Gene regulation Flashcards
What are the levels of control of gene expression?
- transcription
- RNA processing
- Transcriptional regulation
- post-transcriptional regulation
- epigenetic
Transcription produces 3 main types of RNA, what are they?
- rRNA, mRNA, tRNA (also many small RNAs)
What does the typical eukaryotic promoter refer to?
- usually refers to the whole 5’ region including the promoter proximal elements and upstream enhancer elements
What can be used to initiate transcription in eukaryotes?
- TATA box and Initiator sequence (inr)
- TATA box is used in genes that are rapidly transcribed. Less than 50% of all genes have a TATA box. ~ -35 to -25 (consensus sequence)
- Initiator sequence used when genes don’t have a TATA box. Exact sequence determines strength of initiator. Initiator sequence begins at a defined site +1 (an A)
Other than TATA or initiation sequence, what are the other RNA pol II core promoter elements?
BRE - TFIIB recognition element (GC rich)
DPE - Downstream core promoter element
What are the complex of proteins that bind to either TATA or initiation sequence in transcription?
- Called TFs
- Designated TFIIA, TFIIB etc - most are multimeric proteins
- Large (composed of TATA box binding protein TBP and TBP associated factors (TAFs)
- TAFs are involved in RNA pol positioning in non TATA containing genes
What are the steps initiation of transcription?
- TFIID binds to TATA box via the TBP subunit and bends DNA
- TFIBB binds and makes contact with both TBP and the DNA
- A complex of TFIIF and Pol II binds such that the Pol II is positioned over the start site
- TFIIE binds and forms a docking site for TFIIH
- TFIIH binds to 2 subunits that have helicase activity
- TFIIH unwinds the DNA (requires ATP) at the start site (allows release of general TFs, allows pol to move down DNA and synthesise RNA
What are 2 of the sequence specific TF binding elements?
- Promoter-proximal elements occur near the start site - position dependent
- enhancer elements can occur far upstream, in introns or downstream of the genes in higher eukaryotes - position independent
What are the distance-independent promoter elements?
- enhancers are cis-elements (DNA) that bind TFs and increase transcription rates
- silencers are cis-elements that bind silencer proteins and decrease transcription rates
- enhancers and silencers are similar to proximal-promoter elements except they act as a distance by DNA looping (facilitated by architectural proteins)
What are transcription factors?
A TF is a sequence specific DNA binding protein that recognises a short DNA sequence.
What common protein motifs physically interact with specific DNA sequences?
- zinc fingers
- leucine zippers
What is combinatorial control?
- cooperative interactions between cis elements
- enable fine tuning of transcriptional control
- having multiple cis elements in right arrangement can lead to amplified response (synergistic effect)
- leads to the formation of an Enhanceosome
What is the enhanceosome?
A large transcription factor complex that acts synergistically
What is chromatin’s role in gene regulation?
- highly condensed heterochromatin has a general silencing effect
- open euchromatin is transcriptionally more active
- chromatin also acts locally to directly control transcription
What is chromatin composed of?
DNA and histones (plus other chromosomal proteins)
What does the nucleosome contain?
Histones; H2A, H2B, H3 and H4
- histone H1 binds between and keeps the nucleosome intact
How can modifications of histone tails control chromatin condensation and function?
- each histone has a flexible N-terminus extending out from the nucleosome
- H2A and H2B also have flexible C-term tails
- histone tails subject to modification
What’s the histone code?
Only a subset of modifications present on any histone. Histone marks = epigenetic regulation
Do histones from heterochromatin contain different marks than histones from euchromatin?
- YES
- lysine residues are often acetylated in extended conformations
- acetylation neutralizes the +ve charge of lysine important in maintaining the 30nm fibre
- methylation prevents acetylation
What reads the histone code?
- read by proteins with a chromodomain (e.g. HP1 - heterochromatin protein 1)
- HP1 chromodomain binds to tri-methylated H3K9
- HP1 chromoshadow domain interacts with other proteins with the same domain - oligomerization of HP1
- result is condensation of chromatin (heterochromatin)
- HP1 chromoshadow domain also interacts with domain of H3K9 histone methyltransferase
- leads to tri-methylation of adjacent H3K9s -spreading of heterochromatin (chain reaction)
- boundary elements prevent condensation spreading to active chromosome regions
What can gene transcription be activated by?
Histone acetylation
How do transcriptional activators regulate assembly of the initiation complex?
- TF bind cognate sequence
- TF contacts Mediator complex
- very large complex (30 subunits)
- molecular bridge to initiation complex
What does the highly ordered multistep process of transcription involve?
- sequence-specific TFs
- chromatin remodelling complexes
- histone acetylation complexes
- a mediator complex
- general TFs
- RNA pol
Describe the steps of transcription in a yeast HO gene
- Gene decondensed by SWI5 binding at enhancer site upstream of gene and interacting with SWI/SNF
- SWI/SNF remodels chromatin exposing tails
- GCN5 recruited by SWI5 and acetylates histone tails. SWI/SNF continues de-condensation
- SWI5 released but SWI/SNF and GCN5 remains associated by binding acetylated histone tails. Remodelling allows binding of TF SBF to exposed DNA
- Mediator complex binds transcriptional activator SBF
- Preinitiation complex is formed via interaction with mediator complex
What is a super enhancer?
Formed by exceptional amounts of TFs and control the expression of gene critical for cell identity
What are the types of levels of controls of RNA splicing?
- some ubiquitous others tissue-specific
What are the 3 sequences needed for splicing?
- Splice donor including invariant GU at 5’ end on intron
- Splice acceptor including invariant AG at 3’ end on intron
- Branch site near splice acceptor (invariant A) initiates splicing reaction
What sequences regulate RNA splicing?
- enhancers and suppressors that are introns (ISS, ISE) or exons (ESE,ESS)
- can produce different proteins
What percentage of genes are alternatively spliced?
- 90% in humans
What is an example of alternative splicing leading to 2 different proteins?
CDKN2A gene leads to 2 different tumour suppressor proteins:
- P14ARF
- P16INK4A
- different reading frames due to alternative promoter usage and splicing
What is translational regulation?
Mechanism to allow rapid response to environment. Mediated via RNA-secondary structures and RNA-binding proteins
What’s an example of translational regulation?
Iron metabolism - ferritin and transferrin receptor
- IRE (iron-responsive element) in mRNA binds to IRE-binding protein (IRE-BP) when FE concentration low
In Iron metabolism what happens when FE concentration is low in the different IRE locations?
- in 5’IRE in ferritin mRNA block translation
- in 3’ IRE in transferrin-R stabilizes mRNA
What is a microRNA?
A small RNA molecule that forms after transcription, through a series of cleavage and processing steps. MiRNAs bind to mRNAs and regulate their activity level. An example of post-transcriptional gene silencing. A single miRNA can regulate multiple mRNAs
What are Long noncoding RNAs?
Non-protein en-coding transcripts longer than 200bp. Account for 4/5 of transcription. Participate in chromatin organisation and transcriptional and post-transcriptional gene expression. Also act as structural scaffolds of nuclear domains
What resembles mRNAs?
Long noncoding RNAs (5’ capping, splicing, polyA)
