Transcription in eukaryotes and gene repression by noncoding RNAs

Question 1

mRNA Synthesis and Processing in Prokaryotes

and Eukaryotes.

Answer 1

• mRNA has same function in all cells
• significant differences in synthesis and processing of
mRNA in prokaryotes and eukaryotes
Prokaryotes
• transcription & translation occur concurrently in
cytoplasm
Eukaryotes
• transcription occurs in nucleus
• translation occurs in cytoplasm
• processes are not concurrent

Question 2

what happens to prokaryotic mRNA before translation?

Answer 2

mRNA is synthesized as pre-mRNA
pre-mRNA is processed as it’s transcribed
- addition of 7-methylguanosine cap at 5’end
- addition of 3’polyAtail
- splicing to remove interons and bring the coding region togather

Question 3

7-methylguanosine cap at 5’ end

Answer 3

• Protects from hydrolysis by exonucleases (cannot recognize 5’)
• Tells the cell that the 5’ end is intact
• Functions as an ‘attach here’ signal for ribosomes
• added at 5’ phosphate- 5’-5’linakge
• stabilize the mRNA and acids in transport across the nuclear membrane

Question 4

Polyadenylation of the 3’terminus of

eukaryotic mRNA

Answer 4

Approx 200 A residues are added enzymatically to 3’ end of mRNA- the more AAAs the more protection you have
Function:
- Increased stability of mRNA (protection from exonucleases)
- Has a role in export out of nucleus
- Essential for initiating translation of some mRNAs
Catalysed by enzyme poly(A) polymerase (PAP) and
involves CPSF protein (cleavage and polyadenylation
specificity factor).

Question 5

comparison of the structures of prokaryotic and eukaryotic mRNA

Answer 5

prokaryotes:
-unprotected 5’ and 3’
-few coding sequence are transcribed togather, they have similar function (operon)
Eukaryotes
- 7-methylguanosine cap at 5’
- polyadenylation of the 3’ end
- no operon

Question 6

How many RNA polymerase are there in EUKARYOTES

Answer 6

RNA Polymerase I
• Transcribes rRNA genes (except for 5S rRNA)
RNA Polymerase II
• Produces mRNA
• Also transcribes some snRNA genes and miRNAs
RNA Polymerase III
• Transcribes most small RNA genes (tRNA, some
snRNAs, 5S rRNA)

bacteria has one RNA polymerase as they have samll genome

Question 7

Eukaryotic RNA polymerase II

Answer 7

Eukaryotic RNA polymerase II is much larger and more
complex than its bacterial counterpart, as it not only
synthesizes mRNA, but also coordinates its processing.

Question 8

what is the role of CTD?

Answer 8

The Carboxyl Tail Domain (CTD) of RNAP II plays a central role in coordinating all three processing events (5’-capping, 3’- polyadenylation and splicing).

The CTD of RNAP II is located near the site where newly synthesized RNA emerges from the polymerase. It is an ideal place to orchestrate the binding and release of proteins needed to process RNA as it is being synthesized. The CTD plays a central role in coordinating the processing events

Question 9

role of CTD in capping

Answer 9

capping is

• cotranscriptional
• takes place while transcription is still occurring

As the 5’ end of RNA first emerges from RNA polymerase, the CTD recruits capping enzymes that add a 7- methylguanosine residue to its 5’ triphosphate group.

Question 10

Polyadenylation at 3’ terminus

- role of CTD domain of RNAP II

Answer 10

Once transcription started, CPSF is recruited to the CTD and ‘rides’ on it until AAUAAA sequence is
transcribed
(a) At which point CPSF binds to the AAUAAA sequence instead
(b) Additional cleavage factors are recruited that cleave RNA from the polymerase
(c) Polymerase PAP then adds ~200 adenylate residues to the 3’ end of the transcript

Question 11

Polyadenylation at 3’ terminus signal

Answer 11

(Signal 11-30 nt upstream of 3’ end (AAUAAA))

Question 12

Does Polyadenylation and slicing occur together?

Answer 12

Recent data suggests that poly-adenylation of the 3’-end occurs
concurrently and in coordination with splicing of mRNA.

Question 13

Most eukaryotic genes are in the ….. state

Answer 13

Most eukaryotic genes are in the off state

Question 14

Why eukaryotes genes are off in ground state?

Answer 14

1- DNA is highly compacted, DNA is not accessible for binding of RNA
2- binding of RNA polymerase is not enough to initiate transcription - regulatory proteins + activators are required

Question 15

RNAP II activity

Answer 15

• Heavy reliance on transcription regulators
• RNAP II only gives low level of gene transcription
in the absence of regulatory proteins
• Usually multiple activators are required for
maximal promoter activity

Question 16

Regulatory proteins bind to cis-acting regulatory

elements

Answer 16

Cis- acting= they are on the same molecule
– Promoter sequence
– Promoter proximal elements (binding site for general transcription factor)
• Up to 100 bp upstream the promoter
– Upstream activation sequences (UAS) = enhancers
• Can be 10,000 – 100,000 bp from promoter (DNA looping

Question 17

Transcription initiation in eukaryotes

Answer 17

• In eukaryotes, proteins called general transcription factors bind to the promoter and promoter proximal elements.
  Then RNA polymerase binds to transcription factors and promoter to create a transcription pre-initiation
  complex.

Question 18

Transcription initiation in prokaryotes

Answer 18

In prokaryotes, RNA polymerase recognizes and binds
directly to the promoter region via its sigma-factor subunit
-35 and -10 regions in E. coli promoters

Question 19

Transcription in eukaryotes

Answer 19

Promoter recognition involves TATA box located ~25-30 bp upstream from the transcription start site
• 1st event - binding of the TATAbox binding protein (TBP) which is in fact part of the TFIID
• Binding of TBP to the TATA box causes bending of DNA which is though to serve as a landmark that helps to attract the other general transcription factors

Question 20

Formation of pre-inititation complex:

Answer 20

When bound to the TATA box, TBP attracts other
general transcription factors (GTFs) and the
RNA polymerase II, thus forming the pre-initiation
complex.
Transcription can start after Carboxyl Tail Domain
(CTD) of RNAP II is phosphorylated by one of the GTFs

Question 21

General transcription factors

Answer 21

General transcription factors are named so because they are the same for all genes (which is why the promoter-proximal cis-regulatory regions are
conserved).

Question 22

are enhancer the same for all genes?

Answer 22

NO
In contrast upstream activation sequences (enhancers) are unique to a gene or a group of genes, and each serves as a binding site of a unique transcriptional activator

Question 23

true or false

A gene is controlled by one transcriptional factor

Answer 23

False
A single gene may be controlled by multiple different transcriptional activators. A gene may have many
UAS sites which add to give maximal
promoter activity.

Question 24

True or false

10% of all proteins produced by a eukaryotic cells are transcriptional regulators (activators or repressors).

Answer 24

True

Question 25

Proteins required to start transcription

Answer 25

binding of general transcription factors, RNA polymerase, Mediator, Chromatin remodeling complexes, and histone modifying enzymes.

Question 26

Initiation transcription

Answer 26

To initiate transcription of a eukaryotic gene, first a transcription activator must bind to a specific sequence in DNA (called enhancer) and help to attract DNA-bending proteins, transcription factors and RNA
polymerase to the start point of transcription.
In addition, transcription activator attracts ATP-driven chromatin-remodelling complexes – a feature that is not present in prokaryotes.
Some activator proteins act from a distance of several thousand nucleotides.
This is possible because DNA can bend and form loops.
In addition to the transcription activators, eukaryotic transcription initiation also requires the presence of a protein complex known as Mediator. It mediates interactions between activators, chromatin remodelling proteins and RNA polymerase.

Question 27

The role of transcriptional activators in tissue-specific gene expression

Answer 27

Activity of different enhancers of a eukaryotic gene
may be cell-type specific. As a consequence, the same
protein can be expressed at different levels in different
cell types (or not expressed at all).
Thus, despite containing the same DNA, different cell
types synthesize different sets of RNAs and proteins.

Question 28

Tissue Specific Regulation- dpp gene

Answer 28

• Example: the dpp (decapentaplegic) gene of Drosophila.
• Mediates signals between cells.
• dpp gene has numerous enhancers over 50 kb interval, including downstream of gene.
• Each enhancer regulates dpp in a different site in a developing fly.

Question 29

The role of transcriptional activators in regulating gene expression in response to change in physiological conditions

Answer 29

Example: the GAL system from yeast
• Enzymes GAL1, GAL2,… convert the imported
galactose into glucose that can be metabolised
• These genes are silent if there is no galactose in the
growth medium
• Three additional genes (GAL3, GAL4 and GAL80)
encode regulatory proteins
• GAL4 is a transcriptional activator

Question 30

The mechanism of gene repression by microRNAs

Answer 30

Example of regulation of gene expression by noncoding RNAs (RNA interference or RNAi)
Capped and polyadenylated miRNA precursors are synthesized by RNAP II. They are the products of transcription from independent
genes or from introns of protein-coding genes. A ~70-nucleotide precursor hairpin (pre-miRNA) is produced in the nucleus and then exported to the cytoplasm.
There, it is cleaved by an enzyme Dicer to yield ~20-bp
miRNA/miRNA duplex.
This duplex is bound by Argonaute protein and one strand is degraded. Other proteins bind and a complex named RNA-induced silencing complex, or RISC, is formed.

Question 31

Prokaryotes

Answer 31

• Transcription & translation linked in cytoplasm
• mRNA monocistronic or polycistronic
• Termination occurs by dissociation
• mRNA not capped or tailed
• mRNA coding sequence is continuous - no introns
• Single RNA polymerase that binds directly to promoter

Question 32

Eukaryotes

Answer 32

• Transcription in nucleus, translation in cytoplasm
• mRNA monocistronic
• Termination involves RNA cleavage
• mRNA capped and tailed
• mRNA has introns and must be spliced before translation
• Three RNA polymerases that bind to transcription factors