Transcription of eukaryotic genome Transcription of eukaryotic genome > Primary transcripts > precursor messenger mRNA (pre-mRNA) heterogeneous nuclear RNA (hnRNA) = pre-mRNA forming in nucleus precursor ribosomal RNA (pre-rRNA) precursor transfer RNA (pre-tRNA) 5S-rRNA small RNA (snRNA, snoRNA, scRNA) > > > > > Eukaryotic DNA-dependent RNA-polymerase - RNA-polymerase , II, III > Transkription factors IkaryoM > RNA polymerase I > Synthesis of pre-rRNA > Only in nucleolus > Not sensitive to a-amanitin > RNA polymerase II > Synthesis of hnRNA and some snRNA > Sensitive to a-amanitin > RNA polymerase III > Synthesis of pre-tRNA, 5S-rRNA and some snRNA > Compound from 12 subunits Cr jtfon unit of pro polycistronic character trp operon in Escherichia coli-5 genes E D C B A trp mRNA t t t t t B origins of translation 5 proteins for synthesis of Trp F/Tc L LJ nit of > Monocistroni character > Contains: > Promoter > Leading sequence (5-UTR) > Polyadenilation signal > Terminator upstream enhancers WA box' Coding region 3'UTR 1 Promoter Exon 1 Exon 2 5' Exon 3 / Initial transcript 5' cap (still in nucleus) final mRNA (in cytoplasm) I Intron 2 DNA 3' 3' Poly-A tail / AAAAA AAAAA Enhancer Proximal (distal control elements) control elements DNA Upstream A Poly-A signal sequence Termination region Exon Intron Exon Intron Exon Promoter Primary RNA transcript 5 Transcription Exon Intron Exon Intron Exon Intron RNA 1 RNA processing Downstream . Cleaved 3' end of primary transcript Poly-A signal Coding segment mRNA G-d?HgHg>-~ AAA AAA; 3' —y-' ^j-LStart Stop 5 Cap 5'UTR codon codon 3 UTR Poly-A tail http://nitro.biosci.arizona.edu/courses/EEB600A-2003/lectures/lecture24/lecture24.html IV VII XI trp3 1 trp5 1 trp2 Transcription unit for synthesis of Trp in Saccharomyces cerevisiae = togehter 5 genes located on 4 chromosomes > Regulatory elements necessary for transcription initiation > Usually initiate transcription, rarely inhibit it > Their different combination bind to the promoter, then the RNA polymerase bind to DNA strand Types of transcription factors > general TF > present in all and most types of cells > necessary to transcription initiation > basal - low activity, minimal cell requirements > constitutive - increase the basal activity according to cell type; basal cell requirements > special TF > only in cells of specific tissues and in a certain time > applied in inducible transcription PROKARYOTIC PROMOTER ^roYo lymeras® BS starting nucleotide +1 TTGACAT TATAAT region -35 EUKARYOTIC PROMOTER Pribnow box region -10 constitutive and special transcription factors basal transcription factors dCTFI^ < CjFIIQ TATAAAA Hogness box (TATA box) region -34 to -26 0)0 1) Recognises by basal TF TFIID 2) Part of TFIID is TBP protein (TATA binding protein), which is present in all eukyotes Model of TFIID assembly in vivo XTAF4 TAF6 TAF6 TAFS ^TAFS >47 V TBP Kevin J. Wright et al. PNAS 2006:103:12347-12352 Stable Core Sub-complex ^^^4^^^^^^ ^^^^^^^^ Holo-TFIID Transcription of hnRNA 1) Separation of transcription and translation 2) hnRNA is capped by the cap, and methylated (binding to ribosome) 3) In the 3 - region (after STOP codon) the sequence AAUAAA is present, in this location the hnRNA is digested 4) At 3 end is polyadenylated (stabilisation in cytoplasm) 5) After removing introns and joining exons it is transformed to mRNA 1) 2) 3) 4) mmmth Binding of transcription factors on TATA box and others regulation sequences = preinitiation complex Binding of RNAP II on preinitiation complex = closed initiation complex Phosphorylation of CTD domain of RNAP II by trancription factor TFIIH (halicase and kinase activities) -► RNAP II activation and unwinding of dsDNA = open initiation complex Disociation of RNAP II from TFs (except TFIIF) and start of RNA synthesis F« - . _ — CopyriQhi C The McGraw-HiH Companies. Permission required tor reproduction or display igure 11.33 Minimal initiation complex Active -. i transcription complex https://www.youtube.com/watch?v=icZjgZozkB8 http://www.cbs.dtu.dk/dtuco urse/cookbooks/dave/LektO 3bkg.html Activators These proteins bind to genes at sites known as enhancers. Activators help determine which genes will be switched on, and they speed the rate of transcription. Enhance/- Repressors These proteins bind to selected sets of genes at sites known as silencers. They interfere with the functioning of activators and thus slow transcription. Coactivators These "adapter" molecules integrate signals from activators and perhaps repressors and relay the results to basal factors Basal transcription factors In response to injunctions from activators, these factors position RNA polymerase at the start of the protein-coding region of a gene and send the enzyme on its way. ill / transcription Linear transcription units A B Assembly of functional expression units Coordinated expression Expression unit Small genomes i Complex genomes Spatial transcription units Functional organization of the nucleus Promoter assembled from 2 promoters? Dekker J,: Science 319,1793 -1794 (2008) Transcription factory Spatial assemblies (A) Linearly defined expression units in compact genomes and spatially assembled expression units in complex genomes. (B) Association between coordinately expressed genes. (C) Colocalization of genes at subnuclear structures, such as transcription factories. Circles, regulatory elements; rectangles, genes. Arrows indicate direction of transcription. Dekker J.: Science 319,1793 -1794 (2008) Termination of transcription 1) Terminator contains AATAAA sequence = polvadenilation signal 2) Once polyadenilation signal is transcripted into hnRNA, it is recognised by protein complex, which cut hnRNA 10-30 nt towards 3-end 3) Subsequently, RNAP II disociate from DNA and the rest of hnRNA behind the polyadenilation signal is degraded a Termination at mRNA-coding genes in yeast Recruitment of the CPF-CF complex ERNA cleavage and polyadenylation -\r Dissociation of the elongation complex -vr:- Allosteric model CPF-CF 5' complex TSS 4NA £= CTD CFIA Phosphorylated Ser2 5' tion factors DNA Poly(A) signal b Termination at ncRNA genes in yeast Phosphorylated Ser5 [Dissi elon Recruitment of the NNS complex Dissociation of the igation complex RNA polyadenylation and degradation or processing DNA Nrdl-and Nab3-binding sites Nature Reviews Molecular Cell Biology 16, 190-202 (2015) NatUre Reviews | Molecular Cell Biology > In yeast, frequently transcribed genes are localised near by nuclear pores > Also after transcription activation activated regions are transported from central part of nucleus to its surface > Yeast have no lamins which are localised in inner surface of nucleolema > Multicellular organisms have lamins Lamins bind to heterochromatin, they deactivate the gene expression Ikegami, K. a Lieb, J. D. PlosGenetics 6 (2), 1-2 (February 2010) 'ranscriptlon and nucleopon D > Nuclear pore complex (NPC) selectively transmit macromolecules > They are complexes of more than 400 proteins (nucleoporins) which create about 30 subunits B Cytoplasm Nup153 I Mtor mRNA 4 Nucleoporins (e.g. Mfork ■^Cytoplasm Nucleoplasm^ Filament or Granule Nucleoplasm Nucleoporins Nup153 and Mtor form filamentous structures which transport DNA from inner part of nucleus to nuclear pores Ikegami, K. a Lieb, J. D. PlosGenetics 6 (2), 1-2 (February 2010) 'osttranscriptlon RNA pro hnRNA modifications > hnRNP-complexes forming > adding cap to 5 - end > polyadenylation of 3 - end > splicing of hnRNA • Proteins which specifically bind on hnRNA = hnRNP-proteins • Proteins which specifically bind on small nuclear RNA (snRNA) = snRNP-proteins • snRNP-proteins + snRNA = snRNP-particles • hnRNA + hnRNP-proteins + snRNP-particles = hnRNP-complex • snRNP-particles bind on intrones and form spliceosom, which drive the hnRNA splicing • hnRNP-proteins participate on transport of mRNA to cytoplasm hn Ch*üfnaiin SRm16oO nnflKA to«e- mRN AHinRNP/snflNp Splicing snRNP eEJC A/afcvre Reviews Molecular Cell Biology Z, 195-205 (March 2002) mRNA-hnPNP Nature Reviews | Molecular Cell Biology Binding of 7-metylguanosine (m7G) via three phosphate groups to 5'-end of hnRNA by 5'-5' bound Last two 5'-end nucleotides could be aslo methylated m7G plays important role during initiation of translation CH, I - N xx:> H.N N O .N \\ o — P —O-P — o — p —o N NH, pppNpRNA pi 4-4 RNA Triphosphatase (RTPase) ppNpRNA GTP PPi 3 RNA Guanylyltransferase (GTase) GpppNpRNA SAM SAH 3 RNA (guanine-N7)-Methyltransferase (N7MTase) O — CY 0=z P —O <1 0= P —o < m7GpppNpRNA SAM SAH 31 (Cap-0 RNA) RNA (nuceloside-2'-0-)-Methyltransferase (2'0MTase) N "NH, m7GpppNmpRNA (^P 1 RNA) Addition of of 50 - 250 adenosines to 3'-end of hnRNA = polv(A) sequence Catalyses by polv(A)-polvmerase Poly(A)-polymerase is a subunit of complex, which binds on polyadenilation signal of hnRNA Poly(A) tail is important during transport of mRNA to cytoplasm and for its stabilisation RNA capping and polyadenylation coding noncoding sequence sequence Figure 7-16a Essential Cell Biology 3/e(© Garland Science 2010) □ B Introns are cut out from hnRNA and mRNA is created Intron structure: - The rule of GU-AG Rrsnph oito http://www.geneinfinity.org/sp/sp_coding.html Splice donor site Branch site Splice acceptor site Exon I I AG 10s to 10,000s <20 1 Exon — AG ................ GA^ C'TCG'T Pu I NC_A C ................ ccccccccccNc TTTTTTTTTT T Py rich Pu Py G A «t f> IN -» »- N rf in • • » • i 8 2£ iS^^^^^f^w-o-NM Consensus sequences at the DNA level in introns of complex eukaryotes piicm Principle of splicing -transesterification - any energy from ATP or GTP is needed snRNA and snRNP-particles play the crucial role Reaction is catalysed probably by snRNA Intron is cut out in the form of lariat intron RNA i transfer I ? OH o< branch nucleotide A attacks at 5' spbce »te Iff l ,-r! cap @po 5' phoephofyl £ eg O CD g https://www.youtube.com/watch?v =YgmoHtLGb5c phoaphoester iranater © U I 3' OM of exon 1 attacks 5' phosphoryl at 3- spice i txonl A Q •v. \ k* M -pJ-pJ- =-P„-fJ spitcod axons NX -IX § o < DC O) XL O to to 13 T3 CD -Q Q base 5 Phoshory p P J mtnohory i • rtbrae Mm 3' splice Br.A V Branching 'A 2'0H rzzi3"OH Tf^ol y5' exon Mg2' / 9......,o P«>-Rp p-P. o-Intron •.....95" Mg2- Br.A // O Exon ligation MI?.'.....Oh 5' exon \ \ pro-RpO-P ■3' exon O pro-Sn Mg2- 8~ ntron Nature 503, 229-234 (14 November 2013) doi:10.1038/nature12734 Autocatalytic process of introns and exons splicing. • No proteins and enzymes are included in this process. • Digestion and ligation of RNA substrate molecules during self-splicing is catalyse by ribosyme Posttranscription insertion or deletion of nucleotides in RNA strand or conversion of one base to another Resulting in RNA transcript which sequence do not correspond to original sequence of DNA!!! Structural genes undergoing of editation = kryptoqenes RNA editing was described in '80 in Trypanosoma The types of RNA editin 1) Site specific deamination 2) gRNA-directed editing It proceed in specific mRNAs and only in certain tissues or cell types The process is regulated Two forms of apolipoprotein B arise liver intestine Long Short Apo B-100 Apo B-48 Effect of the codon UAA formation dltlng of mRNA for apolipoprotein I pre-mRNA 1 1 I I I IcaaI I I I H I liver yr V imesiine ^^deamination) II caa 1 I I uaa | translation J I Qln I I I Apo B-100 Apo B-48 4 563aa 2 153aa adenine inosine cytosine RNA specific adenosine deaminase (ADAR) > It proceed in ion channels of mammal brain > Single nucleotide change proceeds to exchange of one amino acid > This change permeability of ion channel to Ca2+ ions If the process is inhibited serious damages of brain tissue development are found gRNA (guide RNA) are 40 - 80 nucleotides long Described in the coxll gene in Trypanosome They enable adding of U in specific region of the transcript The resulting mRNA molecules contain additionally huge segments (inserts) which consist of U and opposite miss several U from original (maternal) DNA strand The inserts are such huge that finally up to 50% of edited mRNAs have post-transcriptionally added U The gRNAs joint to mRNA, enable their digestion, adding missing nucleotides and again ligation of digested segments Each gRNA has three regions 1) The first, at the 5 - end (anchor) enables anchoring of gRNA to region of mRNA editing 2) The second direct which nucleotides will be added to edited sequence 3) The third, at the 3- end is the polyU (poly u) C U AA CAUAUGGA 1 J region of region of editing anchoring ExampOt ______________ 5'-, 0( N } AUAUAGUAUAAl- 30 * t? A C G° ^A A U-A - ^A-U □ G—C ^ ax a ij|-A ^ U ° G 20 u A A U A U-A -A-U6° so G-U U-A G-U A-U UUUGAC IJUWDIjOuI^ open square -kethoxal filled circle -DMS filled square -DEPC open circle - CMCT filled arrow + bracket - cobra venom nuclease open arrow + bracket - Tl, T2, SI nucleases (i.e., probes for single-stranded regions) x - frequent reverse transcriptase termination site in untreated control boned bases - anchor sequence and U-tail Position of four U nuoleoti pro-mRNA of the coxll DNA pre-mRNA mRNA protein [ GAGAACCT i C GAGAACC U editing uu uu 1 ..... non-edited RNA 5 C GAGAACCU g RNA (poly U) C U AA CAUAUGGA L 1 J region of region of editing anchoring mRNA region of U adding GAG A ACCU gRNA i G A ACCU i [(polyU) CUCAUAUGGA | AA digestion by endonuclease dUTP adding [ (poly u) D u u ga: : g Qa accu ] (poly u) cu cauaugga J i aa ligation ga a accu cuaacauaugga J Eukaryotic translation > It proceeds in 2-3 compartements, cytoplasm, > mitochondria, and chloroplasts > The first AA is not fMet, but Met, which binds to a specific initiator tRNAjMet, which recognize the AUG codon > The number of initiation factors which are necessary to beginning of translation is higher in eukaryotes > The number of initiation factors which are necessary to beginning of translation is higher in eukaryotes http://www.ncbi.nlm.nih.gov Taxonomy Genetic Codes > similar as a translation in prokaryotes > initiation, elongation, termination > Particular complexes are more complicated > More of translation factors > Genetic code of mammalian mitochondria has different meaning of some codons, 22 tRNA > Eukaryotic cell possesses 45 tRNA with different anticodons > Speed of translation -1-20 AA/s, depends on species and enviroment The cytoplasmic ribosomes Formation of ribosome structure involves also > 150 non-ribosomal proteins > 100 small non-coding RNA Ferreira-Cerca, S. et al. (2007): Analysis of the In Vivo Assembly Pathway of Eukaryotic 40S Ribosomal Proteins, Molecular Cell 28, 446-457, November 2007 > Free ribosomes occur in cytoplasm > synthesis of intracellular proteins > the rest is bounded to the endoplasmic reticulum > rough ER = covered by ribosomes > smooth ER = without ribosomes > synthesis of extracellular proteins > > > 40S subunit with bound tRNAjMet in P-site and initiation factors recognise m7G cap of mRNA Subsequently, this complex moves to 3-end until finds the initiation codon AUG Large 60S subunit binds to 40S subunit using the energy from hydrolysis of GTP Nature Reviews Neuroscience 5, 931-942 (December 2004) doi:10.1038/nrnl557 Met-tRNAMet GTP elF5B»GTP-^ + 60S \l 9lF5B«GDP *-^|^-* W^B^/''' m7GZ □orvgation Nature Reviews I Neuroscience B B B rj S ům ns latii© in Nature Reviews Molecular Cell Biology 11, 113-127 (February 2010) doi:10.1038/nrm2838 elMF complex > J? 2 elF2 ternary comp lei formation 31 z^^w.-IRNA mRNA 60S eRFtandeRF3 \ 40S J ' Ribosome recycling i 1 3 43S complex formation elF2 ABCtl C 9 elF3 ( ) o elFS^ _ I elF4A 4 mRNA activation g elF4B-0^| •JLAU&™UGA_V/V^N 'm'G olFi lTlf f I EPA 40S 3 XA> ATP ▼ ADP Pi n PABP'A 43Spreinttiation complex 5 Attachment to mRNA Seil -AUG ^6 5' to 1' scanning TI p aT 40S / \A.AA ' 17 Initiation codon re hydrolysis of elF? and P release recognition, bound GTP 48S initiation complex 60S J ♦eiFSB &1?J Post TC Elongation 80S initiation complex V 4os 9 Hydrolysis of clFSB-bound GTP Nature Reviews Molecular Cell Biology VV7( AG The canonical pathway of eukaryotic translation initiation is divided into eight stages (2-9). These stages follow the recycling of post-termination complexes (post-TCs; 1) to yield separated 40S and 60S ribosomal subunits, and result in the formation of an 80S ribosomal initiation complex, in which Met-tRNAMetj is base paired with the initiation codon in the ribosomal P-site and which is competent to start the translation elongation stage. These stages are: eukaryotic initiation factor 2 (elF2)-GTP-Met-tRNAMetj ternary complex formation (2); formation of a 43S preinitiation complex comprising a 40S subunit, elF1, elF1 A, elF3, elF2-GTP-Met-tRNAMet; and probably elF5 (3); mRNA activation, during which the mRNA cap-proximal region is unwound in an ATP-dependent manner by elF4F with elF4B (4); attachment of the 43S complex to this mRNA region (5); scanning of the 5' UTR in a 5' to 3' direction by 43S complexes (6); recognition of the initiation codon and 48S initiation complex formation, which switches the scanning complex to a 'closed' conformation and leads to displacement of elF1 to allow elF5-mediated hydrolysis of elF2-bound GTP and P; release (7); joining of 60S subunits to 48S complexes and concomitant displacement of elF2-GDP and other factors (elF1, elF3, elF4B, elF4F and elF5) mediated by elF5B (8); and GTP hydrolysis by elF5B and release of elFIAand GDP-bound elF5B from assembled elongation-competent 80S ribosomes (9). Translation is a cyclical process, in which termination follows elongation and leads to recycling (1), which generates separated ribosomal subunits. The model omits potential 'closed loop' interactions involving poly(A)-binding protein (PABP), eukaryotic release factor 3 (eRF3) and elF4F during recycling (see Supplementary information S5 (box)), and the recycling of elF2-GDP by elF2B. Whether eRF3 is still present on ribosomes at the recycling stage is unknown. Termination of translation > Only one termination factor = eRF > Disociation of ribosome from mRNA needs the energy from GTP https://www.youtube.com/watch?v=qlwrhUrvX-k Extracellular end membrane protein* > all extracellular and membrane proteins have on their N-end so named signal peptide 15-25 AA long > signal peptide joints the proteins to signal recognition particle (SRP) > SRP stops translation on ribosome > binding of SRP to membrane receptor results in removing the signal peptide signal peptidase, and translation starts again THE CELÍ, Fourth Edition, Figuro 10.8 O 2O0» ASM Praas and Sinauar AstoMM*. Inc. Crystal structure of the eukaryotic ribosomes was described in resolution 4.15 A The most interesting thing was founding that both unit of ribosome fit as ratchets and during translation turn around themselves OveraE wiew of the x-my structun View from E-site View from A-site A Ben-Shem et al. Science 2010:330:1203-1209 Maaas View from side of 40S A Ben-Shem et al. Science 2010;330:1203-1209 Science Waaas 1 ^MiiT- .-'"f^^^houlder 18S rRNA (blue) m zC ' 5 as 8 - /oSsr ""V 5 m & ES3 JLIJV- ltES41 5S rRNA (magenta) 25S rRNA (yellow) 5,8S (red) Ben-Shem A. et al. Science 2010:330:1203-1209 Maaas The ribosome controls movement of tRNA and mRNA, structures described in resolution ~ 3.2 A. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling. Dunkle et al. Science 2011:332:981-984 Crystal structure of the large 60S eukaryotic ribosomes was described in resolution 3.5 A Klinge et al. Science 2011; 334 (6058): 941-948 Crystal structure of the ribosomes bound endoplasmatic reticulum was described in resolution 31 A Pfeffer et al. (2012): Structure 20,1508-1518