Regulation of gene expression Flashcards
How does tretinoin reduce acne?
- Acne occurs due to increased cell-cycle and increased
inflammation - Tretinoin normalizes cell cycle and reduces expression of nitric-oxide synthase which is pro-inflammatory
Are all genes expressed all the time?
No, only the necessary genes are expressed
What does the expression of certain egens depend on?
Amount, activity, functional protein depends on regulation at multiple levels
Housekeeping gene vs regulated gene
Housekeeping genes - expressed in cells all the time as they are necessary for these cells to survive
Regulated genes are the genes that are expressed only when they are necessary for a cells function at that point of time
What restricts access to promoter?
Chromatin structure
Regulatory proteins are usually ___
Regulatory proteins are usually multimeric
What does nucleus separates in terms of gene expression in eukaryotes?
Transcription (happens in nucleus) and translation (happens in cytoplasm) are separated by
nucleus
Define a gene
Gene is a string of DNA sequence that codes for a protein or a functional entity
Where is regulatory sequence found on the gene
It is found at 5’ end- left side
The sequences on the right side are called ___ sequences
The sequences on the left side is called _____ sequences
The sequences on the right side are called downstream sequences
The sequences on the left side is called upstream sequences
What is a promoter?
Promoter is a DNA sequence that regulates/promotes gene expression
What is a transcription factor?
Transcription factor is a protein that affects the regulation and transcription initiation of a gene by binding to a regulatory sequence near or within the gene and interacting with RNA polymerase and/or other transcription factors
What can serve as a molecular signal that participates in gene regulation?
Hormone
Covalent modification
Allosteric regulator
Interacting protein
Where does RNA polymerase binds to the gene?
RNA polymerase binds at promoter sites generally found near points at which RNA synthesis begins on the DNA template
What is a coactivator?
Acoactivatoris a type of transcriptional coregulator that binds to an activator (a transcription factor) to increase the rate of transcription of ageneor set of genes
Do coactivators and corepressors bind to promoter region directly?
NO
Do regulatory proteins bind to promoter region directly?
yes, it is absolutely necessary
What happens after regulatory proteins bind to promoter region?
After binding innate enzyme (RNA polymerase) binds and then functions
What is negative regulation Describe 2 ways of negative regulation
Negative regulation- bound repressor inhibits transcription . A) Gene is off. Molecular signal causes dissociation of regulatory protein from DNA. Transcription can take place.
The signal could be anything- hormone, covalent modification, a protein
B) Gene is on. Molecular signal causes binding of repressor to DNA. Transcription is inhibited
What is positive regulation Describe 2 ways of positive regulation
Positive regulation- bound activator facilitates transcription.
A) Gene is on. Molecular signal causes dissociation of an activator from DNA, inhibiting transcription
B) Gene is off. Molecular signal causes binding of activator to DNA, inducing transcription. is made to dissociate by a signal. . Gene is turned off
Define regulatory sequence
Regulatory sequence is a DNA sequence involved in regulating the expression of a gene e.g. a promoter o operator
Promoters in prokaryotes vs eukaryotes
Prokaryotes commonly have a string of genes that a regulated by common promoters
In eukaryotes each gene has its own promoter
Define an operator
Operators are the binding sites for repressors. They are generally near the promoter
Where are activator binding sites? What are they?
Upstream there are activator binding sites- where positive regulators bind
How do regulatory proteins bind to dna?
When a regulatory protein has to bind to DNA, its amino acids have to interact with nucleotides of DNA
These interactions are specific and depend on hydrogen bonding
Hydrogen bonds occur between amino acids of a protein and nucleotide base of DNA
What are the most common AA involved in hydrogen bonding of regulatory proteins to DNA?
Asn, Gln, Glu, Lys and Arg
Asparagine, Glutamine, Glutamic acid, Lysine, Arginine
What are DNA binding motifs/domains?
Regions of proteins that bind to DNA
What are the 3 common typed of DNA binding motifs?
Helix-turn-helix
Zn finger domain
Leucine zipper domain
Describe Helix-turn-helix
Has about 20 amino acids in two short α-helical segments
One of the two α-helical segments is called the recognition helix, because as it is the one that contains the amino acids
interact with the DNA.
Describe Zn finger domain
Many transcription factors in eukaryotic systems have this
In a zinc finger, about 30 amino acid residues form an elongated loop held together at the base by a single Zn2 ion
Weaker binding
Proteins may have >1Zn finger To make up for the weak binding and provide sufficient interaction
Can also act as RNA binding motif Thus participate in regulation of RNA binding
Describe leucine zipper
This motif is an amphipathic helix with a series of hydrophobic amino acid residues concentrated on one side forming a straight line along the hydrophobic
surface
Leu occurs at every 7th position
Regulatory proteins with leucine zippers often have a separate DNA-binding domain with a high concentration of basic (Lys or Arg) residues that can PARTIALLY interact with the negatively charged phosphates of the DNA backbone-> Weak interaction
Apart from DNA binding motifs what else do regulatory proteins have? What does this result in?
Regulatory proteins have protein-interaction domains, in addition to DNA binding motifs-> allow to interact with other proteins - that’s why they can be multimeric
Promoters are located near the___
Promoters are located near thetranscription start sites
Regulatory proteins function by ___ to regulate gene expression
Regulatory proteins function by combinations (mix-match) to regulate gene expression
Define nucleosome
Nucleosome is DNA coiled around a core of histones
What are the 2 states of a chromatin
depending on how tightly nucleosomes are arranged:
Euchromatin is loosely arranged. This one is accessible
Heterochromatin - chromatin is tightly woven; is transcriptionally inactive
What is histone made up of?
Of proteins which are present in a dimer- 2 copies of each are present
H1- conditionally
H2A, H2B, H3, H4- always
What is H1? Its function
It’s a linker protein, brings several nucleosomes together
No H1= transcriptionally active region
What is chromatin made up of
DNA + histones (H1, H2A, H2B, H3, H4)
Transcriptionally active chromatin – deficient in __
Transcriptionally active chromatin – deficient in H1
Describe Transcriptionally active chromatin
Transcriptionally active chromatin- enriched with histone variants such as H3.3 and H2AZ
What do SWI/SNF enzymes do?
SWI/SNF enzymes deplete H1 and replace histone variants
Name and describe possible modifications of histones (5)
Methylation (lysine and arginine) Phosphorylation (serine and threonine) Acetylation (lysine) Ubiquitination Sumoylation (lysine)
Which part of histones is modified? Which type of bonding is it?
Histone tails
Covalent
Modification can only occur when chromatin is ___
Modification can only occur when chromatin is open
How is histone acetylation regulated?
Regulated by enzymes HATs and HDACs
- (HDAC) is an enzyme that removes the acetyl group from histone proteins on DNA, making the DNA less accessible to transcription factors.
- HAT -Histone acetyltransferase- adds acetyls
What is the role of histone acetylation?
- The acetylation and deacetylation of histones figure prominently in the processes that activate chromatin for transcription
- Acetylation may also prevent or promote interactions with other proteins involved in transcription or its regulation
Name and describe a modification of DNA
DNA can be methylated in CpG sequences
Methylation occurs at CpG C is followed by a G. C is methylated.
P is phosphate bond that occurs between the 2 nucleotides
This inhibits access to promoter region
These genes have inaccessible promoter - not expressed
What is the next step after TF bind?
Assembly of preinitiation complex
What are enhancers?
Regions of DNA that can be bound by activators to increase the likelihood that transcription of a particular gene will occur
What are transcription factors?
Transcription factors are proteins which recruit RNA polymerase and control and regulate the transcription of DNA into mRNA.
What are the three main portions that make up a promoter?
core promoter, proximal promoter, and distal promoter
Where is TATA box found?
In the core promoter region
Where are enhancers found?
A bit upstream of gene
What are activators?
Proteins that bind to enhancers
They decide which genes need to be activated
Activators are able to initiate ____ at promoter site
Activators are able to initiate histone nucleosome modification at promoter site
What are the 2 common regulatory sequences in eukaryotes?
TATA box and initiator element/sequence (Inr)
TATA box and Inr- which are present in eukaryotes? Prokaryotes
TATA is present in eukaryotes and prokaryotes
Initiator sequences are unique to large mammals such as humans
Do all genes contain TATA and Inr
Not all genes have TATA and Inr. Some have just TATA or Inr, some bo
What is the 1st protein that binds to TATA box?
TATA binding protein (TBP) -it is a TF that identifies TATA box in promoter region
What are basal TF?
General TF that are common for all gene expressions
What is the second protein that binds after TBP?
TF2B- a coactivator
What are the coactivator complexes and where do they function?
TBP and TFIIB
Coactivator complexes function at or near the promoter’s TATA box.
What is preinitiation complex made up of? Where is it formed?
pol II along with basal TFs
It is formed at TATA box and Inr site
___ stimulates pol II to start transcription
TFIIH stimulates pol II to start transcription
What are mediators? Why are they needed?
Mediators are any proteins that link enhancers to promoter region
Enhancer is usually far away from the gene
How do mediators link enhancers to promoter region ?
Either structurally or functionally
Structurally - mediators stack up so much, causing a physical contact between enhancer and promoter
Functionally- mediators begin their process at enhancer region and become modified. The protein floats to promoter region from the region where activation was taking place
Mediators bring in the information from enhancer region to promoter region
Mediators can be ___ or ____ regulators
Mediators can be positive or negative regulators
What do positive mediators do?
Positive mediators stabilize RNA Pol II and help in formation of pre-initiation transcription complex
What do negative mediators do?
Negative/repressor mediators inhibit the formation of active pre- initiation complexes.
In what ways can mediators inhibit the formation of active pre- initiation complexes.
Can inhibit:
- binding of proteins to UAS (Upstream activating sequence)
- binding of HMG proteins
- binding of nearby transcription factors
What are HMG proteins?
HMG proteins enable bending of DNA to enable far part regions closer
What is lncRNA?
Long non-coding RNA- lacRNA or lncRNA
Can act as enzymes that enable proteins that are not translated to play role in transcription
Give an example of a non-protein repressor
lncRNA
How do TF recognize DNA sequence?
Transcription factors recognize DNA sequence based on their AA sequence
How is core promoter defined?
Core promoter is defined as anything -40 to +40 nucleotides before and after start site
How is the start site denoted?
+1
_ means either A or T
_ means either G or C
W means either A or T
Y means either G or C
Nuclear receptors are ___ that are regulated by __
Nuclear receptors are TF that are regulated by hormones
How can nuclear receptor be further regulated, apart from hormones?
Can further be regulated by covalent modification - phosphorylation, methylation
All TF that bind to DNA have ___ domains
All TF that bind to DNA have DNA binding domains
What is a DNA binding domain?
This domain will have short AA sequence that will interact with DNA element
protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA
Which 3 domains do nuclear receptors have?
DNA-binding domain
Hormone binding domain
transcription activation domain
Which domains do all TF have
DNA-binding domain
transcription activation domain
What is the role of
transcription activation domain?
This domain helps all the mediators and TF to help in the formation of pre-initiation complex
Which type of domain do nuclear receptors have a domain type in the DNA binding domain region ?
Zinc finger
What are the 2 main types of nuclear receptors?
cytoplasmic and nuclear
Where are the two types of nuclear receptors found?
Type I- cytoplasm
Type 2- nucleus
Which type of ligands do type 1 nuclear receptors have?
Lipid soluble hormones that can cross the cell membrane and enter the cytoplasm easily by diffusion
In which state are type I nuclear receptors found in the cytoplasm? Why?
Type I nuclear receptors are found in the cytoplasm in an inactive state
Remain inactive being bound to proteins Hsp70- chaperon proteins
What do chaperon proteins do?
Chaperon proteins take protein from one place to another or hold protein in inactive state
What happens when a ligand binds to type I nuclear receptor?
When ligand binds to the receptor- it dimerizes, but has to find an active receptor
Dimerized receptor goes into nucleus and acts as TF - act as activators and interact with other TF; help in the process of pre-initiation complex and regulate target gene
How can nuclear receptor be inhibited? What does this result in?
can be inhibited by other repressor such as protein repressors or lncRNA
These do not allow nuclear receptors to bind to their elements - no transcriptional activity
In which condition are type II nuclear receptors found?
Found in nucleus either bound to promoter region already or floating in nucleoplasm
When they are in an inactive state, they are bound to a corepressor
What happens when a ligand binds to type II nuclear receptor?
Binding of hormone (ligand) will remove the corepressor and brings in the activator allowing transcription to take place
Where do nuclear receptors bind to?
to hormone response elements
What is RNA processing?
RNA processing- introns are removed from mRNA
Does one gene mean 1 protein?
No - alternative splicing
__% of genes with multiple exons undergo alternative splicing
95% of genes with multiple exons undergo alternative splicing
What are the ways of regulation of gene expression at translation?
- Micro-RNAs (miRNAs)
- siRNA
Describe micro-RNAs
Micro-RNAs (miRNAs) silence genes by binding to mRNAs and cleaving it (via endonuclease Drosha or Dicer) or by blocking i. Bind to mRNA due to sequence complementarity
These are short, non-coding RNAs
They are endogenous
Describe siRNAs
Short inhibitory RNAs- they are synthesized, not endogenous
siRNA are also called stRNA
Long RNA is synthesized
Because of it’s length it can form hairpin structure- RNA double stranded on itself
OR
Double stranded RNA can be made separately
These double stranded RNA are targeted by dicers which bring them back to single stranded RNAs that are short in sequence
As this sequence can be complementary mRNA- they can bind (nucleotide to nucleotide hydrogen bonding as in DNA)
Once these siRNA/stRNA bind to target mRNA- they can lead to cleavage of mRNA or inhibit translation
TFIIh vs TFIIB
TFIIH enables pol II to start transcription
TFIIB is a coactivator that binds afte TBP
