Lecture 17 Eukaryotic Transcription 1 Flashcards
Eu/prokarytic transcription compared
Eukaryotes have introns - non coding regions between exons (the coding regions) that need to be spliced out
5’ guanosine cap
Poly A tail lots of Adenine at 3’ end
Order of events for eukaryotic processing
Transcription starts at UTR
Addition of m⁷ Gppp cap (5’ cap)
3’ cleavage and addition of poly A tail
Intron splicing (excision)
Exon ligation
5’ cap and poly A tail stabilise the mRNA for intron removal to be possible without disintegration
Step 1: adding 5’ cap
Modified guanosine base has an additional methyl group otherwise similar to guanosine
So 7-methylguanosine is bonded to 5’ end of primary transcript by 3 phosphate groups. This protects it from degradation and allows cellular machinery to recognise this molecule as a transcript.
Makes incomplete mRNA recognisable as breaks lead to chain ending in one phosphate. Broken chains cannot receive a new cap because they can only be added onto di or tri phosphate.
5’ guanosine cap stabilises and prevents non-transcriptional or damaged mRNA from translation
Step 2: adding poly A tail
Identifies molecule as mRNA
Aids translation
Addition of 150-250 A’s to 3’ end of transcript.
Recognition sequence for polyadenylation - addition of multiple adenine groups
Pre tail zone amino code of AAUAAA followed by 10-30 nucleotides with a CA end here is the cleavage site
followed by a GU or U rich section
that is cleaved off degraded in nucleus and reused
Cleavage reveals an OH group on the CA terminal
Poly A polymerase enzyme adds Adenine tail of upto 250 aminos at 3’ end
Step 3: intron splicing
Must be correct as codons are read as 3 groups of bases. If exons connect incorrectly even by one base a frame shift will cause missense.
Specific signaling controls where it happens
5’ splice site consensus sequence followed by recognition sequence identifying intron
3’ splice site from end of intron to beginning of Exon another consensus sequence
Splicing of transcript by spliceosome
Requires 5 snRNP particles
SnRNP = snRNA+proteins
Adenine in intron reacts with guanine at splice junction. Assisted by snRNP’s U1 and U2 that help find cleavage site.
Cleavage of 5’ end disconnecting from 5’ exon, free end of intron binds to same adenine forms lasoo still connected to 3’ exon.
Aided by snRNp’s U 4,5&6 aid 5’ exon 3’ OH end to find and bind to 3’ exon
Lariat (intron loop) released so intron is spliced and 5’ and 3’ exon ligased
RNA polymerase ll coordinates these 3 steps (adding 5’ cap/ adding 3’ poly a tail/intron splicing)
Each gene has a pattern order for intron splicing not in order of 1/2/3/4 e.g. 2 may be taken out first
C terminal domain of RBP large protein part of RNA Poly carries factors for steps 1/2/3 and adds them onto RNA as it is produced
Alternative splicing produces different mRNA
Controlled by different splicing molecules
A)Constituent: standard pre-mRNA with introns removed
B)Alternative splicing:
exon skipping/inclusion
Alternative 5’ splice site
Alternative 3’ splice site
Intron retention - one intron not spliced out
Mutually exclusive exons - whole piece cannot be spliced so you get 1/3/4 or 1/2/4 but can’t get all exons
Alt. Processing varies proteins that can be produced from one gene. Under diff temp. cell types or stress conditions.
In drosophila alt. Splicing can generate 2 diff types of transcript whose protein later goes on to determine if individual is male or female
In drosophila
Alt. Processing varies the proteins that can be produced from a gene : thyroid/ brain cell use of pre-mRNA
In thyroid cells pre-mRNA cleavage and polyadenylation take place at end of Exon 4
Producing an mRNA that contains exons 1/2/3/5/4. Translation produces hormone calcitonin
In brain cells 3’ cleavage takes place at end of Exon 6.
During splicing exon 4 is eliminated with the 5 introns
Producing mRNA with exons 1/2/3/5/6
Translation yields calcitonin-gene-related peptide
Eukaryote transcription factors
Equivalent of bacterias
-10 box pribnow TATAAT
Eukaryotes have
-25 TATA box
General transcription factors needed all the time for any transcription to occur
Specific transcription factors - cis element promotor motifs - short 6-8 bp recognition sequences for specific transcriptional activators/repressors
In response to stimulus or environmental change
Transcription factors are usually activators (but may be repressors)
Direct specific expression (cell type, developmental stage, stress etc.)
Transcription factors can coordinate control a bit like operons
1) a stressor (e.g. drought) activates transcription factors
2)binding of active transcription factors to dehydration response elements (DREs) stimulates transcription of genes
3) which produce different proteins participating in stress response
General transcription factors
Transcription factor llD arrives first guides other factors needed to position RNA pol ll at TATA -25 box and start transcription - similar to sigma factors in prokaryotes.
TFllD is made up of Tata box binding protein (TBP) for sequence location and 13 Tata box binding factor associated factors (TAFs)
Forms PIC - preinitiation complex to start transcribing
Binding of TATA binding protein (TBP) leads to bending of DNA helix
C terminal of Tata binding protein (TBP) interacts with TATA box, binding results in helix bending opening up double helix
(As CAP protein does in prokaryotes)
Young and Kornburg : how to get activator dependent transcription
Found that TFs(activators) + Pol ll + GTFs added to promoters in a cell free (in vitro) system doesn’t start activator dependent transcription.
You need a co-activator
The mediator co-activator complex was identified - mediates other components
Mediator co-activator multiprotein complex
Mediator recruits RNA pol ll to the gene
4 sub modules
Tail - binds to transcription factors
Middle- hinge allows folding
Kinase- made up of 4 proteins that phosphorylate/dephosphorylate and modify activity
Head - makes multi connections to RNA polymerase
Bends into loop to start transcription
