Transcription in eukaryotes and gene repression by noncoding RNAs Flashcards
mRNA Synthesis and Processing in Prokaryotes
and Eukaryotes.
• 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
what happens to prokaryotic mRNA before translation?
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
7-methylguanosine cap at 5’ end
- 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
Polyadenylation of the 3’terminus of
eukaryotic mRNA
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).
comparison of the structures of prokaryotic and eukaryotic mRNA
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
How many RNA polymerase are there in EUKARYOTES
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
Eukaryotic RNA polymerase II
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.
what is the role of CTD?
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
role of CTD in capping
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.
Polyadenylation at 3’ terminus
- role of CTD domain of RNAP II
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
Polyadenylation at 3’ terminus signal
(Signal 11-30 nt upstream of 3’ end (AAUAAA))
Does Polyadenylation and slicing occur together?
Recent data suggests that poly-adenylation of the 3’-end occurs
concurrently and in coordination with splicing of mRNA.
Most eukaryotic genes are in the ….. state
Most eukaryotic genes are in the off state
Why eukaryotes genes are off in ground state?
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
RNAP II activity
• 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
Regulatory proteins bind to cis-acting regulatory
elements
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
Transcription initiation in eukaryotes
- 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.
Transcription initiation in prokaryotes
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
Transcription in eukaryotes
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
Formation of pre-inititation complex:
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
General transcription factors
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).
are enhancer the same for all genes?
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
true or false
A gene is controlled by one transcriptional factor
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.
True or false
10% of all proteins produced by a eukaryotic cells are transcriptional regulators (activators or repressors).
True
Proteins required to start transcription
binding of general transcription factors, RNA polymerase, Mediator, Chromatin remodeling complexes, and histone modifying enzymes.
Initiation transcription
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.
The role of transcriptional activators in tissue-specific gene expression
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.
Tissue Specific Regulation- dpp gene
- 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.
The role of transcriptional activators in regulating gene expression in response to change in physiological conditions
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
The mechanism of gene repression by microRNAs
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.
Prokaryotes
- 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
Eukaryotes
- 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
