Promoters and Cis Elements Flashcards
What are the main differences between prokaryotic and eukaryotic transcription?
- location
- prokaryotes only have exons
- capping, splicing and polyadenylation only occurs in eukaryotes
- PTMs only happen in eukaryotes
- transcription and translation are uncoupled in eukaryotes
What genes does RNAP I transcribe?
5.8S, 18S and 28S rRNA genes
What genes does RNAP II transcribe?
- all protein coding genes
- snoRNA genes
- miRNA genes
- siRNA genes
- lncRNA genes
- most snRNA genes
What genes does RNAP III transcribe?
tRNA genes and 5S rRNA genes
What is differential expression mainly due to in eukaryotes?
RNAP II
What are the 4 protein factors involved in eukaryotic transcription?
- RNAP and GTFs
- specific TFs
- co-activators/co-repressors
- mediator
Why are GTFs required?
RNAPs cannot initiate transcription on their own
What are the 4 components for transcription initiation?
- regulatory regions of the gene e.g. promoter, cis elements
- PIC
- specific TFs
- co-activators and chromatin remodelling proteins
What is the eukaryotic PIC made up of?
RNAPs and GTFs
What is the promoter?
the binding site of a gene for basal transcriptional machinery for initiation of transcription
What are cis-elements?
the binding sites of DNA for specific TFs or other regulatory proteins which affect the rate of transcription (enhancers/silencers)
What is the TATA promoter for?
non-housekeeping genes
What is the TATA-less promoter for?
housekeeping genes
What is the TATA box similar to?
the -10 Pribnow box of prokaryotes
What does the TATA box allow?
correct positioning of the polymerase to start transcription about 30 bases downstream from TATA box
What is the initiator sequence (INR)?
a TATA-less promoter that is the start site of transcription (in some genes, it can initiate transcription alone)
What is the BRE element?
the binding site for TFIIB
What does DPE allow?
cooperative binding to TFIID
How can the rate of transcription be altered?
by binding of activators or repressors to specific sequences
What are enhancers?
binding sites for transcriptional activators that increase the rate of transcription
What are silencers?
binding sites for transcriptional repressors that decrease the rate of transcription and sometimes prevent a region from being transcribed at all
Where can enhancers and silencers be located?
- near or far away from the transcription unit (upstream or downstream)
- in introns
What is GUS?
a reporter gene from bacteria that encodes beta-glucuronidase, which can convert colourless substrates to blue and fluorescent colours for quantification
What can DNA bending cause?
regulatory sequences interacting with the promoter
What are common properties of type 1 (TATA) promoters?
- sharp TSS
- TATA-box enrichment
- disordered nucleosome configuration
- mostly no CpG islands
What are common properties of type 2 (TATA-less) promoters?
- broad TSS
- ordered nucleosome configuration
- CpG islands
What happens at a sharp TSS?
transcription initiation from a single nucleotide
What happens at a broad TSS?
transcription initiation from multiple nucleotides
How can it be determined whether the TSS is sharp or broad?
a primer extension assay which uses the property of DNAP to incorporate specific dNTPs based on template DNA sequence
What is chromosomal effect?
the fact that expression of a transgene may be affected by the local chromosome structure or cis elements around the transgene integration site
How is the chromosome effect overcome?
by use of LCR, insulator or MAR in transgenic studies
What is the chromosome conformation capture technique used for?
to map chromatin interactions genome-wide which then provide insights into the spatial organisation of chromosomes and the mechanisms by which they fold
What do LCRs consist of?
multiple DNase I hypersensitive sites
What are DNase I hypersensitive sites?
short regions of chromatin that are highly sensitive to cleavage by DNase I that typically occur in nucleosome free regions and arise as a result of TF binding
What are LCRs required for?
correct expression of the whole gene clusters
What do LCRs do?
control the transcription of targeted genes by direct interactions with promoters through formation of looped structures
How do LCRs function?
by recruiting chromatin-modifying coactivators and transcription complexes
What does LCR deletion cause?
condensation to heterochromatin
What is MAR?
the DNA region attaching to the nuclear matrix, which can be experimentally isolated
What are MARs important in?
regulation of gene transcription within the chromosome loop
What are the 2 roles of insulators?
- prevent enhancer effect to neighbouring genes
- provide a barrier against the spread of heterochromatin
What are insulators?
specialised chromatin structures that contain DNase I hypersensitive sites
What are the 5 steps of transcriptional initiation?
- TFIID binds to the TATA box
- binding of TFIID facilitates binding of TFIIB which recruits RNAPII with TFIIF
- TFIIE joins the complex to facilitate recruitment of TFIIH
- TFIIH uses ATP to open up the double helix so that transcription can occur and phosphorylates the CTD tail of RNAPII
- RNAPII is released from the GTFs and starts moving along the template DNA
What does TFIID contain?
TBP and ~11 TBP-associated factors
What does TBP do?
recognise and bind the TATA box which causes significant bending and opening of DNA
What do TAFs do?
recognise promoter and initiator elements and interact with gene-specific regulatory proteins
What is TFIIB and what does it do?
a single subunit protein that binds BRE in the promoter to enable the interaction between TFIID and RNAP II-TFIIF and also positions RNAP at the initiator
What is the TFIIB N-terminal involved in?
the switch from abortive initiation to initiation-elongation transition
What is TFIIF made up of and what is it similar to?
2 subunits similar to sigma factor in prokaryotes
What does TFIIF do?
guide specific binding of RNAPII to the complex assembly at the promoter and also help with the elongation of nascent RNA
What does TFIIE do?
- control TFIIH functions
- enhance promoter melting
- stimulate transcription
What is TFIIH?
a release factor made up of 9 subunits
What are the 2 enzymatic functions of TFIIH?
- ATP-dependent helicase activity helps to melt promoter
- kinase activity phosphorylates CTD of RNAPII
Where is the CTD in RNAPII and what does it consist of?
RPB1 made up of multiple heptamer repeats that can be phosphorylated
What do rRNA genes have?
a bipartite promoter consisting of a core promoter and an upstream promoter element
What 2 factors does RNAPI require?
- UBF - required for high frequency initiation, maintains open chromatin structure and stimulates SL1 and promoter release of RNAPI
- SL1 - has 4 subunits required for recruitment of RNAPI
What does a ribosome contain?
4 rRNAs (3 produced by RNAP1 and 1 from RNAPIII)
What do both cleavage and chemical modifications of rRNA precursor require?
smoRNA as guide RNAs based on base pairing
What does RNAPIII have?
3 types of promoters
What do the internal (type 1 and 2) promoters of RNAPIII consist of?
2 consensus sequences (box A/B, or A/C), which are downstream of the start site
What does assembly of initiation complexes for RNAPIII require?
type 1 and 2 promoters
What are TFIIIA and TFIIIC?
assembly factors
What is TFIIIB?
the true initiator factor for RNAPIII
What is the difference between type 1 and 2 RNAPIII promoters?
the requirement of TFIIIA (type 1 requires but type 2 doesn’t)
What are common features of transcription initiations by the 3 RNAPs?
- GTFs bind at the promoter before RNAP can bind and form the PIC
- positioning of all 3 RNAPs requires TBP
- RNAPs are all large proteins with ~12 subunits with 3 in common (RPB5, 6 and 8)
What are the GTF events for each RNAP?
- SL1 binding to UBF before recruiting RNAPI
- TFIID recognising a promoter for RNAPII
- TFIIIB binding adjacent TFIIIC to localise RNAPIII
