Transcription in Eukaryotes

Question 1

How many polymerases do Eukaryotes have?

Answer 1

5

Question 2

What toxin can inhibit the activity of polymerases II and III?

Answer 2

α-amanitin found in Death Cup mushroom

Question 3

Were are Eukaryotic promoters usually located?

Answer 3

Downstream from the initiation site

Question 4

What are similarities and differences between the polymerases?

Answer 4

All polymerases are related to each other and share several subunits. Each specialises in transcribing specific genes and uses different promoters.

Question 5

What is the role of the TBP and what Polymerases use it?

Answer 5

TATA-binding protein is utilised by polymerases I, II and III. It binds and bends DNA marking the starting point for transcription.

Question 6

What does RNA polymerase II transcribe?

Answer 6

All protein-coding genes and snRNAs

Question 7

What are the RNA Polymeras II core transcription factors and their recognition elements?

Answer 7

TBP - recognises the TATA box
TFIID - recognises the Initiator(Inr) region and downstream promoter elements (DPE)
TFIIB - recognises TFB recognition element (Bre)

Question 8

What processing of RNA is used to yield mature rRNA?

Answer 8

• splicing of the primary transcript
• methylation of hydroxyl group on the 2nd carbon on ribose
• conversion of uridine to pseudouridine

Question 9

How RNA polymerase is positioned at the promoter?

Answer 9

o TFIID binds TATA box
o TFIID recruits TFIIA and TFIIB
o This complex recruits TFIIF, which stabilizes binding of RNA polymerase II

Question 10

How is preinitiation complex (PIC) formed?

Answer 10

TFIIE and TFIIH bind to RNA polymerase II on the DNA

Question 11

What happens as a result of PIC formation?

Answer 11

The polymerase C-terminal domain (CTD) is phosphorylated by TFIIH and the DNA melts

Question 12

What are the 2 activities of TFIIH and what are their roles in transcription?

Answer 12

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

Question 13

How is mRNA processed after transcription?

Answer 13

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

Question 14

How is 5’ cap added?

Answer 14

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

Question 15

How termination is initiated and what does it trigger?

Answer 15

Termination sequence at the end of the transcribed gene triggers the transfer of polyadenylation enzymes onto the RNA

Question 16

Describe the process of polyadenylation.

Answer 16

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

Question 17

What are splicing signals?

Answer 17

Consensus sequences at the exon-intron boundries which guide the spliceosome during RNA splicing.

Question 18

Describe the splicing reaction.

Answer 18

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

Question 19

What is the structure of spliceosome?

Answer 19

Spliceosome comprises of ~150 proteins and 5 snRNAs (U1, U2, U4, U5, U6)

Question 20

What is the advantage of the splicing mechanism

Answer 20

• expands informational content of the genome

- allows for alternative tissue-specific expression

Question 21

What is the cause of β Thalassemia?

Answer 21

The cause of this acute anaemia is a genetic error in splicing of β-Globin RNA transcript

Question 22

What are the regulatory points of eukaryotic transcription? What are the 2 types of the influence on the transcription?

Answer 22

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

Question 23

What mechanisms of transcription regulation are used in tissue-specific regulation?

Answer 23

Both distal and proximal control of transcription activation.

Question 24

What is the function of chromatin remodelling complexes?

Answer 24

They remodel the chromatin, uncovering the transcription sites, allowing for binding of various TFs that recruit the polymerases

Question 25

What are the 2 main mechanisms of epigenetic gene expression regulation?

Answer 25

• Histone modifications (acetylation, methylation, phosphorylation)
• DNA methylation

Question 26

How do histones hold on to DNA and what can be done to change that?

Answer 26

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

Question 27

How does DNA methylation influence gene transcription? What regions tend to be methylated and why?

Answer 27

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

Question 28

What does hyper- and hypomethylation mean?

Answer 28

Hypermethylation – gene silencing

Hypomethylation – gene activation