Transcription in Eukaryotes Flashcards
How many polymerases do Eukaryotes have?
5
What toxin can inhibit the activity of polymerases II and III?
α-amanitin found in Death Cup mushroom
Were are Eukaryotic promoters usually located?
Downstream from the initiation site
What are similarities and differences between the polymerases?
All polymerases are related to each other and share several subunits. Each specialises in transcribing specific genes and uses different promoters.
What is the role of the TBP and what Polymerases use it?
TATA-binding protein is utilised by polymerases I, II and III. It binds and bends DNA marking the starting point for transcription.
What does RNA polymerase II transcribe?
All protein-coding genes and snRNAs
What are the RNA Polymeras II core transcription factors and their recognition elements?
TBP - recognises the TATA box
TFIID - recognises the Initiator(Inr) region and downstream promoter elements (DPE)
TFIIB - recognises TFB recognition element (Bre)
What processing of RNA is used to yield mature rRNA?
- splicing of the primary transcript
- methylation of hydroxyl group on the 2nd carbon on ribose
- conversion of uridine to pseudouridine
How RNA polymerase is positioned at the promoter?
o TFIID binds TATA box
o TFIID recruits TFIIA and TFIIB
o This complex recruits TFIIF, which stabilizes binding of RNA polymerase II
How is preinitiation complex (PIC) formed?
TFIIE and TFIIH bind to RNA polymerase II on the DNA
What happens as a result of PIC formation?
The polymerase C-terminal domain (CTD) is phosphorylated by TFIIH and the DNA melts
What are the 2 activities of TFIIH and what are their roles in transcription?
o TFIIH kinase activity - triggers phosphorylation of the polymerase C-terminal domain.
Phosphorylation of the tail -> conformational change -> RNA polymerase II escapes the PIC(releasing all TFs) and start transcription
o TFIIH helicase activity - melting of DNA to form a transcription bubble
How is mRNA processed after transcription?
1) A cap ( 7-methyl guanylate) is added at 5’end
- RNA polymerase CTD carries the enzymes for 5’-capping
2) Cleavage at Poly(A) site
3) Polyadenylation
4) RNA splicing
How is 5’ cap added?
1) Phosphatase removes γ (gamma) phosphate at the 5’-end of mRNA precursor
2) 5’-end of mRNA transcript receives a GMP group from GTP in a reaction catalysed by guanyltransferase
3) The base of the guanylate group is methylated at N-7
4) In some cases, ribose groups can be additionally methylated
How termination is initiated and what does it trigger?
Termination sequence at the end of the transcribed gene triggers the transfer of polyadenylation enzymes onto the RNA
Describe the process of polyadenylation.
o Poly-A signal is transcribed onto mRNA
o CstF (cleavage stimulation factor) and CPSF (cleavage and polyadenylation specificity factor) bind to mRNA poly-A signal
o CstF cleaves mRNA and diffuses away
o PAP (poly-A polymerase) binds at the 3’-end to protect DNA adds ~200 A bases
What are splicing signals?
Consensus sequences at the exon-intron boundries which guide the spliceosome during RNA splicing.
Describe the splicing reaction.
o Spliceosome facilitates the attack of Adenine at the branching site on phosphodiester bond on exon-intron border
o A new phosphodiester bond is formed between Adenine and the last nucleotide of intron
o Exons join together forming a phosphodiester bond
o (2 trans-esterification reactions)
o Intron lariat is degraded
o This process requires ATP hydrolysis
What is the structure of spliceosome?
Spliceosome comprises of ~150 proteins and 5 snRNAs (U1, U2, U4, U5, U6)
What is the advantage of the splicing mechanism
- expands informational content of the genome
- allows for alternative tissue-specific expression
What is the cause of β Thalassemia?
The cause of this acute anaemia is a genetic error in splicing of β-Globin RNA transcript
What are the regulatory points of eukaryotic transcription? What are the 2 types of the influence on the transcription?
1) Initiation of transcription - assembly of the GTFs at the promoter
2) Activation of transcription
- Proximal activation of transcription - TFs binding at promoter-proximal regulatory elements and stimulation of the transcription rate - e.g. hormone-dependent gene activation
- Distal activation of transcription - enhancers, insulators, silencers, locus control region binding regulatory regions far away from the gene promoter
What mechanisms of transcription regulation are used in tissue-specific regulation?
Both distal and proximal control of transcription activation.
What is the function of chromatin remodelling complexes?
They remodel the chromatin, uncovering the transcription sites, allowing for binding of various TFs that recruit the polymerases
What are the 2 main mechanisms of epigenetic gene expression regulation?
- Histone modifications (acetylation, methylation, phosphorylation)
- DNA methylation
How do histones hold on to DNA and what can be done to change that?
Histone tails are positively charged and therefore interact with the negatively charged DNA, blocking the transcription of covered sequences. The histone tails can be acetylated to loosen the chromatin structure and allow for the transcription of the genes
How does DNA methylation influence gene transcription? What regions tend to be methylated and why?
DNA methylation prevents gene transcription. CpG islands (CpG-rich regions) tend to be unmethylated and found near transcription sites of highly expressed (house-keeping) genes
What does hyper- and hypomethylation mean?
Hypermethylation – gene silencing
Hypomethylation – gene activation
