Post-transcriptional control of gene expression Flashcards
RNA purification for RNA:protein ints
- synthesise biotinyllated oligo
- incubate with proteins
- recover RNA:protein complexes with streptavidin beads
- detect bound proteins by WB
Protein purification for for RNA:protein ints
- analogous to ChIP: purify protein with RNAs using antibodies
- generate cDNAs from mRNAs using reverse transcriptase
- detect cDNA of interest by PCR
Cross-linking RNA and proteins for RNA:protein ints
stabilise ints by crosslinking with UV light
3 parts of eukaryotic mRNA processing
5’ capping
removal of introns (splicing)
3’ polyadenylation
function of mRNA 5’ cap
increase splicing efficiency
need for export to cytoplasm
need for efficient translation initiation
protect mRNA from 5’ exonucleases
5’ cap formation
- remove terminal phosphate from DNA 5’ end by RTPase (RNA terminal phosphatase)
- GMP transferred from GTP by RNA guanylyl transferase (RGTase), gives G5’ppp5’N
- guanine is methylated by RNA-(guanine-N-7)-methyltransferase. = cap 0. further mods in mammals
- RTPase and RGTase are part of same polypeptide in multicellular orgs
early, cotranscriptional event
nuclear cap bound by CBC, cap-binding complex
specificity of 5’ capping
- all and only pol II transcripts
- only di-/ tri- phosphate ends
- cotranscr, by factors associated w pol II CTD
pol II a CTD
- heptad tandem repeats
- residues that can be phosphorylated
- close to RNA exit channel of pol II
- initiation: dephosph
- elongation: early S5 phosph, later S2 phosph
CTD = landing platform for cotranscriptional factors
experiment that shows pol II CTD needed for capping
- cells transfected witha version of amanitin resistant RNAP II
- one version has normal length CTD, one version has fewer repeats
- inhibit endogenous RNAP II with amanitin (so see activity of mutant only)
- quantify capped and uncapped mRNAs
- fewer capped mRNAs produced with mutant CTD than WT
2 experiments to show capping enzymes associate w phosphorylated pol II CTD
1
pass nuclear extract thr affinity column, WT CTD/ mutant CTD/ phosphorylated CTD
- measure capping act in each of samples retained
- only retained in column w phosph CTD
2
fission yeast: make CTD that cannot be phosph at S5, replace endogenous gene w mutant - no cell growth
fuse mammalian capping enzyme to CTD - rescues mutation
5’ cap structure
N7 methyl guanosine, attached to mRNA 5’ through 5’-5’ triphosphate bond
pol II transcription termination
doesn’t terminate at precise positions
evidence for run-on transcription
incubate nuclei with NTPs + radioactive UTP. RNAs are completed, cleaved and hybridised to DNA probes spaced along the gene and downstream regions
==> radioactive signal continues downstream of mature 3’ mRNA end, signal decreases in 5’ to 3’ direction
structure of polyA tail
many A residues, shortened as mRNA ages so linked to RNA decay
functions of polyA tail
protection from 3’ exonucleases, control degradation rate, need for transcr initiation
cis sequences needed for polyadenylation
AAUAAA upstream of cleavage site
U// GU rich DSE downstream of cleavage site
how was polyadenylation shown to occur in 2 stages
- cleavage needs AAUAAA, 10 As are added
- polyA doesn’t need AAUAAA but does need 10 As, longer polyA tail added
2 stages of polyadenylation
- cleavage
- polyadenylation
trans factors needed for polyadenylation
- CPSF = cleavage polyadenylation specificity factor, binds AAUAAA and CStF, need for both cleavage and polyadenylation
- CStF = cleavage stimulation factor, binds GU/ U, need for cleavage only
- polyA polymerase adds A residues, also need for cleavage
(identified by MS)
evidence that 3’ end processing is cotranscriptional
mutant CTD cells are defective in 3’ end processing
CPSF and CStFbind CTD in affinity columns
example of alternative polyadenylation
Sex-Lethal (SXL) in drosophila: RBP expressed only in females, regulates polyadenylation
target: e(r), has 2 alternative polyadenylation sites. males use first site, females use second
females: SXL binds e(r) premRNA and competes w CStF for binding first GU element - so second site used. in males, CStF binds proximal site.
importance: female specific 3’ UTR has transcriptional repression sequences so e(r) not produced.
genome-wide view of polyadenylation
- high throughput seq: fragment RNAs, purify fragments w polyA, sequence and identify those w boundary betw polyA and gene sequence
3’ end processing assay
incubate RNA substrate with nuclear extracts and ATP
- presence of ATP:cleavage, polyA
- ddATP: cleavage but not polyA
- RNA that mimics cleaved substrate is polyAed.
discovery of splicing: R loop analysis
- hybridise mRNA to dsDNA in conditions that favour RNA:DNA interactions, 1 DNA strand displaced = R loop
- visualise by EM: can distinguish single vs double strands by width
- use on adenovirus DNA: mRNA had tails protruding on both ends. 3’: polyA, 5’ unexplained
- 5’ anneals the mRNA from a separate part of the genome, mRNA=composite
cis elements for splicing
- 5’ splice site consensus
- most introns start w GU and end with AG (GU/AG rule)
- 3’ end: branch point consensus with conserved adenine, polypyrinidine tract downstream of branchpoint
3’ splice site = YAG
trans factors for splicing
snRNPs = smalll nuclear RiboNucleoProteins: RNA associated w protein, RNA = functional
U1 snRNP: basepairs with 5’ splice site
U2 snRNP: basepairs with branchpoint
U2AF: U2 auxiliary factor (protein). large 65 subunit binds polypyrimidine tract, 35 subunit binds 3’ splice site.
splicing: mutations
mutation within consensus sequence can inactivate splicing/ use cryptic splice sites
mutation outside of consensus - no effect
5’ SS/ branchpoint mutations can be reversed by complementary mutations in U1/ U2 = evidence for base pairing
in vitro analysis of splicing
- synthesise splicing substrate (2 exons, 1 intron)
- incubate radiolabelled RNA w nuclear extracts, ATP
- take samples at intervals, analyse RNA by denaturing electrophoresis, autoradiography
debranching - normal migration of previously circular intermediates
see product acc over time
chemistry of splicing
2x transesterification
- 2’ hydroxyl of A of branchpoint attacks phosphate at 5’ of intron, 5’ exon released and lariat formed
- 3’ hydroxyl of 5’’ exon attacks phosphate at 3’ of intron. exons are ligated, intron released as lariat. lariat then degraded.
splicing code
will a splice site be used?
splicing enhancers and repressors form splicing code, regulates efficiency of splice site usage
introns often contain regulatory sequences as well as core splice sites
will a splice site be used?
- strength - similarity to consensus
- enhancers/ repressors with bound proteins
- RNA secondary str
alternative splicing - why?
pairing dif combos of splice sites - tissue specific/ developmental specific differences
- regulation of gene expr
- different protein isoforms
most human genes
alternative splicing: drosophila SXL
SXL expressed in females but not males: include exon 3 in males, includes a stop codon so no SXL produced.
skip exon 3 in females so can produce ful length protein
exon 3 = ‘poison exon’
so control gene expression
alternative splicing: drosophila DSCAM
many mutually exclusive exons
38k possible isoforsm (more than drosophila genes!)
protein variability for recognition of specific neurones
splicing and genetic disease
many genetic conditions causes by mutations affecting splice sites, eg inactivation/ generating new, regulatory regions
eg HGPS: a silent point mutation activates a cryptic splice site, creates a protein with a deletion lacking a protease cleavage site, incorrectly processed.
splicing: ATP
actual chemistry: no ATP needed as number of phosphodiester bonds conserved.
ATP needed for spliceosome assembly
translation cycle - what happens in each stage
initiation - recognise initiation codon … up to first peptide bond formation
elongation - formation of all peptide bonds
termination - release of polypeptide, ribosome dissociation
stages of translation cycle
- small subunit then large associates with mRNA
- can associate without mRNA, dissociation factor binds and prevents this reassociation to form an inactive complex. (IF3/ e-IF3) - released in initiation
tRNA binding sites of ribosome
A: aminoacyl-tRNA
P: peptidyl-tRNA
E: exit from ribosome
what is needed for prokaryotic translation initiation (7)
- mRNA with RBS
- ribosomes
- initiator tRNA
3 IFs: - IF1: bind A site, prevent tRNA access
- IF2: complexes with initiator tRNA and GTP
- IF3: dissociation factor
- GTP
prokaryotic translation initiation
- IF1 and IF3 bind the 30S subunit, this then binds mRNA at the RBS
- IF2 forms a ternary complex with GTP and charged initiator tRNA. joins the 30S, forming 30S initiation complex
- 50S joins, GTP hydrolysis by IF2 and all IFs released. 70S initiation complex
prokaryotes - initiator tRNA
formyl methionine tRNA
experiment to identify translation initiation sites - prokaryotes
- bind ribosomes to mRNAs, elongation inhibited
- digest unprotected mRNA with RNase
- isolate and sequence the protected fragments
in vitro evidence for base pairing between the SD and 16S rRNA
- radiolabel an RNA fragment containing initiation codon , incubate with ribosomes, initiation factors, initiator, with initiation allowed but elongation inhibited.
- treat with SDS (denatures proteins but doesn’t affect RNA:RNA ints)
- non denaturing gel electrophoresis
labelled RNA comigrates with 16S rRNA
in vivo evidence for base pairing between the SD and 16S rRNA
compensatory mutations in 16S rRNA suppress mutant SD
eg: hGH gene under constitutive promoter w mutant SD
E coli 16S pre-rRNA gene under control of heat shock promoter, with complementary mutation and resistant to spectinomycin.
- 37C: no hGH, mutated SD nonfunctional
- 42C: get hGH, must be due to mutant 16S rRNA
spec: inhibits WT 16S rRNA
polycistronic mRNAs: prokaryotes
possible to recognise multiple RBS on a simgle mRNA as recruitment is direct
locations of factors - early initiation translation
P site: initiator
A site: blocked by IF1
locations of factors - late initiation translation
P site: initiator
A site: empty, ready to accept first aa-tRNA
locations of factors - elongation translation
P site: peptidyl-tRNA
A site: next aminoacyl-tRNA, tRNA paired to next codon to be translated
initiator is only aminoacyl-tRNA to enter P site
basic steps of translation elongation - prokaryotes
- aminoacyl-tRNA binds A site
- peptide bond formed as polypeptide transferred from P site to aminoacyl-tRNA of A site
- ribosome translocation
deacylated tRNA ejected through E site
translation factors - prokaryotic elongation
EF-Tu - brings aa-tRNA to A site
EF-G - need for translocation
EF-Ts - recycling of EF-Tu
binding of aminoacyl-tRNA to A site
- EF-Tu binds GTP and aa-tRNA, gives ternary complex
this binding masks aa-tRNA, prevents from reacting with peptidyl-tRNA - ternary complex into A site. incorrect codon-anticodon match: release ternary complex w/o GTP hydrolysis
correct match: GTP hydrolysis and release of EF-Tu GDP. aminoacyl can now move towards peptidyl-transferase centre (accomodation)
peptide bond formation - translation
accomodation - aminoacyl and peptidyl ends brought together, catalysed by 50S peptidyl transferase activity
translocation - prokaryotic translation
ribosome moves 3 nts along - needs EF-G-GTP. can only bind if the ternary complex has been ejected ie the peptide bond has been formed
GTPase needed for translocation
translation termination prokaryotes
release factors recognise termination codons, induce hydrolysis of peptide chain class I: direct recognition of term codon (RFI, RF2) class 2: RF3, binds ribosome with GDP, GTP replaces GDP, conformational change, relase of RFs (?)
importance of GTP hydrolysis for translation
GTP hydrolysis needed for correct order/ fidelity of process, rather than being coupled to chemical modifications
eg EF-Tu conformation changes between GTP and GDP bound forms, only the GTP-bound form can bind aa-tRNAs and mask the amino acid.
Ribosome structure
Core = RNA, stabilise through ints with peripheral proteins and base pairing
A, P, E located between both subunits
23S: peptidyl transferase = ribozyme
structural view of translation termination
RFs mimic tRNA shape, one domain induces peptidyl-tRNA hydrolysis, one domain recognises termination codon, via a tripeptide called the peptide anticodon.
translation initiation site consensus features
- AUG start codon
- Shile-Dalgarno seq = RBS = AGGAGG, complementary to 3’ of 16S rRNA
translation initiation - prokaryotes - model
SD pairs with complementaryregion of 16S rRNA, identify initiation codon. fMet-tRNA anticodon pairs with AUG
regulating translation initiation - prokaryotes (6)
- similarity of SD to consensus
- translational coupling
- proteins
- temp (thermosensors)
- small ncRNAs
- small molecules (riboswitches)
regulating translation initiation - prokaryotes - repressor proteins, MS2 example
experiment
MS2 coat protein binds replicase gene initiation site w high affinity, stabilising a stem loop which represses initiation of translation
so replicase only translated during early infection, not once coat protein accumulated.
regulating translation initiation - prokaryotes - translational coupling
eg MS2
- polycistronic mRNAs: sometimes ribosome initiation of a downstream codon is dependent on translation of an upstream cistron.
eg MS2 - secondary str around SD of replicase gene needs to be unwound by ribosomes translating the coat protein gene (upstream) for accessibility
regulating translation initiation - prokaryotes - secondary structures
bacteriophage MS2 mRNA
- coat protein gene: SD folded into secondary str
- mutations carried out which stabilised or destabilised the secondary structure without changing SD
- fraction of unfolded mRNA corrleates with translation efficiency, so SD accessibility important
thermosensors
secondary structures which block SD access and are temp sensitive
2 types of transcription termination - prokaryotes
rho dependent, rho independent
rho dependent transcr term
Rho binds a C rich region, translocates along RNA until it reaches the polymerase and induces dissociation.
a hairpin delays RNAP so rho can reach it.
rho independent transcr term
GC rich hairpin, 6xUs
hairpin causes polymerase pausing, U-A RNA:DNA pairing is weak so dissociate
attenuation
eg?
way to repress gene expression with premature termination of transcription
relies on transcription and translation being coupled
eg for amino acid biosynthetic operons
trp operon
riboswitches
guanine-binding riboswitch
- control gene expression in response to small molecules via attenuation or translational control
- eg guanine binding riboswitch - highly specific binding of guanine, controls attenuation: w guanine, a terminator is formed, so there is premature termination of transcription.
at which stage is translation in bacteria mostly regulated
initiation
eukaryotic vs prokaryotic translation (6) 3 similarities, 3 differences
generally similar
- start codon
- initiator tRNA
- small ribo subunit binds first
- euk - larger ribosomes
- initiation very different, more complex in euk
- eukaryotes need both GTP and ATP, prok only need GTP
eukaryotic vs prokaryotic translation initiation
prok: small subunit binds SD and initiation codon
euk: small subunit binds cap, scans mRNA until first initiation codon
initiation in eukaryotes - scanning model
43S binds methylated cap, scans to 3’ and first AUG is initiation codon. 60S joins, elongation starts
evidence for scanning model - eukaryotes
- initiation at new AUGs inserted betw cap and actual
- stable secondary str between cap and AUG inhibit translation
- cap needed: low efficiency of uncapped/ cap analogue mRNAs
mechanism of translation initiation - eukaryotes
cap binding complex on 5’ end of mRNA
joined by 43S initiation complex
forms 48S initiation complex
43S initiation complex
40S ribosome subunit, initiation factors, initiator tRNA
cap binding complex
eIF4F - cap recognition. contains eIF4A, a helicase, and eIF4E, recognises cap. eIF4G
eIF4B then joins cap and stimulates eIF4A activity
scanning - eukaryotic translation initiation
- ribosome translocation
- unwinding of RNA secondary structure - by helicase, needs ATP
GTP is hydrolysed once the AUG is recognised, eIFs are released.
role of polyA in translation
polyA coated in PABP: polyA Binding Protein
interacts with eIF4G, causes mRNA to have a circular conformation which stimulates translation
internal initiation
picornaviruses - uncapped mRNA with many AUGs and secondary str - mRNA translated by direct ribosome binding to an internal ribosome entry site, IRES
some picornaviruses inactivate host cap-dependent protein synthesis
evidence for internal initiation
dicistronic reporter experiment - insert part of viral 5’ UTR (contains IRES) between 2 cistrons, can drive internal initiation of downstream cistron
why must eukaryotic mRNAs be monocistronic?
since ribosomes scan from 5’ cap and detach at termination, cannot access downstream cistrons.
global regulation of translation in eukaryotes: eIF2a phosphorylation
eIF2-GDP needs recycling to the GTP bound form by a GEF, eIF2B
phosphorylation of eIF2a sequesters eIF2B in a comlex, so eIF2 cannot be recycled, translation initiation is blocked globally
global regulation of translation in eukaryotes: eIF4E-eIF4G interaction
eIF4E binds cap, recruits eIF4G - interaction modulated by 4E-binding proteins - compete with 4G
their binding affinities are regulated by phosphorylation
gene specific regulation of translation in eukaryotes: RNA binding proteins
one protein binds 3’ UTR: specificity. one protein binds the cap and one bridges the others. this forms a loop which blocks translation by inhibiting eIF4F recruitment.
RNA degradation - how
- cap and polyA tail normally protective from exonucleases - start by removing one of these
eg polyA nucleases shorten polyA.
then can get decapping and 5’-3’ degr, or 3’-5’ degr
3’-5’ - degradation by the exosome
redundancy of pathways
RNA degradation - why
keeping mRNA levels constant requires a balance between transcription and decay
removal of defective RNAs
transcript specific regulation of RNA decay - factors
cis elements often in 3’ UTRs
trans factors - sequence specific RBPs/ miRNAs
how to measure the half life of an mRNA
- block transcription - changes in mRNA levels are due to degradation only
- follow mRNA changes with a time course
mRNA localisation - why/ how
to specific subcellular compartments, causing asymmetric localisation of encoded proteins. eg need for embryogenesis, yeast differentiation
determining mRNA localisation with FISH: ASH1
- incubate fixed cells with flourescently labelled probe complementary to the mRNA, observe by fluorescence microscopy
determining mRNA localisation with cis elements: ASH1
- make reporter with ASH1/ control 3’ UTR
- folow localisation by FISH and probe complementary to reporter seq
localisation of B-actin mRNA: in vivo tagging
- tag mRNAs by adding MS2 coat protein binding site
- MS2 protein fused to GFP expressed, binds MS2 binding site on mRNA, follow in vivo by fluorescence microscopy
3 types of eukaryotic small regulatory RNAs
what protein do they work with?
miRNA
siRNA
piRNA
argonaute family
biogenesis of miRNA
- transcribed as long precursors by pol II
- cleaved by nuclease drosha
- export to cytoplasm, further cleavage by dicer
- miRNA loaded onto RISC (contains argonaute proteins), passenger strand is degraded and guide kept
biogenesis of siRNA
- from dsRNA precursors, eg generated from viral infection, also when exogenous RNA introduced in RNAi
- cleaved by Dicer and loaded onto RISC
regulation of gene expression by miRNA and siRNA
siRNA: base pairs with target mRNA, leads to endonucleolytic cleavage and decay of target
miRNA: causes translational repression
genome wide view of translational control - ribosome profiling
- measuring translational rates for every mRNA in the cell
- mRNAs can be translated by multiple ribosomes at a time, the number of ribosomes estimates efficiency of translation
- purify mRNA with ribosomes
- use a ribonuclease which degrades all mRNA except fragments protected by ribosome binding
- sequence protected fragments, map to genome
- number of protected fragments corresponding to an mRNA reflects how many ribosomes are bound, so how efficiently mRNA is being translated
identifying RBP binding sites: CLIP
analogous to ChIP, RNA and proteins are crosslinked using UV light, purify the protein of interest with antibody, analyse by deepseq and map to genome
functional stidies of RBPs (splicing)
splicing sensitive DNA microarrays: have probes for exon-exon and exon-intron junctions so can quantify frequency of each splicing event.
exonucleases vs endonucleases
exo - decreade from ends
endo - cut in the middle
how to block transcription
- RNAP II inhibitor
- ts mutants of RNAP II components
- clone gene of interest in with regulateable promoter
