12 - Eukaryotic Transcription Flashcards
do bacterial transcription and translation occur in the same compartment
- what compartment
yes
- the cytoplasm
where eukaryotic transcripts are processed an transported to
- give an example
processed in the nucleus (nuclear envelope)
- transported via nuclear pore to the cytosol
e.g. mRNA precursors processed in nucleus and transported to the cytosol for translation
Three main RNA Pol in eukaryotes
RNA Pol 1
RNAPol 2
RNA Pol 3
general structure of eukaryotic RNA Polymerases
contain:
- B’ and B-like subunits
- alpha-like subunits
- common subunits
- enzyme-specific subunits
Conserved elements of eukaryotic RNA Pol II promoters
- lnr - initiator element
- TATA box promoter has a TATA box
- TATA-less promoter has a DPE (downstream core promoter element)
- enhancer - can be several kb away
- maybe a CAAT box or a GC box between -40 and -150
differences between bacterial and eukaryotic transcriptional promoters
- spacing is different, and CAAT box (or GC box) of eukaryotic promoters can be on either strand
- the -10 and -35 sequences interact with a part of the holoenzyme (sigma subunit)
- whereas the TATA, CAAT and GC motifs are recognised by other proteins, and not RNA Pol II
- meaning these are fundamentally different RNA replication mechanisms
Eukaryotic transcription - initiation mechanism
- TFIID (a transcription factor) guides RNA Pol II to its promoters
- TFIID contains the 30kDA TATA-box binding protein (TBP)
- TBP binds tightly to the TATA box (10^5 times more than nonconsensus sequences)
- bending of DNA caused by binding widens the minor groove
- complex forms called the basal transcription apparatus
formation of this complex in order: - TFIIA binds TBP
- TFIIB binds TBP
- TFIIB recruits TFIIF, RNA Pol II, TFIIE and TFIIH
- TFIIH is bi-functional
- it is a helicase that opens the DNA double helix
- and also is a kinase that phosphorylates the C’ terminal domain (CTD) of the RNA Pol II L’ subunit
- Phosphorylation of CTD marks transition from initiation to elongation
general nomenclature of transcription factors
e.g. TFIID
- TF (Transcription Factor)
- II (Pol II)
- D (A, B, C, D)
TFIIH function in eukaryotic transcription
- TFIIH is bi-functional
- it is a helicase that opens the DNA double helix
- and also is a kinase that phosphorylates the C’ terminal domain (CTD) of the RNA Pol II L’ subunit
What marks transition from initiation to elongation in eukaryotic transcription
- phosphorylation of CTD (C’-Terminal Domain) by TFIIH
eukaryotic transcription - elongation mechanisms
- TFIIB, TFIIE and TFIIH dissociate from basal transcription apparatus complex
- RNA is synthesised
- RNA Pol II progresses
- it frees the promoter and TFIID/TFIIA complex for further recuitment
Inhibitor of RNA Pol II
- process of inhibition
- alpha (a)-amanitin
- found in death cap fungi/mushroom
- it is a cyclic octapeptide
- binds tightly to active site of RNA Pol II
- reduces rate of RNA production to a few nucleotides per hour
- constrains the flexibility RNA Pol II requires to translocate DNA through its active site
enhancers of eukaryotic transcription
- how enhancers affect DNA looping model
- they increase the transcription level of genes
- can be active in a tissue-specific manner
- so can play developmental roles in the body
- DNA looping model
- proteins bound to a distant enhancer interact with components of transcription initiation complex
- this loops out the intervening DNA
The DNA looping model overview
- Mediator protein acts as bridge between activator proteins that bind enhancer control elements and non-phosphorylated CTD of RNA Pol II in its initiator state
- enhancer control elements bind activator proteins
- silencer control elements bind repressor proteins
enhancer and silencer gene function in eukaryotic transcription
enhancer - turn genes ‘on’ - allow transcription of genes
- activator proteins required to bind enhancer control elements
silencer - turn genes ‘off’ - repress transcription of genes
histone acetylation in eukaryotic transcription
- histone tails protrude outwards from a nucleosome
- acetylation of histone tails promotes a loose chromatin structure that permits transcription
- allows regulation of transcription
control of gene activation in enhancer proteins for eukaryotic transcription
- since activator proteins are required to bind enhancer control elements in transcription
- transcription can only be enhanced if appropriate/specific activator proteins are present
- this allows cell-specific control of gene expression stopping
Processing of eukaryotic pre-mRNA - simple
RNA Pol II transcripts (mRNAs) are modified in three ways:
- 5’ end capped by a nucleotide triphosphate through an unusual linkage - accompanied by methylation
- 3’ end of mRNA is trimmed and poly-A tail is added
- Introns are removed by splicing (mRNA splicing)
Addition of a 5’ cap on mRNA in Pol II Eukaryotic trancription - process
Addition of poly-A tail on eukaryotic mRNA
How do capping and tailing enzymes find the mRNA transcripts in eukaryotes
Major differences between bacterial and eukaryotic mRNA transcription
Bacterial:
- primary transcript is often a mature mRNA and is used directly for translation
- transcription and translation occur in the same compartment - the cytoplasm - so may be coupled
- bacterial mRNAs are often polycistronic; they encode more than one protein
Eukaryotes:
- primary transcript is not mature and has to be processed before translation
- transcription is nuclear, translation is cytosolic - no coupling of the two processes
- eukaryotic mRNAs are monocistronic; they encode just one protein
