I. DNA & RNA | 14. Structure of the eukaryotic genes, initiation and termination of transcription in eukaryotes Flashcards by Aurora H

How many types of eukaryotic polymerases?

Eukaryotes have 3 types of RNA polymerases (I, II, III)

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What is the difference in RNA polymerases between eukaryote and prokaryote?

1/ Eukaryotes have 3 types of RNA polymerases (I, II, III), while prokaryotes have only 1 type (α2ββ’).

2/
- In prokaryotes, transcription + translation occurs simultaneously i
- in eukaryotes, RNA is first transcribed in the nucleus and then translated in the cytoplasm.

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RNA polymerase type I
-> What is the synthesized RNA?

Large pre-rRNA

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RNA polymerase type I
-> What is the synthesized RNA?

Large pre-rRNA

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RNA polymerase type II
-> What is the synthesized RNA?

hnRNAs
snRNAs

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RNA polymerase type III
-> What is the synthesized RNA?

1/ pre-5s rRNA
2/ pre-tRNAs
3/ U6 snRNA
4/ SRP RNA

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RNA polymerase type I
-> What is the mature RNA product?

28S, 18S and 5.8S
rRNAs

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RNA polymerase type II
-> What is the mature RNA product?

1/ mRNAs (from hnRNAs)
2/ snRNAs

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RNA polymerase type III
-> What is the mature RNA product?

5s rRNA
tRNAs
U6 snRNA
SRP RNA

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Structure of eukaryotic genes

1/ Exons
2/ Introns
3/ Promoter sequences

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What are exons?

coding sequence, transcribed + translated.
-> Coding for the amino acids in the polypeptide chain

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What are introns?

non-coding sequence, transcribed but NOT translated (almost always have GU sequence in their 5’ end and AG in their 3’ end)

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What are Promoter sequences?

DNA sequences that define where transcription of a gene by RNA polymerase II begins.
-> Typically located directly upstream or at the 5’end of the transcription initiation site.

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Eukaryotic RNA polymerase II-dependent promoters are composed of 4 sequences which are ___

1) TATA box-TBP
2) BRE–TF2B (BRE = B recognition element)
3) DPE–TF2D (DPE = downstream promoter element)
4) INR–TF2D (INR = initiator element)

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Eukaryotic RNA polymerase II-dependent promoters
-> Characteristics of TATA box

1/ Usually located 25-35 base pairs upstream of transcription start site
2/ TATA binding protein (TBP) binds to TATA-
box sequence -> unwinds the DNA
3/ Adenine (A) + thymine (T) = easiest place to unwind double helix, since only 2 H-bonds
between them

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Eukaryotic RNA polymerase II-dependent promoters
-> Characteristics of TATA box

Eukaryotic RNA polymerase II-dependent promoters
-> Characteristics of BRE - B recognition element

BRE–TF2B
 BRE = B recognition element
 Transcription factor IIB (TF2B) binds here

Eukaryotic RNA polymerase II-dependent promoters
-> Characteristics of DPE (downstream promoter element)

DPE–TF2D
- DPE = downstream promoter element
- When the promoter appears in the downstream region (+)

Eukaryotic RNA polymerase II-dependent promoters
-> Characteristics of INR - initiator element

INR–TF2D
- INR = initiator element
- First nucleotide is transcribed here = transcription start point

What are Terminator sequences?

a DNA sequence just downstream of the coding segment of a gene, which is recognized by RNA polymerase as a signal to stop transcription

What are the 2 Signals participate after eukaryotic transcription?

 Upstream sequence signal for addition of cap: signal for addition of cap to the 5’ end of mRNA (7-methyl-guanosine).
=> Cap facilitates initiation of translation + stabilization of mRNA

 Downstream sequence signal for addition of poly-A-tail: AAUAAA sequence is a signal for addition of a poly-A-tail.
=> Makes the mRNA more stable + prevents its degradation (NOTE: poly-A-tail added after transcription)

Where does Transcriptional initiation in eukaryotic cells take place?

The eukaryotic transcription initiation must take place on DNA that is packaged into nucleosomes and higher-order forms of chromatin structure.

The eukaryotic RNA polymerase requires ___
-> Why?

general transcription factors, because they are required at most promoters of genes transcribed by RNA polymerase II

Initiation of transcription by RNA polymerase II requires initiation factors, that help to __ (2)

Position RNA polymerase correctly to the promoter
Aid in pulling the 2 DNA strands so that the template strand can enter the active site of the enzyme

What are the 6 common General transcription factors:?

- TFII (TF2) = transcription factors for polymerase II - TF2D = TATA-box recognition - TF2B = accurate positioning - TF2F = stabilizes the structure of initiation - TF2E = regulation of TF2H - TF2E = unwinds DNA double helix, ATP hydrolysis

How does Initiation complex form?

1. The TBP (subunit of TF2D) is the first protein to bind to a TATA-box promoter -> TAF (TBP-associated factors) initiate transcription from promoters that lack a TATA-box; The TBP will start the assembly of the initiator complex 2. TF2B bind at BRE (assists RNA polymerase II in melting the DNA strands at the transcription start site + interacts with template strand near the polymerase II active site) 3. TF2B and RNA polymerase II binds – position the polymerase over the start site 4. TF2E creates a docking site for the TF2H 5. Binding of TF2H completes the assembly of the transcription pre-initiation complex. 6. CTD (C-terminal domain) of RNA pol II gets phosphorylated by TF2H. 7. After the assembly of the pre-initiation complex: - TF2D (+TFB) and TF2A remain at TATA-box as the RNA polymerase II transcribes way from the promoter region - TF2B, F and H dissociate completely 8. RNA polymerase II binds to DNA -> start transcription

During the formation of the initiation complex -> Binding of TF2H completes the assembly of the transcription pre-initiation complex. -> the subunit of TF2H uses (1)_____ to unwind the DNA at the transcription start site, exposing the template strand to (2)____

1/ ATP energy 2/ the polymerase

In eukaryotic cells, the DNA is packaged into nucleosomes, which are further arranged into higher-order chromatin structures. This is why the TATA-box of the core promoter is well hidden in the packed DNA. => Therefore, the TFs need some help to get to the TATA-box -> How do they achieve this?

By recruiting Activators, Mediators, Chromatin modifying enzymes

proteins that must bind to a sequence on the DNA known as the enhancers. -> Its function is to help attract RNA polymerase II to the initiation point of transcription

large complex of proteins which allow activator proteins (enhancers) to communicate properly with the RNA pol II and TFs. -> Mediator complex can activate/inhibit transcription

includes enzymes, chromatin-remodeling- complexes and histone-modifying-enzymes, which can increase access to the DNA by loosening up the tight structure of nucleosomes through covalent modification in the chromatin

Features of Termination of eukaryotic transcription

The last part of mRNA is the 3’-poly-A-tail, which will be the mark for the termination of transcription. As RNA polymerase II reaches the end of the gene, a similar mechanism ensures that the 3’-end of the pre-mRNA is appropriately processed

5 steps of Termination of eukaryotic transcription

1. RNA polymerase II reaches a sequence that encodes for the 3’-end 2. CstF (cleavage stimulation factor) and CPSF (cleavage + polyadenylation specificity factor) are 2 proteins that will travel together with the RNA polymerase tail 3. RNA is cleaved from the RNA polymerase II and CstF and CPSF bind to their recognition sequences on the newly cleaved RNA molecule, resulting in additional proteins assembling with them to create the 3’-end of the mRNA 4. PAP (polyA polymerase) adds approximately 200 adenine nucleotides to the 3’-end produced by the cleavage of ATP 5. RNA polymerase II continues to transcribe the sequence even after the 3’-end of the synthesized mRNA is cleaved -> the newly synthesized RNA that emerges lacks a 5’ cap. (Since it is unprotected, the 5’-3’ exonuclease will degrade nucleotides along the polymerase tail)