Post-transcriptional control of gene expression: mRNA processing, translation, decay Flashcards
(35 cards)
Transcription termination in bacteria (Rho dependent and independent)
Termination triggered by signals in mRNA leading to dissociation of RNA polymerase and RNA release.
Rho-dependent: transcripts have GC rich hairpin (variable length) and ~6U residue run at 3’. Termination can be disrupted by mutations that destabilise the stem, mutations stabilizing DNA-RNA hybrid after stem loop, deletion of PolyU or in vitro transcription with ITP instead of GTP (I=C pairs form 2H bonds). Hairpin causes polymerase pause, rU and dA base pairing weak, allowing template DNA to dissociate-> termination
Rho-dependent: Rho= ring-shaped hexamer w/ RNA-dependent ATPase activity and may act as helicase. Rho binds C rich region, translocates along RNA until reached RNAP (which has been paused by downstream hairpin), induces its dissociation.
Methods to study post-transcriptional control (6 points)
Cis-acting seq in RNA molecules recognised by trans-acting factors (protein/mRNA). Cis seqs ID’d by consensus seqs or by isolating+ characterising RNAs bound to certain proteins. Mutagenesis tests base importance.
Trans acting factors ID’d by isolating factors bound to cis seqs. EMSA, footprinting+ modification interference can be used for RNA-protein as well as DNA-protein interactions (see ctrl/gene exp). Other methods based of purification of specific RNAs/proteins (affinity methods)
RNA purification: RNA oligo synthesised in vitro using biotinylated UTP (other labels possible), incubated with cell extract/ pure protein, complexes recovered with streptavidin beads (bind biotin- pulldown assay), then bound proteins Western blotted.
Protein purification (analogous to ChIP): protein purified w/ associated RNAs w/ antibodies (immunoprecip.), mRNA->cDNA, regions of interest detected by (RT)PCR or using DNA microarrays/ seq.
Cross-linking: many RNA-protein interactions require stabilisation by irradiation (UV light- in vivo or vitro) to ID. UV-> formation of stable covalent bonds between RNA+ protein. (NB formaldehyde fixes DNA-protein)
Genome-wide studies complement detailed analysis of individual model transcripts: info on how general molecular findings are, coordination of gene exp for gene groups. ID and quantify 1000s molecules in parallel. Mainly microarrays and deep seq (AKA massive parallel/ high throughput/next gen). Use to: ID targets of RNA-binding proteins systematically, measure use of introns/exons, determine mRNA decay rates+ translation rates.
euk pre-mRNA processing: capping
In nucleus, often co-transcriptional.
5’ end capping with N7-methyl-guanosine via 5’-5’ triphosphate bond, not encoded in DNA. Increases 5’ proximal intron splicing, export to cytoplasm, efficient translation initiation, protection from 5’ endonucleases.
Formation v early+ co-transcriptional: upsilon Pi from 5’ triphosphate end pre-mRNA removed by RNA terminal phosphatase (RTPase). GMP transferred from GTP by RNA guanylyl transferase (RGTase- part of same polypeptide as RTPase in multicellular organisms, form heterodimer in yeast)-> G5’ppp5’N, Pi released. Added guanine methylated at N7 (methyl donor= S-adenosylmethionine) to form Cap0 (stops here in yeast, but in mammalian cells come mRNA nts also modified). Cap bound in nucleus by cap-binding-complex dimer but by other proteins in cytoplasm.
transcript capping specificity (evidence)
Capping specificity: mRNAs digested by endonucleases are not capped. Only di/tri-Pi ends capped. All Pol II transcripts capped (convert Pol II promoter-> Pol I/III promoter-> no cap) as capping done by enzyme associated w/ Pol II CTD. CTD also activates mammalian RNA guanylyl transferase. Evidence:
Pol II CTD required for capping: cells transfected with amanitin (Pol II inhibitor)-resistant Pol II, either w/ normal CTD (52 repeats) or mutant (5 repeats)-> cells incubated w/ amanitin so endogenous Pol II inactive-> capped+ uncapped mRNAs quantified. Wild type CTD cells» capped mRNA than mutant.
Capping enzymes associate w/ Pol II CTD, S5 Pi-tion recruits capping enzymes:
* Nuclear extract passed through an affinity column, either wild type CTD/ mutant CTD/ Pi CTD. Capping activity only in Pi CTD column
* Mutant CTD w/out S5 replaces endogenous CTD gene in yeast-> cells can’t grow. Can be rescued if mammalian capping enzyme fused to CTD
Run-on transcription
Run-on transcription: Pol II transcription continues past stop for 100-1000s bp downstream w/ no discrete termination site (run-on transcription). Mature 3’ end made by cleavage of extra nts+ polyA addition:
If incubate nuclei+ NTPs+ alpha-P32 labelled UTPs in vitro. New transcription initiation inhibited, but elongation continues with UTPs. New labelled RNA cleaved – 50-100bp fragments, hybridised to DNA probes along gene+ downstream. Find signal downstream of STOP which decreases gradually 5’-3’.
Poly-adenylation
3’ PolyA not DNA encoded. ~240 in mammals, gets shorter in transport of cytoplasm+ ageing (rate transcript-specific). Protects from 3’exonucleases, ctrls degradation rate, needed for translation initiation.
Formation: co-transcriptional in vivo, using signals at 3’ mRNA end: conserved AAUAAA consensus 12-30nt upstream from cleavage site+ U/GU rich seq up to 30nt downstream of cleavage site (DSE). Formation studied w/ in vitro assays using radiolabelled RNA, nuclear extract and ATP to show:
* Cleavage+ PolyAtion independent: radiolabelled RNA substrate cleaved+ PolyA’d in ATP presence. When only ddATP present, cleaved but not PolyA’d. RNA that mimics cleaved substrate is PolyA’d but not cleaved.
* PolyAtion is in 2 stages: 1st requires AAUAAA, leads to addition of ~10A. 2nd requires this short A tail, leads to extension of PolyA. Assay shows that a cleaved substrate is polyA’d only with wild type AAUAAA. Substrate with short PolyA and mutated AAUAAA adenylated further.
Co-transcriptional: in experiments like those for capping enzymes, cells expressing CTD mutants defective in processing. CPSF and CstF bind CTD affinity columns.
Alternative cleavage and PolyA: Type I RNAs have 1 constitutive cleavage/PolyA site. Some RNAs have multiple signals in terminal exon (type II) or in different exons (type III). PolyA site linked to alternative splicing.
High throughput seq maps cleavage/PolyA sites: fragment RNA, purify PolyA fragments, seq to find attached cleavage+ PolyA site.
Site selection integrates multiple conditions: proliferating cells prefer proximal cleavage, have shorter 3’UTRs, less binding sites for negative regulators.
Splicing pre-mRNA: R-loop experiment
Exons code proteins+ UTRs, smaller+ fewer than introns. Average 9 per gene.
Sharp+ Roberts used adenovirus (infects mammalian cell, produces capped, spliced and PolyA mRNAs) in
R loop experiment: mRNA hybridized to dsDNA, displacing one DNA strand-> loop of ssDNA (R loop) visualised with electron microscopy, ss+ds distinguished by width. mRNA had tails protruding at both ends: 3’ was clearly PolyA, 5’ investigated further: If R loops incubated with non-contiguous DNA in viral genome, they annealed, showing mRNA was composite molecule from multiple regions (hence 5’ protrusion).
Cis elements’ role in splicing
Cis elements in splicing: Most introns start w/GU, end w/AG. 5’ splice site (/donor) at intron/exon boundary 5’ of intron, consensus (C/A)AG|GURAGU. 3’ site (acceptor) at intron-exon boundary 3’ on intron, has branch point consensus w/ conserved A about 18-40nt upstream of splice site, 18-40nt polypyrimidine tract downstream of branch point and splice site (YAG)
Trans factor role in splicing (snRNPs)
Trans factors in splicing/ snRNPs (small ribonucleoprotein particles) have small nuclear RNA (U1/2…) that often directly base pairs substrate associated w/ several proteins for several reactions. U1 base pairs 5’ splice site; U2 base pairs branch point; U2AF (auxiliary factor) contains large subunit (binds polypyrimidine tract) and small subunit (associates 3’ splice site).
Cis/trans factor interactions in splicing
Cis/trans interactions: Mutations in highly conserved positions (can trigger use of nearby cryptic sites), removal of first 8nt/U1, mutating 5’ splice site/branch point (rescued by complementary mutations in U1/2- base pairing evidence), antibodies against U1, removal of U snRNPs or extending distance between splice sites over ~70nt inactivate splicing. Mutations in introns/exons outside consensus usually don’t have effect.
Analysis of splicing by isolation of intermediates
Analysis of splicing by isolation of intermediates: synth splicing substrate with 2 exons+1 intron in vitro, radiolabel, incubate w/ nuclear extract+ ATP, take samples at different times, remove proteins and run RNA on denaturing gels, autoradiograph. Circular intermediates migrate anomalously (slower) but treatment with debranching enzyme hydrolyses 2’-5’ binds, linearising lariats. Results imply 2 successive transesterifications:
* 2’OH branch site attacks 5’ intron Pi-> release 5’ end of exon, form lariat intermediate (5’ intron end+ 2’ adenosine branch point bonded)
* 3’OH of 5’ exon attack 3’ intron Pi-> both exons ligated, intron lariat released, then debranched+ degraded. #Phosphodiester bonds conserved so no ATP used in these steps.
The spliceosome and its function
Spliceosome: gels also show that pre-mRNA becomes part of large ribonucleoprotein complex (spliceosome) containing U1, 2, 5, 4/6 and other proteins, whose assembly requires ATP, and which brings all components in place for the splicing reaction as above. Assembly inferred from non-denaturing gels(PAGE)/ glycerol gradient with radioP RNA in nuclear extract as follows:
Early complex: U1 base pairs 5’ splice site, U2AF recognises polypyr. tract+ 3’ splice site (SR proteins needed)
A complex requires ATP: U2 base pairs branch point, A of branch point protrudes.
B complex: base paired U4/6+ U5 recognise pre-formed complex,.
Catalytic spliceosome: U4/6 pairing breaks, U1 leaves (displaced) from 5’SS+ U6 base-pairs 5’SS and U2.
5’SS-U6-U2 aligns substrate pre-mRNA for 1st splicing step and generates catalytic site (U2+U6). Splicing is RNA-catalysed. Some protein splicing factors belong to ATP-dependent RNA helicase family, may catalyse some RNA conformational changes.
Splicing regulatory elements
Exonic/intronic splicing enhancers/silencers (ESEs, ISEs, ESSs, ISSs) are gene specific, recognised by regulatory proteins that modulate interactions between splicing sites and the spliceosome, and regulate constitutive or alternative splicing. The form a “splicing code” read by sequence specific RNA-binding domains that bind cis elements, modulating recruitment of spliceosome. Most introns have 2+ of these.
Activator e.g.: SR proteins (modular) with RNA binding and protein-interaction (RS) domains. Can activate splicing by, e.g., promoting U2AF recruitment through interactions between RS and UA2F35.
Repressor e.g., hnRNPs: RNA binding and protein interaction domains. Repress splicing by several mechanisms, e.g., PTB protein prevents binding of UA2F.
Alternative splicing purpose and examples: SV40 Y antigen and DSCAM gene
Generate more gene product variation and help regulate gene exp. Switches in splice pattern often tissue/developmental stage specific. EST databases and NGseq helps analyse alt splicing by aligning all seqs from 1 gene, show most human genes have alt splicing in non-all-or-nothing manner.
E.g.s:
* SV40 T antigen: 2x5’ SSs compete- if 1st selected, get shorter mRNA without exon 1 STOP and get large T antigen (late infection, induces transformation), otherwise small t antigen (early infection, represses apoptosis), as includes STOP in downstream part of exon 1.
* Drosophila DSCAM gene: over 38k isoforms (some of 24 exons constitutive, some chosen from large # of mutually exclusive exons). DSCAM proteins needed for neuronal patterning, so alt splicing-> variability for recognition of specific neurons.
Drosophila alternative polyA type II SXL and alternative splicing
- E.g.: Drosophila alternative PolyA type II (SXL) and alt splicing:
SXL RNA-binding protein regulated splicing+ PolyA+ translation, expressed only in females. Exon 3 has STOP, spliced out in females but not in males, so only females produce full SXL protein. This case is an on/off gene exp switch.
One target= enhancer of rudimentary (e(r )) w/ 2 PolyA sites: proximal used in males and distal in females. Females have longer 3’UTR w/seqs that induce translational repression so e(r ) not produced in females.
Another target: transformer (SS choice regulated by SXL)- transformer RNA has 2 alt SSs. In males no SXL, U2AF65 binds proximal SS-> premature termination. FemaleSXL bind proximal pyrimidine tract, forcing U2AF to bind distal SS-> full length transformer produced.
What factors determine which splice site is used? (+ heritable conditions involving alt splicing)
Factors in if a splice site is used: strength of splice sites/ consensus; enhancers/repressors and proteins (RBPs) bound to them; RNA secondary structure (can mask SS). Exons always included= constitutive, not incl= skipped.
Heritable conditions caused by mutations affecting splicing by inactivating 5’+3’SS/ enhancers/ repressors or generating new SSs, e.g. Hutchinson-Gilford progeria syndrome causing premature ageing+ death caused by (silent gly->gly) point mutation/exon 11/prelamin A gene activating a cryptic splice site-> protein 50aas short (including protease cleavage site)-> not processed properly, acts as dominant negative.
Translation machinery
Prok, then Euk
Ribosomes 30S+50S, ~66% RNA 40+60S, ~60% RNA
Activating enzymes 20 (1 per aa)
(activated) tRNAs ~80
Initiation factors IFs 1,2,3 (3 polypeptides total) eIFs 1, 1A, 2(3pp), 2B (5pp), 3 (2+pp), 4A, 4B, 4E, 5, 5B (30 polypeptides total)
Elongation factors EF-Tu, EF-Ts, EF-G EF1alpha, EF1betaupsilon (2pp), EF1-2
Termination factors RF1 (UAA, UAG), RF2 (UAA, UGA), RF3, RF4 eRF1, eRF3
Ribosome cycle
When not translating, subunits reversibly associate depending of [Mg2+]+[K+], physio conditions favour assoc.
Dissociation factor (IF3/eIF3) binds small subunit, prevents reassociation, primes for initiation.
Small subunit associates mRNA, then large, the subunits stably associate w/each other (dissociation factor released), can be dissociated (irreversibly) by unphysiological treatment (e.g., EDTA)
Subunits released separately at termination.
1(e )IF3 per 10 ribosomes- the rest translating or associated.
Prokaryotic translation initiation
30S/IF1/IF3 complex forms, interacts mRNA at ribosome binding site. IF2, GTP+ charged initiator tRNA-> ternary complex, which joins 30S on mRNA-> 30S initiation complex w/ initiator tRNA @ P site, IF1 in A.
50S joins-> GTF hydrolysis by IF2, initiation factors released-> 70S initiation complex. IF2 recycled (GDP switched for GTP). IF1 vacates A site, ready to accept next aminoacyl-tRNA.
NB mRNA polycistronic, initiation allows recognition of multiple ribosome binding sites.
Aminoacyl-tRNA charged w/ formylmethionine (1st aa) = initiator. Formyl rapidly lost by hydrolysis (peptide deformylase). In 50% proteins, initial Met removed (methionine peptidase)
IDing translation initiation sites in prokaryotes (include in vitro and in vivo evidence)
IDing translation initiation sites: typical mRNA has 2+ AUG, polycistronic, sometimes initiated @ GUG/UGG. Experiment: bind ribosome subunits to mRNA, inhibit elongation. Digest unprotected mRNA, isolate ~35nt protected fragments of mRNA+seq. Shows consensus in protected fragments- all have: initiation codon+ polypurine stretch ~10nt upstream of it (Shine-Dalgarno/RBS) AGGAGG, complementary to seq on 3’ end 16SrRNA from 4-9bases. SD-rRNA pairing allows initiation codon ID. fMet-tRNA anticodon pairs AUG.
In vitro evidence of SD/16S pairing: 37nt RNA w/AUG radiolabelled, incubated w/ ribosomes, f-Met-tRNA, initiation factors (no elongation). SDS denatures proteins, dissociated RNA-protein interactions (RNA-RNA pairing still intact). Separate on native polyacrylamide. Labelled RNA vo-migrates w/ 16S rRNA-> base paired.
In vivo dedicated ribosome experiment: compensatory mutations in 16SrRNA supress mutant SD seq. Plasmid w/ hGH gene (constitutive promoter, mutant SD seq) and E coli pre-rRNA gene (42o inducible promoter, streptomycin (rRNA inhibitor) mutant, complementary SD- binding seq mutant) introduced. Incubate w/ streptomycin (confirms suppression specifically due to mutated 16S rRNA). At 37, no hGH (mutated SD not functional). At 42, hGH produced (mutant 16S rRNA supressed mutation in hGH SD)
Prokaryotic translation: elongation (hint: mention ternary complex, ribosome translocation, and effects of ribosome mutations)
P site contains peptidyl-tRNA (or fMet-tRNA for 1st round). EF-Tu binds GTP with aminoacyl tRNA (ternary complex analogous to that formed by IF2), masking aa group in aa-tRNA, preventing reaction w/ peptidyl-tRNA. Ternary complex joins A site pairs w/ next codon (checking process called decoding)- if no match, ternary complex released (no GTP hydrolysis); if match, ribosome conformational change triggers GTP hydrolysis+ EF-Tu-GDP release. Aa end of tRNA moves toward peptidyl transferase centre (accommodation).
GTP hydrolysis+ EF-Tu-GDP bring together aminoacyl+ peptidyl ends. Transfer of polypeptide from P site to aminoacyl-tRNA in A site-> polypeptide bond (catalysed by ribosomal peptidyl transferase on 50S).
Ribosome translocates with help of EF-G (GTPase activity stimulated by binding ribosome, only binds ribosome when no EF-Tu). GTP hydrolysis stimulates ribosome translocation. Translocation-> new peptidyl-tRNA goes to P and deacylated tRNA ejected through E site.
EF-T binds EF-Tu, displaces GDP, is displaced by GTP (recycles EF-Tu from GDP back to GTP bound form which then binds another aa-tRNA). EF-G recycled without ancillary factors (GTP affinity>GDP)
Ribosome mutations: Ser-4, 5, 12 residues next to decoding site and important for speed. Mutations in Ser4/5 at E site cause ribosome to go faster+ make more mistakes, ser12 at A site makes it go slower, perhaps because slightly change sterics at tRNA entry site. Ribosome speed kept quite fast perhaps by ribosome sensing shape/charge of aa sidechains and being able to exclude clearly incorrect aa-tRNAs faster.
Prokaryotic transcription termination
Release factors (RFs) mimic tRNA (1 domain close to PTS, induces peptidyl-tRNA hydrolysis, 2nd domain directly recognises termination codon), recognise STOP, 2 classes:
Class I (RF1+2) recognise STOP directly (mimic tRNA) w/ different specificity (3 stop codons!), induce ribosomal hydrolysis of peptidyl-rtRNA. Mutational analysis shows specificity conferred by single tripeptide/”peptide anticodon”- 3D structures confirm.
Class II (RF3) GTP-binding protein w/ GTPase activity; bind ribosome in GDP bound form, GTP displaces GDP while on ribosome, conformational change releases class I factor, GTP hydrolysis-> release RF3.
Throughout, GTP hydrolysis not coupled to chemical mods but ensures correct order and fidelity of translation.
The prokaryotic ribosome
rRNA: Secondary and tertiary rRNA structures mostly by WC pairs. Most proteins contact rRNA directly. Interface between subunits has almost no proteins. Tertiary structures stabilised by interactions w/proteins, long-range base pairs (nts far on 2o structure), non-canonical bps.
Ribosomal proteins: many globular w/long extensions that would be disordered in isolated protein, often buried in RNA, crucial for RNA folding.
Structure: mRNA pairs anticodon in 30S, peptidyl transferase site in large subunit. E, P, A sites in order.
Ribozyme activity: 23S rRNA has peptidyl-transferase activity: after extraction oaf most proteins from 50S rRNA still active, but if rRNA damaged, activity lost. 23S rRNA made in vitro can catalyse peptide bond formation (low efficiency). No proteins are within 1.8A of active site.
Regulation of prok translation
Initiation efficiency partly controlled by SD/16SrRNA pairing strength. Also, accessibility of SD+ in turn mRNA secondary structures can be modulated by translation of other ORFs in polycistronic mRNA (translational coupling), proteins, temperature, non-coding RNAs, small molecules (es).
