Structure of eukaryotic genome, replication and gene expression in eukaryotes Molecular Biology i 1. Structure of eukaryotic genome >Eukaryotic cells: >cell wall consists of cellulose (plants) or chitin (fungi), animal cells have no cell wall Mhey contain organelles (mitochondria, plastids) >nucleus is divided by mitosis >nucleus consists of chromatin: >dsDNA > histories >non-histone proteins >chromosomes contain linear dsDNA 2 Mitochondria >Eukaryotic cells: reproduction: asexual (unicellular organisms), sexual replication, transcription and translation are more complicated processes than in prokaryotes >genes usually contain introns Reticulum http://pulpbits.net/7-eukaryotic-cell-structure/structures-of-eukaryotic-cells/ >Genome of eukaryotic cells: >animal cells: nucleus and mitochondria >plant cells: nucleus, mitochondria a plastids >chromosomal (nuclear) DNA (nDNA) >mitochondrial DNA (mtDNA) >chloroplast DNA (ctDNA) >plasmids 4 > Chromatin > material which forms the nucleus > consists of DNA and two types of proteins: histones and nonhistone proteins > level of chromatin condensation is dependent on cell cycle phase > depending on the level of condensation and the ability to be stained by basic dyes, we distinguish two types of chromatin: euchromatin (weakly stainable, decondensed, transcriptionally active) and heterochromatin (strongly stainable, condensed, transcriptionally inactive) https://wwwxliffsnotesxom/study-guides/biology/biochemistry-ii/eukaryotic-genes/structure-of-chromati 5 >Heterochromatin: >Constitutive >constantly in heterochromatin state >centromeres, telomeres >one X chromosome in women > Facultative >switches between heterochromatin and euchromatin states during ontogenetic development http://histology.med.yale.edu/histological_features_of_cells/histological_features_of_cells reading.php >Chromatin components: >Histones: > small basic proteins (positively charged) which bind to negatively charged DNA >5 types: H1, H2A, H2B, H3, H4 > high content of arginine and histidine >Non-histone proteins: > RNA polymerases and other enzymes of the transcriptional apparatus >HMG1 and HMG2 (high mobility group proteins) >HMG3 and HMG4 bind to the histone core especially in transcriptionally active regions http://biology.kenyon.edu/courses/biolll4/Chap01/chrom_struct.html 7 >Nucleosome: >basic unit of chromatin >histone octamer (H2A, H2B, H3,H4)2 >1 molecule of histone H1 >DNA segment of 200 bp, which is wound around the histone octamer twice >nucleosome fibre (10 nm) is visible by EM octamer of core histories: H2A, H2B, H3, H4 (each one x2) core DNA histone H1 linker DNA Stryer, Lubert (1995). Biochemistry (fourth ed.). New York - Basingstoke: W. H. Freeman and Company. ISBN 978-0716720096 8 Discovery of nucleosome stacking in 2014 9 Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 2014 Apr 25;344(6182):376-80. doi: 10.1126/science.1251413. >Chromatin domains: >loops of 30nm chromatin fibre attached to protein scaffold (60-150 kbp) >one molecule of topoisomerase II in the base of each loop - change of topology during replication and transcription >each domain has one ori locus >one human chromosome contains approximately 2000 domains protein scaffold connecting region 10 > Mitotic chromosomes: >originate by condensation of 30 nm chromatin fibres Mormed during mitosis or meiosis condensation of 30 nm chromatin fibres into 600 - 700 nm fibres, which form the chromosome structure >in chromosomes, chromatin is in highly condensed state and is transcriptionally inactive ii >Mitotic chromosomes: Two chromatids Chromosome as seen via an electron micrograph 12 http://ib.bioninjaxom.au/standard-level/topic-l-cell-biology/16-cell-division/dna-supercoiling.html >Chromosomal (nuclear) DNA: > 1 linear molecule of dsDNA > the number of bp per one chromosome in haploid cell is 1,34x 107 to1,5x1010 > only 1.5 % of mammal genome contains protein-coding genetic information > most structural genes are about 1 x 104 to 2 x 106 bp long - considerable part consists of regulatory elements 13 >Repeats in nuclear DNA: >short tandem repeats- not in prokaryotes >dispersed repeats- not in prokaryotes >25-50% of structural genes are unique sequences Mhe rest occurs as gene repeats >Gene repeats: >Gene family: group of genes with similar sequence, from the same original gene, generally with similar functions (genes for hemoglobin subunits) or pseudogenes (dysfunctional genes) >Tandem gene repeats: directly adjacent, separated by spacers (intergenic sequence), genes for 5S-rRNA, genes for tRNA, genes for histones ^Dispersed gene repeats: copies are dispersed in different locations in the genome (genes for tRNA, snRNA, etc.) 15 >Chromatin organisation in the nucleus - fractal globules E. Lieberman-Aiden et al., Science 326, 289-293 (2009) 2. DNA replication in eukaryotes replication of mitochondrial and chloroplast DNA replication of nuclear chromosomes: >semiconservative (each new dsDNA contains one parental and one newly synthesised strand) and semidiscontinuous (leading and lagging strands) initiation, elongation, termination J \J J Genetic materií >only in S-phase of cell cycle 17 >Replication of nuclear chromosomes: >proceeds in several places at once (in contrast to prokaryotes) >chomosome is a set of replicons, many ori sites (30-50k in mam >euchromatin is replicated earlier than heterochromatin 0_„ 5', 3'- 5' 3' 3' DNA .3' -5' ■5' 5', .3' ,3' 5' http://www.biology-pages.info/T/Telomeres.html Incomplete ends >DNA replication process: >Eukaryotic DNA polymerases: >DNA polymerase a- in complex with primase synthesizes Okazaki fragments, does not possess 3'-5' exonuclease activity (no proofreading activity) >DNA polymerase ß- synthesizes short fragments during DNA repair >DNA polymerase y- synthesis of mitochondrial DNA >DNA polymerase 5- synthesis of leading strand and completing the lagging strand >DNA polymerase z- leading strand synthesis and DNA repair >Eukaryotic replication fork: Origin of replication lagging strand https://biology.stackexchangexom/questions/31585/does-dna-polymerase-always-go-the-same-direction primase Ccd45 GINS Mcm2-7 helicase leading strand PCNA 21 H3-H4 partitioning during nucleosome assembly >Histone partitioning: >H3-H4 dimers are formed by parental or newly synthesized histone monomers >newly synthesized dimers are associated together or mixed with parental dimers >H2A-H2B dimers are added and nucleosome is formed Newly synthesized histories Parental niKleosomes a H3-H4 ■ dirrwr 1 H3-H4 j\ dimer D. Ray-Sallet et al., Science 328,56-57 (2010) >Nucleosome assembling is associated with hydrolysis of ATP SWR1-mediated histone replacement Nucleosome «£j»P H2A.Z-H2B dimer H2A-H2B dimer E. Luk et al.. Cell 143.725-736. Nov 24.2010 23 Origin >Replication of linear molecules: Mhe end replication problem Melomerase = ribonucleoprotein - RNA is a template, protein has catalytical function O DNA replication is initiated at the origin; the replication bubble grows as the two replication forks move in opposite directions. 0 Finally only one primer (red) remains on each daughter DNA molecule. The last primers are removed by a 5'—* 3' exonuclease, but no DNA polymerase can fill the resulting gaps because there is no 3' OH available to which a nucleotide can be added. O Each round of replication generates shorter and shorter DNA molecules. Leading strand Lagging strand 3' 5'. 5' Gap 5' 3' 5' 3' 5" 3" c 2012Pearson Education, Inc. https://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-19/CB19.html 3 5 Gap 3 5 3' 5' ■ 5' .3' ■ 5' 5' > Telomeres: >ends of eukaryotic chromosomes >repeats of TTAGGG/CCCTAA sequence (in vertebrates), several thousands of repeats >single-stranded overhang of 50 - 200 nucleotides >protection of chromosomes against degradation (by exonucleases) or fusions Melomeres associate with nuclear membrane 25 >Filling of missing 3'-ends: >telomerase elongates 3'-ends >formation of hairpin and RNA primer >replication of complementary strand and removing of hairpin PROCESS: TELOMERE REPLICATION Missing DNA on 5 lagging strand 1. End is unreplicated. Telomerase with its own RNA template iBSMfikMMfiflfl 5 2. Telomerase extends unreplicated end. 3. Again, telomerase extends unreplicated end. DNA polymerase RNA primer A A U C C C LmIIIImmIIII ■ Sliding clamp 4. Lagging strand is completed. 3 ©2011 Pearson Education. Inc. http://masteringyourwaytomedschool.blogspot.ez/p/bio-1000-dna-shortening.html 26 >Sequences of telomeres: TABLE 11.5 Telomeric Repeat Sequences Within Selected Organisms Group Examples Telomeric Repeat Sequence Mammals Humans TTAGGG Slime molds Physarum, Did/mium TTAGGG Dtctyostelium AG<1-8) Filamentous fungi Newospora TTAGGG Budding yeast Saccharomyces cerevisiae TG(1_5j Ciliates Tetrabymena TTGGGG Paramecium TTGGG(T/G} Eupiotes IIIIGGGG Higher plants Arabidopsis TTTAGGG http://reasonandscience.heavenforum.org/t2263-the-telomerase-enzyme 27 >Protection of chromosome ends: Double-strand break Activates DNA damage response pathways Cell cycle arrest ATM kinase ATR kinase DNA repair Homology-directed repair (HDR) Nonhomologous end joining (NHEJ) Protected from DNA damage response pathways T. De Lange, Science 326, 948-952 (2009) 28 >Mammalian telomeres: Telomere t-loop Strand invasion of 3' overhang T. De Lange, Science 326, 948-952 (2009) 29 >Different outcomes of end-protection problem: Mammals (r _A___ J OTHERWISE J i^j^gj Dicentric chromosomes ■ and genome instability Ku j HDR OTHERWISE Terminal deletions and telomere length changes OTHERWISE G/S orG2/M arrest and apoptosis/ senescence Budding yeast Rif1 Cdc13 Stn1/Ten1 OTHERWISE NHEJ Dicentric chromosomes and genome instability HDR OTHERWISE Telomere length changes OTHERWISE G2/M arrest OTHERWISE ? 30 T. De Lange, Science 326, 948-952 (2009) >Termination of DNA replication in eukaryotes: > replication of prokaryotic chromosome ends in specific sequences -termination zones >other factors involved in termination of DNA replication in eukaryotes -topoisomerase II participates and the process is regulated by ubiquitination TERMINATION ZONE (TERs) Fork fusion .::. Fork pausing Resolution OPolu •Pole/8 OPausing element DOI: http://dx.doi.Org/10.1016/j.molcel.2010.07.024 3. Transcription in eukaryotes >Primary transcripts: >precursor mRNA (pre-mRNA) >heterogenous nuclear RNA (hnRNA)= pre-mRNA forming in nucleus >precursor ribosomal RNA (pre-rRNA) >precursor transfer RNA (pre-tRNA) >5S-rRNA >small RNAs (snRNA, snoRNA, scRNA) >Eukaryotic DNA-dependent RNA-polymerase: >RNA-polymerase I, II, III >Transcription factors >Eukaryotic RNA-polymerases: >RNA polymerase I: >synthesis of pre-rRNA >only in nucleolus >insensitive 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 >RNA polymerase II a, Representative regions of the cryo-EM density for EC1 with the refined model superimposed. Depicted are (from left to right) RPB1 helix al9, RPB8 strand (38, the DNA-RNA hybrid, and the active site aspartate loop with the bound catalytic metal ion A and the 3'-nucleotide of the RNA transcript, b, Ribbon model. The views correspond to the previously used 'top' and 'side' views of yeast Pol II and are related by a 90° rotation around a horizontal axis. Black spheres indicate the location of residues that are not identical between bovine and human Pol II, bovine indicated second (three out of seven residues, the remaining four are disordered). The final model lacked several short surface loops and flexible N-terminal residues. Structure of transcribing mammalian RNA polymerase II Nature volume 529, pages 551-554 (28 January 2016) 35 >Transcription unit: >monocistronic character >contains: > promoter > leading sequence >polyadenylation signal > terminator Enhancer Proximal (distal controj elements) control elements Poly-A signal sequence Termination region DNA Upstream JA. Exon Intron Exon Intron Exon Promoter Primary RNA transcript I #4 Transcription Exon Intron Exon Intron Exon Intron RNA RNA processing Downstream Cleaved 3' end 1 of primary transcript Poly-A signal mRNA Coding segment Start Stop 5'Cap 5'UTR codon codon 3'UTR Poly-A tail http://nitro.biosci.arizona.edu/courses/EEB600A-2003/lectures/lecture24/lecture24.html 36 >Transcription factors: >necessary for transcription inititation >bind to promoter sequence >General TFs: >present in all cells, necessary for initiation >Basal - low activity, minimal cell requirements >Constitutive - inrease the basal activity according to the cell type >Special TFs: >only in specific cells in specific time Munction in inducible transcription >Eukaryotic promoter: > transcription by RNA polymerase II constitutive and special transcription factors basal transcription factors C TFIID TATAAAA Hogness box (TATA box) ■34 to -26 bp 38 >Binding to TATA box: TATA-box Binding Protein (TBP) >recognised by basal transcription actor TFIID >TBP Protein {TATA binding protein) is a part of TFIID, present in all eukaryotes http://www.web-books.com/MoBio/Free/Ch4E.htm 39 >Transcription of hnRNA: 1. decoupling of transcription and translation 2. hnRNA is capped and methylated (binding to ribosome) 3. in the 3'-end (after stop codon) AAUAAA sequence is present, hnRNA is cleaved in this region 4. hnRNA is polyadenylated on 3'-end (stabilization in cytoplasm) 5. introns are removed and exons are connected to form mRNA 40 ^Initiation of transcription: 1. binding of transcription factors to TATA box and other regulatory sequences - preinitiation complex 2. binding of RNAP II - closed initiation complex 3. phosphorylation of CTD domain of RNAP II by transcription factor TFIIH (helicase and kinase activity) - RNAP II activation and unwinding of dsDNA - open initiation complex 4. dissociation of RNAP II from other TFs (apart from TFIIF) and start of RNA synthesis transcription complex 41 >Parts of eukaryotic promoter: 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. Coding region -P- Coactivators These "adapter" molecules integrate signals from activators and perhaps repressors and relay the results to basal factors http://www.cbs.dtu.dk/dtucourse/cookbooks/dave/Lekt03bkg.html 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. >Spatial assemblies during transcription: (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. Dekker J: Science 319, 1793-1794 (2008) >Termination of transcription: Merminator contains AATAAA sequence - polyadenylation signal >polyadenylation signal in hnRNA is recognized by protein complex, which cleaves hnRNA 10-30 nt towards 3'- end >RNAP II dissociates from DNA and the rest of hnRNA is degraded a Termination at mRNA-coding genes in yeast Recruitment of the CPF-CF complex RNA polyadenylation and degradation or processing DNA Nrdl-and Nab3-binding sites Nature Reviews Molecular Cell Biology 16, 190-202 (2015) Nature Reviews i Molecular Ceil Biology 44 termination of transcription: a Termination at mRNA-coding genes in metazoans CTD Phosphorylated 5', I Recruitment of *X£er2 1_ |CPSF-CF complex I 5', TSS RNA cleavage and polyadenylation Dissociation of the elongation complex -—\rw» FI-CFII * "j!E> XRN2 Torpedo model DNA PAS R-loop b Termination at genes encoding snRNAs in metazoans Recruitment of termination or processing factors "Phosphorylated Ser2 Phosphorylated Ser7 Dissociation of the elongation complex NfT'' snRNA 3' end processing |^>^. , 5 snRNA gene 3' box Nature Reviews Molecular Cell Biology 16, 190-202 (2015) Nature Reviews | Molecular Cell Biology 45 >Transcription and nucleoporins: >in yeast, frequently transcribed genes are located near nuclear pores >after activation of transcription, activated regions are transported to nuclear surface Multicellular organisms contain lamins, which are localized in inner surface of nucleolema (not yeast) Ikegami, K. a Lieb, J. D. Plos Genetics 6 (2), 1-2 (February 2010) 46 >Transcription and nucleoporins: >nuclear pore complexes (NPC) selectively transmit macromolecules >complexes of more than 400 proteins (nucleoporins) in 30 subunits >nucleoporins Nup153 and Mtor form filamentous structures which transport DNAfrom inner part of nucleus towards nuclear pores http://cellbio.emory.edu/lab/powers/Research.html Ikegami, K. a Lieb, J. D. Plos Genetics 6 (2), 1-2 (February 2010) 47 >Posttranscriptional RNA processing: >hnRNA modifications: >Formation of hnRNA-protein compl >Adding cap to 5'- end >Polyadenylation of 3'- end >Splicing of hnRNA >Formation of hnRNP complexes: >Proteins which specifically bind hnRNA- hnRNP proteins >Proteins which specifically bind to small nuclear RNA (snRNA)- snRNP proteins >snRNP proteins + snRNA= snRNP particles >hnRNA+ hnRNP proteins + snRNP particles= hnRNP complex >snRNP particles bind to introns and form spliceosome >hnRNP proteins participate on transport of mRNAto the cytoplasm >Formation of hnRNP complexes > Adding cap to 5'-end of hnRNA: >Binding of 7-methylguanosine (m7G) via three phosphate groups to 5'-end of hnRNA via 5'-5' linkage >Also two or three 5'-end nucleotides can be methylated >m7G plays and important role in initiation of translation NH2 J N J > 1 this position can be methylated also in cap 1 O O CH2 opOPOP O CH? 0" O G addod by 5' 5' linkage present in cap 1 (CH3 group in two terminal 5' nucleotides) present in caps 2 (CH3 group in three terminal 5' nucleotides) primary transcript H H -CH? NH \ O OH :3' http://www.biocydopediaxom/index/genetics/expression^^ of_caps_m7g_and_tails_polya_for_mrna.php 52 >Polyadenylation of 3'-end: >Addition of 150-250 adenosines to 3'-end= poly(A)sequence >Catalysed by poly(A)-polymerase >poly(A)-polymerase is a part of complex which binds to polyadenylation signal on hnRNA >poly(A)-end is crucial during transport of mRNAto cytoplasm and its stabilization RNA capping and polyadenylation coding noncoding sequence sequence protein Figure 7-16a Essential Cell Biology 3/e (© Garland Science 2010) >Splicing of hnRNA: >lntrons are cleaved out of hnRNA to form mRNA > Structure of intron: >GU-AG rule (donor and acceptor sites) > Branch site Splice donor site Branch site Splice acceptor site Exon Exon 7AG GT £AG A G ............... ccccccccccNcAG G TTTTTTTTTT T A Pu Pu Py Py rich Consensus sequences at the DNA level in introns of complex eukaryotes http://www.geneinfinity.org/sp/sp_coding.html 53 >Splicing of hnRNA: Mransesterification - no energy from ATP or GTP is needed >snRNA and snRNP particles play crucial role >intron is cut out in the form of lariat RNA excised larut Q baae fcion 5 c*v>*hofy p J P 3 pmphofy ntv -,r http://www.bx.psu.edu/~ross/workmg/RNAProcessingChl2.htm 54 >Role of Mg2+ ions during splicing: Nature 503, 229-234 (14 November 2013) doi:10.1038/nature12734 55 >Self-splicing: >Rare autocatalytic process of hnRNA splicing >No proteins (enzymes) are needed >Digestion and ligation of RNAduring self-splicing is catalyzed by ribozymes (ribonucleic acid enzymes) 56 >RNA editing: >posttranscriptional insertion or deletion of nucleotides in RNA or conversion of one base to another >results in RNA transcript which does not correspond to original coding sequence in DNA >Two types: 1. site-specific deamination 2. gRNA (guide RNA)-directed editing >Deaminaton C -> U: >ln specific mRNAs only in certain tissues or cell types >Two forms of apolipoprotein B >Liver: long Apo B-100 >lntestine: short Apo B-48 > Formation of stop codon UAA Cytidine deaminase >mRNA editing in apolipoprotein B gene: pre-mRNA I Qln -1 I I Apo B-100 4 563 aa Apo B-48 2 153 aa 59 >Deamination A -> I: >ln ion channels in mammalian brain >Single nucleotide conversion changes the coded aminoacid ^H2 >This changes the permeability of ion channel to Mg2+ ions adenosine inosine ribose-5-phosphate or 2'-deoxyribose-5-phosphate ribose-5-phosphate or 2'-deoxyribose-5-phosphate 60 >gRNA-directed editing: >gRNAs (guide RNA) are 40-80 nucleotides long >First described in coxllgene in Trypanosome >Enable adding of U in specific regions of transcripts >gRNAs bind to mRNA, enable their splicing, adding the missing nucleotides and linking of the spliced fragments >Resulting mRNAs contain large segments of added U and lose several U from original sequence insertions of U can be present in up to 50 % edited mRNAs 61 >gRNA structure: >each gRNA has 3 regions: 1. first, in 5'-end (anchor), enables binding of gRNA to region of mRNA editing 2. the second directs which nucleotides will be inserted 3. polyLl sequence at 3'-end 3 5 region of editing region of anchoring 62 > Sequence of gRNA and unedited mRNA unedited RNA GAGAACCU r gRNA (polyU) CUAA CAUAUGGA L 1 J region of editing region of anchoring 63 >Process of editing I: location of U addition mRNA GAG A ACC gRNA mRNA gRNA (polyU) CUAA CAUAUGGA I_I_I UCAUAUGG i digestion by endonuclease 64 >Process of editing II Addition of dUTP U U mRNA gRNA 3' mRNA gRNA (poly U) CU CAUAUGGA [AAJ ^ ligation GA G A ACCU (poly U) CUAACAUAUGGA > Transcription: >Codons: u Second nucleotide C A o o 3 C — U A uuu Phe uuc UUA s*% UCU UC° Ser UCA UCG UAU _^ UAC ^ UAA STOP UAG STOP UGU Cys UGC W UGA STOP UGG A cuu cue f% CUA ^ CUG ecu ccc Pro CCA CCG CAU ~ ~ H.s CAC CAA CAGDifferences from prokaryotic translation: >it takes place in 2-3 compartments - cytoplasm, mitochondria, chloroplasts >the initial AA is not fMet but Met, which binds to a specific initiation tRNAiMet that recognizes the initiation codon AUG >the number of initiation factors required for translation initiation is higher than in prokaryotes 68 >Translation in eukaryotes: >Similar to translation in prokaryotes initiation, elongation a termination >Individual complexes are more complicated >Higher number of initiation factors >Genetic code in mammalian mitochondria has a different meaning for some codons, 22 mitochondrial tRNA genes >Eukaryotic cells possess 45 tRNAs differing in anticodons translation takes place at 1-20 AA/s, depending on organism and conditions >Cytoplasmic ribosomes: > Also involved in the formation of ribosomes: > 150 non-ribosome proteins 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 70 >Bound and free ribosomes: >Free ribosomes occur in the cytoplasm intracellular protein synthesis >Others are bound to endoplasmic reticulum (ER) >Rough ER is covered with ribosomes >Smooth ER is ribosome-free Ribosome http://www.assignmentpointxom/science/biology/about-ribosome.html 71 initiation of translation: >40S subunit with tRNAiMet bound at the P site, initiation factors bind to the m7G mRNAcap >The whole complex moves in the 5-3 direction before it hits the AUG initiation codon >Hydrolysis of GTP, the 60S subunit binds 72 (a) Formation of the cap-binding complex 40S (b) Formation of the ternary translation initiation complex Suppression of translation initiation 40S mRNA IK)- () D "vl oh no o H ° I o on V \ A i C 5'pA-O G-6 6-G"f G. HO OH 6- 4 1 B = Ade G- -c 2B = Gua l G- "v; U' rr^G rn. ,..A C G Crr^G G-C-G--6. ? lh;G i G A-C C-G m-G u A-U M :>-G_C«i G-C C' A c._A_U http://mol-biol4masters.masters.grkraj.org/html/Protein_Synthesis4-Eukaryotic_M tRNA carrying first amino acid UAC Anticodon Ribosomal subunits Small ejf n n n n n n n n n n r njLnjUriJUUTJlJU^Jl^^ " n n n^^n n n n n g( mRNA AUG Start codon UAG Stop codon INITIATION © During initiation, the components^ of the translational apparatus come < together with an mRNA, and a tRNA carrying the first amino acid (AAt) binds to the start codon (AUG). ELONGATION i Q During elongation, amino acids are brought to the mRNA by tRNAs and are added, one by one, to a growing polypeptide chain. Recycling of translational components 1 n ri 11 n n Completed polypeptide 5> ,n n n n n n n pjjjh n n n n n n uuuuuuuu^ui TERMINATION UAG Stop codon 0 During termination, a stop codon in the mRNA is recognized by a protein release factor, and the translational apparatus comes apart, releasing a completed polypeptide. © 2012 Pearson Education, Inc. https://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-22/22_07.jpg 75 >Termination of translation: >Eukaryotic translation termination factor eRF1 (release factor) recognizes all 3 stop codons >Energy from GTP is required to release the ribosome from mRNA https://www.youtubexom/watch?v=C4QiMqBSDe4 76 >Extracellular and membrane proteins: >all extracellular and membrane proteins have a 15-25 AA signal peptide at the N-terminus >signal peptide binds to the signal recognition particle (SRP) >SRP stops translation on the ribosome >binding of SRP to the receptor on the membrane leads to cleavage of the signal peptide by signal peptidase and the translation continues 77 >Extracellular and membrane proteins: membrane >Translocation of extracellular protei THE CELL, Fourth EdrtHto, Figure 10« O 3008 ASM Fnm and Snauw ■ JlOCMn. Ha The Cell, Fourt edition, Figure 10.8. 2006 >Formation of membrane proteins The Cell, Fourth edition, Figure 10.12, 2006 THE CELL. Fourth EaWxxi. Figur* 10-12 O SOX ASM Piro md Smut Ail a CUM, Hie. 80 >Translation and folding: >ln some proteins, the ribosome shift briefly stops following synthesis of short oligopeptide >This will allow precise targeting of the protein to the exit tunnel in the large subunit >Precise targeting of the protein to the tunnel is associated with proper folding Joseph D. Puglisi, Science 2015, 348,399-400 81 Peptidyl Transferase Center Decoding Center http://www.crcl.fr/311-Projets-en-(5B.crcl.aspx?language=en-(5B