thus RNA poly cannot bind
when lactose is present, the formation of allolactose is formed and will bind to the repressor. this leads to conformation change and thus RNA poly can bind.
What happens if LacZ, LacA, and LacY are mutated?
they will be non-functional proteins
what if LacP is mutated?
if the promoter is non-function than RNA poly cannot bind and the gene is not going to be expressed.
What if LacO is mutated.
If the operator is mutated, that means that the repressor cannot bind, thus the system cannot be turned off
What if LacI is mutated?
The repressor would not be functional, thus unable to bind to the operator
What if LacI(s) is mutated?
super repressor… cannot dissociate from the operator …. thus it is always off
What does cAMP do in regard to lac operon activation?
cAMP binds the activator protein CRP (cAMP receptor protein) or CAP (catabolite activator protein), which can then bind lacP to help activate transcription
What bind to the lac promoter?
CRP!
RNA poly can then initiate transcription if not glucose is present
What are cis regulatory elements?
DNA sites where proteins and trans regulatory elements will bind
What are trans regulatory elements?
proteins and transcription factors which will bind to the DNA CIS elements
What are the cis elements?
- the basal promoter sequence: bind transcription factors that are associated with RNA poly
- proximal control regions- bind transcription factors and found near the promoter
- enhancer sequence: are far away from the promoter
Why have multiple levels of control? such as basal and upstream promoters?
tight regulation
transcription factors that bind to enhancer/responce elements must have what three domains?
- DNA binding
- dimerization domain
- Activation/ repressor domain
Why different genes have the same response element?
- provides a mechanism to coordinate gene regulation
- allows multiple genes to be regulated together
- allows different genes to be turned off or on at the same time
What are the three modes of repression transcription factors can have?
- competition
- quenching
- blocking
Explain the competition mode of repression?
the repressor protein will compete with the activator protein in order to bind to the enhancer region
if repressor binds transcription is reduced.
explain quenching (mode of repression)
Occurs when the repressor protein bind to and interferes with the DNA binding domain of an activator protein
explain blocking (mode of repression)
This occurs when the repressor protein binds to the activation domain of an activator protein and prevents it from interacting with the basal transcriptional machinery
What is hypoxia?
It is the reduction in the normal level of tissue oxygen tension
can occur during pathophysiological responces
What is HIF-1?
the activity of that protein leads to the upregulation of genes that are involved with cell survival, glucose metabolism, angiogenesis, cancer progression, and invasion.
in low oxygen environments
What must bind to the HRE gene?
HIF1(alpha) and HIF1 (beta) transcription factors must dimerize, then bind to a specific DNA sequence called the Hypoxia Response Element
Where is HRE found?
it is found up stream on many different genes
What kind of gene is HIF-1 alpha
inducible
O2 sensitive
what kind of gene is HIF-1 beta?
constitutive
What is the role of prolyl hydroxylase?
In high oxygen environments the HIF-1 alpha is hydroxylated and quickly degraded in the proteosome
In no to low oxygen, HIF-1 alpha is stabilized, moved to the nucleus and dimerizes with HIF-1 beta to activate multiple genes that enhance oxygen delivery
What is the glucocorticoid receptor do?
This is a zinc- finger type
The activated GR complex up-regulates the expression of anti-inflammatory genes in the nucleus and represses the expression of pro-inflammatory proteins in the cytosol
How is the glucocorticoid receptor activated?
- when cortisol is absent the GR is held in the cytoplsm as part of an inactive protein complex
- when cortisol binds to GR, it dissociates from regulatory complex
- The GR forms a dimer
- the dimer moves to the nucleus and binds to HRE
- this leads to gene expression if targeted genes
What is the myc/max system?
it is a regulatory mechanism for switching between gene activation and repression
What is myc?
myc has a transcriptional regulation domain, but it cannot bind to DNA until it dimerizes
does a lot with regulation with the cell cycle
In the absence of Myc?
Max forms a homodimer and it represses gene expression
cell cycle is inhibited
In non-proliferating cells what is the expression of myc and max
Max = expressed
myc = not expressed
In the presence of myc?
a myc/max heterodimer is formed to activate gene expression
cell proliferation
What are the different families of hemoglobin?
- fetal hemoglobin = two alpha and two gamma chains
- hemoglobin A = two alpha and two beta
- hemoglobin A2 = two alpha and two tetra chains
What are the two types of interfering RNA’s
- miRNA = regulated gene expression by inhibiting mRNA translation
mostly endogenous
derived from ds pre-miRNA species - siRNA = regulates expression by mRNA degradation
mostly exogenous
derived from long dsRNAs and ‘random’ processing
What does drosha do?
long pri-miRNAs are processed to pre-miRNA hairpin strucutres by drosha
what does exportin 5 do?
transports pre-miRNA to cytoplasm
What does Dicer do?
further processes them to single stranded RNA and initiates the formation of the RNA-induced silencing complex (RISC)
What does RISC do?
will bind imperfectly to the target mRNA which inhibits translation
what protein processes long dsRNA into siRNA
Dicer
How do miRNA’s regulate gene expression?
The translational machinery cannot interact with the 5 prime cap structure, thus mRNA is not translated
How do siRNA’s regulate gene expression?
Perfect pairing of siRNA with mRNA within the RISC complex will activate RISC endonuclease AGO2 activity & mRNA will be cleaved and not translated
