Lecture 1: RNA polymerase Flashcards
What are the different types of DNA-dependent RNA polymerase in eukaryotes? How are they distinguished?
There are 3 types. They can be distinguished by their sensitivity to α-amanitin. All 3 share some subunits.
1) Pol I transcribes rRNA. It is very insensitive to α-amanitin.
2) Pol II transcribes for protein coding genes. It is very sensitive to α-amanitin.
3) Pol III transcribes for tRNA and snRNAs. It is moderately sensitive to α-amanitin.
What is the core promoter?
The core promoter is the minimum part of the promoter required to initiate transcription.
• It binds general TFs which are specific for each type of RNA polymerase.
• It includes to transcription start site (TSS).
How does the PIC form for RNA pol I?
RNA pol I holoenzyme requires multiple components to form a complex.
• The core promoter is from -45 to +20.
• There are also upstream control elements.
• An upstream binding factor (UBF) dimer binds to the UCEs.
• SL1 binds to the core promoter (TATA-binding protein and 3 TBF associated factors).
• UBF causes loops upstream. UCE and core elements contact.
• RRN3 binds to UBF/SL1 complex and Pol I, bringing them together.
How does the PIC form for RNA pol III?
RNA pol III also requires different parts to form the PIC.
• Unusually, the control regions are inside the transcribed section of the gene.
• The core promoter consists of A and B blocks which are from +55 to +80.
• TFIIIC and TFIIIB are both required for RNA pol III recruitment.
• TFIIIC binds to the core promoter.
• TFIIIB consists of B”, TBP and BRF, it links pol III and TFIIIC.
• RNA pol III also binds to an upstream regulatory sequence (URS).
How does the PIC form for RNA pol II?
The preinitiation complex requires a number of transcription factors.
• TFIIA, TFIIB, TFIID (introduces a 90-degree kink in the DNA). TFIIE, TFIIF, and TFIIH (helicase).
• TATA-binding protein (TBP) is a subunit of TFIID and it binds to the DNA.
• TFIIA binds to TBF and aids binding to the promoter.
• TFIIB binds downstream at the TFIIB-recognition element (BRE) and it helps to recruit other factors.
• TFIID-TFIIA-TFIIB complex recruits TFIIF and pol II.
• TFIIE binds and recruits TFIIH.
• TFIIH helps to create the transcription bubble. It has ATPase and helicase activity.
How are subunits conserved?
TATA binding protein (TBP) is conserved for different RNA polymerases. • In RNA pol I it is part of SL1. • In RNA pol II it is part of TFII D. • In RNA pol III it is part of TFIII B. • In archaea it is by itself.
How do core promoters work for RNA polymerase II?
There are no universal core promoter elements for RNA pol II.
• Combinations of promoter motifs can recruit RNA pol II.
• There are two main types: focused and dispersed.
• Focused promoters contain a single TSS or a distinct cluster of start sites over several nucleotides. They are more ancient and widespread across nature, but in vertebrates they are less common.
• Focused promoter genes can be divided into housekeeping, developmental or tissue specific.
• Dispersed promoters contain several start sites over 50-1000 nt. They are usually found in CpG islands.
What are CpG islands?
CpG islands dinucleotides of cytosines followed by guanines.
• The cytosine is often methylated on its 5 position.
• CpG frequency is a fifth of what we expect due to deamination to T when C is methylated.
• Clusters of CpG are therefore significant.
• CpG islands are clusters of C/G dinucleotides that aren’t methylated.
• TF binding sites are clustered near the TSS at CpG island promoters.
What are enhancers?
Enhancers activate promoters in response to external signals.
• They can be upstream or downstream.
• They can be several thousand bases away.
• They are often composed of the same sequence elements found in promoters.
• Sequence elements in enhancers are found within a modular structure containing binding sites for several different TFs.
• Two proto-enhancers are required for enhancer activity.
• Proto-enhancers can be divided into the fundamental units called enhansons.
• Enhansons bind to TFs.
• Spacing between enhansons is important.
How does the SV40 enhancer work?
SV40 is an enhancer found in the SV40 virus.
• There are A, B and C proto-enhancers.
• A combination of any two of the proto-enhancers will enhance expression.
• The enhancers can bind multiple different transcription factors.
How can we study regulatory sequences using genetics and reporters?
We first use bioinformatics to reveal promoter or any sequences we known are regulatory.
• We can delete or mutate sequences to see their effect on gene expression.
• We can transfer sequences to other genes to see how regulation affects the other genes.
• We can add the sequences to reporter genes. Different genes can give us different information.
• Chloramphenicol acetyltransferase (CAT) can show us how much regulation is occurring. Acetylated and unacetylated forms can be separated by chromatography.
• β-galactosidase can show where a gene is being expressed and how much it is being expressed by using X-gal as a substrate.
• GFP can show where a gene is being expressed.
What is a locus control region?
A locus control region can enhance the expression of linked genes.
• LCRs are cis-regulatory elements.
• They recruit TFs and chromatin modifying enzymes.
• We don’t fully understand how they work.
• The main example is β-globin.
What is a super-enhancer?
A super-enhancer is a region of a mammalian genome which contains multiple enhancers.
• They bind TFs, loop to target genes and activate transcription.
• They have unusually strong enrichment for the binding of transcription coactivators.
