eukaryotic transcription Flashcards
chromatin =
complex of DNA and proteins that forms chromosomes within the nucleus of eukaryotic cells that protect DNA
we know chromatin exists outside of metaphase as…
in experiments cell nuclei treated with deoxyribonuclease (cuts DNA randomly, eventually into single nucleotides) - when run on agarose gel it can be seen that increasing the time of digestion results in DNA becoming smaller as bands travelled further but was not fully cut = some DNA protected by nucleosomes
nucleosomes =
a structural unit of eukaryotic chromosome, consisting of a length of DNA coiled around a core of histones
nucleosomes structure…
- consists of 8 protein histones which form a bundle =(H2A, H2B, H3, H4) x2
- around the outside, there are 1.65 turns of double-stranded DNA helix with roughly 10 base pairs per turn
- basic amino acids (lysine and arginine) positioned close to DNA as they are positive = attract to negative DNA - this holds the proteins and DNA together
- histones H2A and H2B are closely packed to form a dimer with positive N-terminal tails
DNA held to histone by…
strong electrostatic interactions between negative DNA + positive histone tails and positive N-terminal of helices
chromatin exists in two forms…
euchromatin and heterochromatin
euchromatin =
- less condensed
- activated chromatin
heterochromatin =
- highly condensed
- inactivated chromatin
DNA regions likely to be expressed are packaged in…
euchromatin - nucleosomes are less condensed to there is room for transcription activator proteins to bind
converting chromatin :
need to open up heterochromatin via modification to the histone proteins in order to activate genes (euchromatin):
lysine acetylation = addition of an acetyl group neutralises lysine, so the protein an DNA are no longer held together
lysine methylation = editing of a methyl group provides extra signal that another protein can recognise - allows binding to other proteins
linker histones:
= connects two strands of DNA between nucleosomes
e. g. Histone H1
- in heterochromatin (condensed) it connects two strands
- in euchromatin (nucleosomes are activated) it only binds to 1 of the strands, controlled by modification
in eukaryotes, most genes are…
repressed by chromatin by default and they need to be activated when needed (opposite in prokaryotes)
class 1 and class 3 promoters -
class 1 = ribosomal RNA
- relatively abundant as ribosomes are abundant and rRNA is very stable with high-efficiency transcription
class 3 = transfer RNA and other small RNAs
class 2 promoters -
messenger RNA and most small nuclear RNA
- snRNA involved in splicing
- have core promoter = comprises TATA element and specifices promoter start site
- have upstream promoter elements = contain motifs
- have enhancer elements = densely packed with motifs
motifs =
sequences located in the enhancer elements of promoters which act as binding sites for specific proteins
activation of class 2 promoters:
multiple proteins bind in various combinations to allow activation
upstream promoter elements and enhancer elements comprise of motifs of about 6-12 base pairs, each bound by a specific activator protein
activator proteins -
have 2 domains connected by linkers:
DNA binding domain = interacts with specific DNA nucleotide sequences
Activation domain = binds to other proteins
how to show test if a specific activator protein binds to a specific motif to activate the promotor:
- plasmid 1 is set up to replicate and propagate E.coli but also has elements enabling it to be recognised in eukaryotic cells as well
- plasmid 2 is set up that has a reporter gene and a promoter which will only be activated by the specific protein we are testing
- these plasmids are placed within nucleus of mammalian cells - plasmid 1 will produce the specific protein which will be translated in the cytoplasm and as this is a nuclear protein it will be transported back into the nucleus = it will bind to its sepcific binding site on plasmid 2 to drive expression of the reporter gene to produce a protein
- without plasmid 1 or if there is a mutation in the protein binding site, the reporter gene will not be expressed = proves protein is needed for expressing the reporter gene
the DNA binding domain of activator proteins -
= recognises specific DNA sequences
- DNA binding occurs in the major groove od the double helix of DNA
the activation domain of activator proteins -
= binds to other proteins (recruiting them)
- recruitment of histone acetyltransferases
- recruitment of chromatin remodelling factors
- recruitment of coactivators
recruitment of histone acetyltransferases by activator proteins -
activation domain binds to histone acetyltransferases and brings them close to where the activator protein is bound in order to acetylate glycine (removes positive charge) = weakness the contact between the nucleosome and DNA
recruitment of chromatin remodelling factors by activator proteins -
activation domain binds to proteins that act as chromatin remodelling factors and bring them into the activator protein
- DNA binding domain binds to a free bit of DNA next to a nucleosome
- there is a hinge in the middle of the nucleosome
- a translocation domain binds another part of the DNA running around the nucleosome
ATP hydrolysis deforms the DNA and pulls it around the nucleosome, the translocation domain allows it to run through = the DNA ends up moving relative to the nucleosome
nucleosome is moved up and down the DNA = nucleosomes can be cleared away from the binding site on the promoter to create space for RNA pol
recruitment of coactivators by activator proteins -
coactivators do not bind to DNA directly - they bind to activator proteins that bind to DNA
coactivators bind RNA pol 2 and stabilise its binding to the promoter
.general transcription factors -
required for specific initiation and efficient elongation
there are 5 for RNA pol II:
- TBP (TATA-binding protein) = binds in the minor groove
- TFIIH = phosphorylates ser 5 repeats on RPB1 - enabling promoter clearance and interacts with RNA processing factors
- TFIIE
- TFIIF
the sequence of events of transcription at RNA pol II promoters:
1) DNA binding domain of activator protein binds to motifs in the upstream promoter and enhancer elements
2) activation domain of activator proteins recruit:
- histone acetyltransferases = weaken contact between nucleosome and DNA
- chromatin remodelling factors = clear space for RNA polymerase
- coactivators = bind to RNA pol and stabilise its binding to the promoter (doesn’t require a primer)
3) formation of a closed preinitiation promoter complex:
TFIIA, TFIIB, TFIID - bind and recruit RNA pol and TFIIF
4) binding of TFIIE + TFIIH and ATP hydrolysis leads to formation of an initiation complex in which DNA is unwound in the active site = forming a transcription bubble
5) carboxy-terminal domain of largets pol II subunit is phosphorylated by TFIIH, and the pol escapes the promoter = transcription begins
6) elongation is aided by transcription factors and elongation factors
7) transcription is terminated (aided by termination factors) - pol is released, dephosphorylated and recycled
RNA strand initiation of transcription and promoter clearance:
- cycles of abortive initiation - some RNA made but ant get out of the exit pore of the RNA pol so the enzyme has to keep on trying until one gets through… then the RNA pol needs to move away from the complex
- TFIIH phosphorylates pol at the serine 5 of the carboxy-terminal domain repeat sequence - this disrupts the contacts causing a conformational change = initiating transcription
- there is a pause before elongation which could be for proofreading
elongation occurs by…
1) change of phosphorylation sites on carboxy-terminal domain - the serine 5 phosphorylation is removed - instead ATP puts phosphate on serine 2 in the sequence repeats
2) this provides a platform for the binding of elongation factors
3) disassembly and reassembly of nucleosomes (prevents DNA being bare and open to damage)
4) methylation on histone 3 triggers deacetylation for chromatin reassembly
mechanism of elongation:
1) the fork loop separates the two strands of DNA - the coding strand is passed over the outside of the structure and the template strand is held tightly within the active site
2) the wall forces the DNA to make a right-angles turn in the active site
3) after going through the centre of the enzyme the two DNA strands need to re-anneal using the zipper
4) the rudder pushes the RNA helix out towards the lid and the fork so the RNA strand cannot follow the DNA
how does the RNA pol move along:
ratcheting by the bridge helix:
- bridge helix is an alpha helix which has a weak point where it is prone to bend
- the last nucleoside to be added on the RNA needs to be moved out of the active site to allow room for a new nucleoside triphosphate to get in
- thermal oscillation at the bridge helix prevents 3’ end slipping back into the active site - as base stacked on tyrosine has crossed over
- bridge helix retracts forming a straight helix again leaving space for the next nucleoside triphosphate to come into the active site
reactions undergone by the nascent (new) RNA:
- during transcription, incoming nucleotides are attached at the alpha-phosphate by the 3’ hydroxyl of the growing RNA chain - however… the first nucleotide of the RNA will be a triphosphate as it doesn’t get attacked (nothing to remove the phosphate) = capping of the 5’ end occurs instead
addition of a 5’ cap to the nascent RNA:
takes place as soon as the first nucleotides of the RNA emerge from the polymerase
on the carboxy-terminal domain of RNA pol II there are enzymes that act in capping:
- triphosphatase = removes a phosphate
- guanylyltransferase = takes GTP and adds guanine and a phosphate to produce a guanosine-triphosphate cap - is methylatedto form a 5’ 7-methylguanosine cap
5’ cap functions in…
splicing, transport, translation, stability
how pol II transcripts stop:
undergo polyadenylation, not termiation
in the last exon, RNA is cut and a 3’ polyadenylate tail is added
a 5’ exonuclease laches onto newly exposed cut end and degrades the RNA to cause the RNA polymerase to fall off
exons =
coding regions
introns =
non-coding regions
splicing of pre-mRNA:
in eukaryotic transcirption, the entire gene is copied into pre-mRNA - during RNA splicing within the nucleus, introns are removed and exons join to form a coding sequence
- catalysed by spliceosomes
spliceosomes =
complexes of small nuclear RNA bound to proteins, to give small nuclear ribonucleoproteins (snRNPs)
how splicing can result in a single gene coding for multiple proteins -
alternative splicing =
- optional exons called cassette exons can be missed out (exon skipping) or put in (exon inclusion) = therefore, the protein mRNA sequence and the protein will be different
every intron…
- begins with GU (5’ splice site)
- ends with AG (3’ splice site)
- has an A at the branch site
= these nucleotides are conserved
every nucleotide in RNA has got…
a ribose ring with a 2’ OH and a 3’ group linked through a phosphodiester bond with the 5’ group of the next ribosome
two steps of splicing for each intron…
1) at the branch site there is an A where the 2’ OH is activated - the proton is removed leaving a negative charge on the O - the O attacks the phosphodiester link of the 5’ splice site = pushes off the 5’ exon and the A joins on to the start of the intron
2) the 5’ exon pushes back and attacks the 3’ splice site - joining the two 5’ and 3’ exons together = pushes off the intron forming a lariat intron product
- lariat intron product is degraded and the exons form mRNA
splicing components: snRNP
small nuclear ribonucleoproteins = spliceosomes that interact with pre-mRNA to allow splicing
- U1 snRNP base pairs to the 5’ splice site
- U2 snRNP base pairs to the branch site
spliceosome assembly:
1) U1 snRNP binds to the 5’ splice site (GU)
2) Branch point binding protein binds to the branch site (A)
3) U2AF bind to the 3’ splice site (AG)
4) connections are made between the 5’ splice site, the branch site and the 3’ splice site = forms an early stage loop
5) ATP required to push off the branch point binding protein to allow U2 snRNP to bind to the brach site
6) U4 and U6 snRNP enter base-paired to each other and this binds to U2
7) U5 also enters
8) rearrangement occurs which requires ATP:
- U1 is pushed off
- U4 unwound from U6 so that U6 can base pair to 5’ splice site and interacts with U2 - to form the active site
splicing and disease:
- many genetic diseases are caused by mutations in splice sites
- splicing switches caused by changes in the expression of splicing factors are important in disease pathways (e.g.Bcl-X and Ron)
- mutations in some splicing proteins cause certain cancers because they alter splicing patterns
spinal muscular atrophy:
- caused by lack of SMN protein (there are two copies of the gene with identical sequences)
- however, if a child inherits two damage SMN1 gees from parent they develop spinal muscular atrophy = SMN protein only forms from SMN1 gene
- this is because SMN2 has a single nucleotide change in the exon (exon skipping) that changes the sequence
beta thalassaemia -
linked to the incorrect splicing of the beta-globin mRNA leading to defective haemoglobin
Ron -
in tumours, the conc of proteins that affect splicing are altered - increase in a form of Ron which lacks membrane-binding domain and produces uncontrolled signals in the cytoplasm for cell proliferation
Bcl-X -
= gene involved in apoptosis (programmed cell death)
splicing at two different 5’ splice sites cause different forms of the gene which can either be anti- or pro- apoptosis
