Eukaryotic Transcription and Post-Transcriptional Regulation Flashcards
central dogma
DNA to RNA to protein
gene regulation
chromatin mods (histone mod, acetylation/methylation) transcriptional control by TFs/Pol RNA processing control RNA transport and localisation control mRNA degradation translational control protein activity control
transcriptional control
chromatin structure
Pol binding
activation factors
additional binding
chromatin structure modifications
highly packed heterochromatin not expressed
acetylation: acetyl to +ve lysines in histone tails loosens chromatin
methylation: condense chromatin
phosphorylation: phosphate next to methylated AA loosen chromatin
histone code hypothesis
chemical mods to histones and DNA determine chromatin configuration so transcription
chromatin can move within nucleus to…
alter gene expression
active are central
heterochromatin is close to membrane
RNA polymerase binding for transcriptional control
3 types: RNA Pol I ribosomal RNA gene
RNA Pol II protein coding small RNAs
RNA Pol III tRNA rRNA some snRNA so other small RNAs
initiation complex
TATA box (prokaryotes have TATAAT at -10)
consensus sequence
in pol II promoters
-25 to transcriptional start
RNA Pol II initiation complex
pol with TFs fine tune process and stabilise and activate pol
TFIID: TBP (TATA box binding protein) and TAF (regulates DNA binding of pol)
TFIIB: positions RNA pol at start site
TFIIF: stabilises RNA Pol
TFIIE: attracts TFIIH
TFIIH: unwinds DNA and phosphorylates ser5 (in CTD tail of pol II)
TBP
binds TATA box and bends DNA so RNA/TFs can bind and stabilised by TFIIF and E and H help binding
additional binding and activation factors (activator proteins, TFs, proximal control elements and distal, combinatorial)
activator proteins bind to enhancers in promoter region so form mediator complex, sends info from promoter to RNA Pol and tells Pol to transcribe
(some promoters so far from start so needs to bend by DNA-bending proteins)
TFs initiate transcription and help RNA pol
proximal control elements close to TATA while distal enhancers are far away
combinatorial control: comb of control elements active when appropriate activators are present
TFs (e.g.)
contain DNA binding domains
Leucine zipper TF bind to promoters and cause transcription
recognise specific features of DNA
Zinc finger TF bind promoter
TFs work together
can also inhibit transcription
forward genetics
identify gene (function) from phenotype e.g. moles, red skin, skin damage is XPD gene functions in DNA repair
reverse genetics
predict phenotype from gene analysis
analysing gene expression
single gene: RT-PCR, live cell imaging, promoter studies
all genes: microarrays, RNA sequencing
RT-PCR
cDNA to PCR to electrophoresis
1 gene at a time
live cell imaging
microscope analyse gene activity and localisation
shows why gene expressed
can fuse to reporter gene (GFP) to see where mRNA expressed in cell
promoter analysis
to see which enhancer motifs affect transcription
add reporter gene to promoter and transfect to cells and see green colour where expressed
ChIP (chromatin immunoprecipitation)
Abs for TFs so pull out sequence with TF so find where it binds and see if changes from disease/env
or amplify sequence and gap is where TF binds
phylogenetic fingerprinting
find sequence where TF binds
microarrays
study expression of interacting groups of genes
automated
compare patterns
microscrope slide with mRNA hybridised to labelled cDNA so show expression levels
1 spot means 1 gene and darker colour means more gene expressed
RNA sequencing
show which exons transcribed in a sample
count number of RNAs per exon in sample so calculate expression level of a gene
also identify gene splicing variants
what is the point of post-transcriptional regulation of gene expression?
for mRNA stability, translation, protein function
types of processing
capping at 5’ end
poly-A tail to 3’
splicing to remove introns
RNA Pol II and regulation
makes mRNA
CTD tail - long with loops, 4 serines at position 5 is where phosphorylation happens
TFIIH is involved in phosphorylation
RNA Pol onto DNA and transcribes it - tail already phosphorylated on serine 5, processing factors can attach to tail and hop to pre-mRNA
capping
makes mRNA stable and protects from enzymes and important for export and translation
5’ pppNpNp (phosphate and any base)
phosphatase removes 1 phosphate so only 2 p’s at start
G added to this end by guanyl transferase then G methylated
another methylation occurs of the downstream base
poly-A
3’ end polyadenylation
3’ sequence: AAUAAA-CA-GU rich region
- 3’ sequence removed by cleavage factor but need to cleave at correct position so don’t remove important nucleotides
CPSF (cleavage and polya specificity factor) binds AAUAAA
G/U rich regions is in part meant to be cleaved and CstF (cleavage stimulation factor F) recognises it
CFII (cleavage factor) recognise CA, bend it so PAP (poly A pol) can bind and chop off end tail
polyA factor waits will AAUAAA then PAP adds As with ATP and CstF/CFI/CFII removed
PABP (poly A binding protein) binds to A tail and helps add As faster
introns
remove and ligate 2 exons
5’ end splice site and 3’ end splice site
‘A’ branch point in intron where join to form lariat
2 step transesterification
- hydroxyl group at branch point A that binds to phosphate group causes attached lariat to 3’ exon
- loops removed and 2 exons ligated with esterification
snRNPs
small nuclear ribonucleoprotein particles
in spliceosome and 200 other proteins involved
specificity (introns exons)
sequence at exon/intron junction at 5’ end 3’ end and in middle
so splicing gives specificity
splicing
5’ exon/intron junction sequence recognised by U1 snRNP
BBP (branch point binding protein) binds to branch point A
which is joined by U2AF (U2 auxilliary factor) which helps U2 snRNP find A
U4/U6 binds to U1+U2 so conformational change and 1st cut and transesterification forms loop
U4+U1 no longer required
U6 causes 3’ intron/exon cleavage and exons fused afterwards
snRNPs give specificity in splicing
U1 has RNA sequence matching 5’ exon/intron junction
cells don’t rely on snRNPs for splicing because exon length same ish but intron size changes so can’t cut on basis of distance
but conserved in some organism so more accuracy to splicing
ESEs
exonic splicing enhancer
SR protein binds and shield exon
hnRNP
heterogeneous nuclear ribonucleoprotein
bind introns and rip up so branch point found so A branchpoint can reach 5’ exon/intron junction
(because introns quite long sometimes)
alternative splicing
evolution, new mRNAs for new function
recognise new parts maybe
RNA editing (definition, types, why)
alter sequence of pre-mRNAs (not splicing or methylation) after transcription
base insertions
cytosine deamination to uracil
adenine deamination to inosine
(examples lecture 15)
to revise mistakes, plasticity for function, defense)
base insertions
usually uracil
sites missing U specified by guide RNA1
edits by pairing to guide RNA2
e.g. sleeping sickness
cytosine deamination to uracil
AA change so new protein
e.g. apoliprotein B
adenine deamination to inosine
protein change by ADAR (adenosine deaminase acting on RNA)
e.g. glutamate receptor
ribozymes
catalytic RNAs bind to mRNA to cleave and destroy
intron RNA can splice w/o spliceosome
RNAse P ribonuclease in processing tRNA and small RNAs, peptidyl transferase ribozyme of ribosome
in viruses as well
rRNAs of ribosomes
can be therapy for cancer
regulation of nuclear export of mRNAs
only processed mRNA goes through pore to cytosol because pores restrict movement of molecules
needs energy to guide through pore
need export or would be degraded
miRNA
small RNAs in junk part of DNA, for regulation of transcription, defence, inhibit/destroy transcripts
21-25 nucleotides, non-coding, single strand, matching mRNA so targets specific
RISC complex helps (RNA-induced silencing complex)
Pol II in miRNA production causes pri to pre to mature single small RNA and cropping dicing so 1 strand digested and 1 to RISC
