Eukaryotic transcription Flashcards
What makes gene expression more complicated in eukaryotic organisms?
Eukaryotes are multicellular organisms so genes may be expressed in some cells and not others
What are the main differences between Eukaryotes and E.coli?
Spatial separation from translation Different RNA polymerase Transcript processing Histones and chromatin structure Post-transcriptional control More components assemble on the gene along with RNA polymerase
Explain spatial separation
Nucleus & ribosomes
mRNA is exported from the nucleus to the ribosome
Explain the different RNA polymerases
RNA polymerase I: in the nucleus, synthesises rRNA, no sensitivity to alpha-amanitin
RNA polymerase II: in the nucleoplasm, synthesises hnRMA, high sensitivity to alpha-amanitin
RNA polymerase III: in the nucleoplasm, synthesises tRNA, medium sensiticity to alpha-amanitin
When is a transcipt fully mature?
Eukaryotes mRNA is made up of exons and introns (which need splicing out). 5’ cap must be added and a 3’ poly-A tail.
A transcript is fully mature once the introns are spliced and these two additions are made.
- 5’ cap added
- poly-A-tail added
- Introns removed
Addition of the 5’ cap
Added to the first nucleotide of the transcript.
The 5’ cap is a modified guanosine base: 7-methylguanosine joined to the first triphosphate group of the nucleotide by a 5’-5’ triphosphate bridge
This improves stability, helps to recognise the transcript as something that needs to be translated
Addition of the poly-A-tail
Polyadrenolation signal
The remaining transcript is cleaved (and degraded in the nucleus) to reveal a 3’ hydroxyl group and a poly-A-tail is added by poly(A)polymerase.
Removal of introns
Introns have a specific consensus sequence that is either side of the intended splice site.
Splitting is performed by a spliceosome.
The adenosine at the branch point is brought to the first nucleotide to allow the spliceosome to nick the intron out. Splicing factors aid in this- small nuclear ribonuclear protein particles (snRNPs)
Lariat loop formed
Chop off lariat
Link exons
Lariat degraded back down to ribonucleotides
What are some snRNPs involved in splicing?
U1
U2
U1 & U2 bring together the branch point with the last guanine in the recognition sequence at the splice site
U4
U5
U6
These snRNPs form a loop out of the intron which is called a lariat.
Role of RNA polymerase II
Co-ordinates three RNA processing activities.
Brings along CAP factors, splicing factors and polyadenylation factors to attach them in a tail- C-terminal domain
The C-terminal domain can also be phosphorylated to increase the activity of RNA polymerase
How can alternative splicing produce different mRNAs?
Exon skipping = exon left out of transcript Alternative 5' splice site Alternative 3' splice site Intron retention Mutually exclusive exons
= exons left out, introns incorporated, exons shortened
Give an example of how one gene can encode different proteins depending on the cell type, stress, or other conditions
Calcitonin or CGRP are produced depending on if cleavage occurs in the thyroid or brain
What is the name of the box in eukaryotes?
TATA
-25
Where do transcription factors bind?
To the consensus sequence further up the gene to regulate gene transcription and increase RNA polymerases ability to transcribe under specific conditions
Positive and negative transcriptional regulators
Mostly positive regulators which assist RNA polymerase. These may be produced at different developmental stages or in response to stress.
What are consensus sequences called?
cis elements/promoter motifs
How many target genes do transcriptional factors have?
Many
They can achieve and co-ordinate control (like operons) as appropriate genes are switched on at the same time even if ther are at completely different parts along the genome when activated by transcription factors.
Up-regulation of expression at the same time
General transcription factos (GTFs)
TFIID fulfils a function similar to the sigma factor. It is made up of lots of proteins (TATA binding proteins which bind the TATA box and assist in the separation of strands to initiate transcription)
Pre-initiation complex: RNAII assembled with GTFs
What happens in vivo if you mix TF & GTFs & RNA polII?
No transcription occurs
Why does no transcription occur if you mix TF & GTF & RNA pol II ?
Co-activators are needed
What is a mediator?
A molecular bridge that links transcription factors to RNA pol III & GTFs
Describe the structure of a mediator
25-35 individual subunits stuck together
4 sub-modules:
tail: interacts with the transcription factors
middle: connects head and tail as a hinge
kinase: phosphorylates other parts of the complez to activate and de-activate other proteins
head: makes connections with RNA pol II enzyme
Mediator 16
Mediator 14
Mediator 5
Mediator 25
mediator 16 refers to a subunit of a submodule
Cold, UV & darkness tested
Med 16 & Med 14 are essential for switching on genes needed for cold, darkness and UV response
Mediator 5: genes that respond to darkness
Med 25; UV
Why are different subunits of mediators important?
Different subunits have different roles and these may work with different subunit partners to switch on different genes
Mediator 16 & Mediator 14 mutations
Mutants fail to express genes in response to cold, darkness & UV
What is chromatin immunoprecipitation used for?
To see how much of a protein complex is present on a piece of DNA.
Cold responsive genes
How much RNA pol II was at the promoter ready to transcribe when it is warm/cold.
In the cold RNA pol II moves on mass to cold responsive genes
med 16, med 2 & med 14 are all needed for this response and without these cold genes are not expressed.
What are nucleosomes?
Bunches of 8 histone proteins with DNA wrapped around them
DNA + histones =
Chromatin
Histone tails
A run of amino acids that stick out. These tails can be modified by the addition of other small molecules.
Methylation: addition of CH3
Phosphorylation: addition of P
Acetylation
Ubiquitination: addition of 70+ amino acids
What is the name for the modification of histones?
Chromatin re-modelling
What does chromatin re-modelling do?
Exposes transcription factor binding sites, alters accessibility to allow transcription to occur
What does the chromatin re-modelling complex require
ATP
What are the outcomes of chromatin re-modelling?
Newly exposed binding site
Transcription factors can bind
RNA polymerase can bind
Histone acetylation
Acetylated chromatin is open and transcriptionally active
De-acetylated chromatin is compact and transcriptionally repressed
Function of small RNA molecules
E.g. SiRNA (small inhibitory) & MiRNA (micro) == these can result in gene silencing
These can interfere with transcription and translation
What is a strong inhibitor of transcription?
dsDNA - this will down regulate dene expression
miRNA (transcribed from…)
micro RNAs are transcribed from sequences that encode RNA, they may also be transcribed from other parts of a longer sequence
What is a micro RNA?
A microRNA (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression
How do microRNAs work to inhibit translation?
Inverted repeat in the RNA
Stem loop structure formed Pri-miRNA
Cleavage of the stem loop
Dicer enzyme complex chops the ds region from the stem loop
The ds region binds to proteins to form a risk complex (RNA induced silencing complex)
What actually is the micro-RNA?
The double standed section of DNA attached to the loop
Where do siRNAs arise form?
From a long piece of double stranded DNA
What do SiRNAs cause?
mRNA degradation
Describe how siRNA work
Double stranded DNA
dsRNA is cleaved by dicer to produce many small siRNAs
siRNAs combine with proteins to form RISC (risk complex)
Risk complex will target mRNA for cleavage and degradation
Exact base pairing
Compare siRNA & miRNA in terms of origin
siRNA: mRNA, trasposon or virus
miRNA: RNA transcribed from distinct gene
Compare siRNA & miRNA in terms of cleavage
siRNA: rna DUPLEX OR SINGLE STRANDED rna that forms long hairpins
miRNA: single stranded RNA that forms short hairpins of dsRNA
Compare siRNA & miRNA in terms of size
siRNA: 21-25 nucleotides
miRNA:21-25 nucleotides
Compare siRNA & miRNA in terms of action
siRNA: degradation of mRNA
miRNA: inhibition of translation
Compare siRNA & miRNA in terms of targets
siRNA: genes from which they were transcribed
miRNA: genes other than those from which they were transcribed
What are the similarities between bacterial and eukaryotic gene control?
have cascades of gene regulation
have DNA binding proteins
have negative and positive control
What are the 5 main differences between bacteria and eukaryotic gene control?
levels of regulation role of chromatin structure operons initiation of transcription transcription & translation
compare levels of regulation in bacteria and eukaryotes
bacteria: primarily transcription
eukaryotes: many levels
compare the role of the chromatin structure in bacteria and eukaryotes
bacteria: absent
eukaryotes: important
compare the operons in bacteria and eukaryotes
bacteria: common
eukaryotes: uncommon
conpare the initiation of transcription in bacteria and eukaryotes
bacteria: simple
eukaryotes: complex
compare transcription and translation in bacteria and eukaryotes
bacteria: occurs simultaneously
eukaryotes: occurs separately
