Gene expression Flashcards
What does expression of genes depend on?
Tissue, developmental stage and intra/extracellular signals
What genes are always expressed at constant levels?
Housekeeping genes
What are 2 types of inducible genes?
Switched on in specific cell types (dependent on TFs)
Switched on in response to environmental cues
What does gene expression regulate (binding)?
Ability of RNA polymerase to bind promoter
How is bacterial transcription initiated?
Binding of sigma factor to promoter, enable RNA polymerase to bind and form preinitiiation complex.
What determines whether sigma factors binds to prokaryotic promoter
Complementary of promoter region
Type of sigma factor
Repressor/activator protein
How is bacterial DNA organised?
Operons - set of genes (for similar function) under control of one promoter
What is the benefit to having no operons in eukaryotic DNA?
Genes encoding proteins for different steps in a pathway have their own promoter so expression can be modulated separately
Protective, one mutation unlikely to damage entire process
What do operons contain?
Regulatory DNA sequences that are binding sites for regulatory proteins to control how much the operon is transcribed
What do regulatory proteins do in prokaryotic transcription?
Repressors bind to operators, reducing transcription
Activators bind to DNA binding site and increases transcription
Does prokaryotic DNA contain introns and exons?
No
How can expression be altered based on modifications to chromatin, what part of chromatin is affected?
Chemical modifications to N terminal histone tails in nucleosomes by activators/repressors to change accessibility of TFs to DNA
What happens in DNA methylation, what’s the result?
Methyltransferase converts cytosine to 5 cytosine
Methyl group disrupts binding of protiens needed for transcription
Chromatin becomes more compact and less accessible
How are TSG often silenced?
Hypermethylation
What is the difference between constituitively heterchromatic and facultative heterochromatic?
Constitutive: permanent
Faculative: can be converted to euchromatic
Which have active genes heterochromatin or euchromatin?
Euchromatin
Describe structure of chromatin in heterochromatin
Chromatin more condensed due to methylation
What does methylation of chromatin also facilitate?
Binding of additional proteins
What is constitutively heterochromatic?
Centromeres and telomeres (contain many tandem repeats)
Example of facultative heterochromatin, how is it altered?
Inactive X chromosome inactivated to become heterochromatic state (barr body) by recruiting enzymes to modifty histones
What is acetylated in gene expression?
Lysine residues of histone tails
How does acetylation affect gene expression? (mechanism)
Neutralise + charge of histone tail so -ve charged DNA less tightly wrapped around histone /nucleosome and more accessible
What does acetylation recruit?
Chromatin remodelling complexes - shift nucleosomes and induce chromatin conformational change
TATA box binding protein associated factors: affecting rate of transcription
How can respositioning nucleosome affect gene expression?
Nucleosome respositioned to make promoter more accessible
Compare active DNA with less active DNA (NUCLEOSOMES) how can you prove this?
Contains fewer nucleosomes
More susceptible to digestion by DNase
Where are housekeeping genes and relatively rarely expression genes placed in relation to nucleosomes?
Housekeeping genes between
Rarely expressed genes in between
Example of gene that can’t be transcribed as DNA is bound to nuclear lamina
Hox genes
What does RNA polymerase required to associate with promoter sequence?
General transcription factor
What is transcription?
First of several steps of DNA based gene expression, in which a particular segment of DNA is copied into mRNA by the enzyme RNA polymerase
What do promoter regions signify?
Initiate transcription at the start of a gene
What is the role of GTFs?
Bind promoter sequences and create scaffold to recruit polymerase
Does RNA polymerase directly bind promoter?
No
What allows recruitment of specific polymerase to a given promoter?
Each promoter recognised by different subtype of GTF
How does eukaryotic gene expression show combinatorial control?
Many TFs work together to recruit proteins to form transcription complex and recruit RNA pol
In prokaroyitc DNA, what’s the difference between the interaction between RNA pol and DNA?
RNA pol recognises the DNA itself, not the transcription apparatus
What initiates formation of initial basal transcription complex?
TATA binding protein binding to TATA box
What sort of DNA region is TATA box?
Cis regulatory element
What ensures correct and regulated gene expression in response to tissue specific and proliferation cues?
Activators and co activators/mediators binding outside core promoter elements
Describe what can influence gene expression outside of promoter region
Distal regulatory elements can decide if gene activated (trans)
How far away can distal regulatory elements be
1MB
What sort of regions are in distal regulatory elements?
Insulator
Silencer
Enhancer
Describe regulatory regions in eukaryotic DNA compared to prokaryotic
More complex
What is the different between cis and trans regulatory elements?
Cis-regulatory elements are often binding sites for trans-acting factors - they are present on the same molecule of DNA as the gene they regulate.
Trans-regulatory elements can regulate genes distant from the gene from which they were transcribed.
What proteins bind to enhancer regions?
Activator proteins
What do activator protiens do?
Associate with the core promoter-TF complex to start transcription.
What is the role of a mediator?
Involved in the looping of chromatin, to allow interaction of regulatory elements with cis regulatory elements and GTFs
What interactions allow distal regulatory elements to affect gene expression?
Protein protein interactions folds DNA into loops allowing enhancer and promoter to interact activating transcription complex
What do enhancers do?
Stimulate transcription
What do silencers do?
Inhibit transcription (switch off promoter)
What does an insulator do?
Only activates promoter to right not left (i.e. limit how enhancer binds gene expression)
An example of a mechanism where extracellular control of gene expression
Hormones, diffuse into cells binding to receptor. Complex diffuses to nucleus and binds receptor elements in DNA that upregulate/down regulate gene expression
What do cis acting TFs do?
Bind promoter regions e.g. TATA box, GC box acting on gene immediately following promoter region
How else apart from transcription is gene expression controlled?
At levels of mRNA processing, translation and mRNA stability
What is the equivalent of TATA box in bacteria?
Pribnow box
What forms the transcription complex in prokaryotic cells?
Sigma factor and RNA pol
What are the 3 parts of promoter region?
Core promoter
Proximal promoter
Distal promoter
What does RNA polymerase do?
Synthesizes RNA from a DNA template.
Initiates RNA transcription,
Guides the nucleotides into position
Facilitates attachment and elongation
Has intrinsic proofreading and replacement capabilities
Termination capability
What are properties of RNA polymerase?
Doesn’t require primer so catalyses initiation and elongation of RNA
Completely processive (single enzyme transcribes complete RNA)
Proof reading mechanism
Where are the 3 isoforms of RNA pol found?
1 - nucleolus
2 - nucleoplasm
3 -nucleoplasm
What does RNA pol 1 produce?
rRNA
Does RNA pol require a primer?
No
What does RNA pol 2 produce?
mRNA, snNA, miRNA
Describe what happens in initiation?
Binding of TBP to TATA box,
Sequential recruitment of TFIIB, TFIIA and TFIIF to promoter forming preintiitaiton complex.
Complex recruits RNA pol 2, TFIIE and TFIIH.
This initiation complex unwinds double helix
Describe normal rate of transcription activation by basal transcription activation?
Slow without activation factors
What happens in elongation?
In 5-3 direction
No primer
Normal base pairing
U not T
Nucleophilic attack by 3-OH at end of growing chain on alpha phos of incoming ribonucleoside triphosphate forming phosphodiester bond
What happens in protein independent termination?
Self complementary RNA palindromic GC rich region forms hairpin in RNA structure due to base pairing, hairpin followed by Oligo U sequence
This destabilises association between RNA and DNA so they dissociate along with RNA pol 2 and double helix reforms
What does RNA pol 3 produce?
tRNA, some snRNAs
What RNA units join to form mRNA?
Ribonucleoside triphosphates
Nucleoside triphosphate vs nucleotide?
dNTP - base, sugar 3x phosphate
NT - base, sugar, phosphate
During transcription, a complementary copy is made of..
The antisense strand (therefore copy of sense strand)
