UNI PHAR Molecular Biology -2 The structure and function of biopolymers during the transitions of genetic information Department of Molecular Biology and Pharmaceutical Biotechnology (45-308) Mgr. Marie Brázdová, Ph.D. (FAF, BFU AVČR) Mgr. Denis Šubert (BFU AVČR) Mgr. Daniel Renčiuk (BFU AVČR) PharmDr. Jakub Treml, Ph.D. (FAF) brazdovam(a>phariTi.muni.cz, 1 Genetic information is coded by DNA The experiment combining two strains of Streptococcus pneumoniae bacteria. S strain I smooth pathogenic bacterium causes pneumonia RANDOM MUTATION R strain o 0o o u rough nonpathogenic mutant bacterium live R strain cells grown in presence of either heat-killed S strain cells or cell-free extract of S strain cells TRANSFORMATION ♦ ^^fe A Some R strain cells are X transformed to S strain cells, whose daughters S strain are pathogenic and cause pneumonia CONCLUSION: Molecules that can carry heritable information are present in S strain cells. (A) S strain cells r fractionation of cell-free extract into classes of purified molecules RNA protein DNA 1 1 lipid carbohydrate 1 1 1 1 1 molecules tested for transformation of R strain cells oo R strain o0 R strain S strain °o R strain o° R strain CONCLUSION: The molecule that carries the heritable information is DNA. (B) Molecular Biology of the Cell (© Garland Science 2008) MB 2 2023 0. Avery C. MacLeod M.McCarty STUDIÉS ON 1HE CHEMICAL XrVTlTlE OF TTTK SUBSTANCE IHDUC1KG TRAN5 fOKJlAriOM OF PWĚUMOCŮOCAL TYPES FyiinriKW oř ^UWHPQTATGQfi pv a Dcsco^rijMMticxfcC ACHt FultujqJ iMUttfcD Tli-Ctf PbíEMOCOCCUB TTFt HI Bi ŮBWALD T. AVGlLV, H.LJ COLItf M. HfrJLEOO, H.P.. **a U.WTf.YK U^V.T TT." M D. ■ JVfin litt ti-řipiiil z/ T.w S&ťStfclltr JmnVyfe for Hí-fcal JkremrAJ PtAI» I ^"Rfia-ivůd í-k ijluliliráciiiri, Fiírembec I, LVU) BirtJflptfj fiivt lnoj atbenpttd Hy cbeoiLÉil tw.t.i tn indLjCE in tpjjtur ůfgaalůmů pwdfcerabl* iiui spůtlác dímuctt fťokl tfwtctíwj omjIJ tt tmu-in kcící ■|_L,.r-- .!:-in -L:ri. AíDang EZULrodr^nuou tt* crarf AEtrapLt -ol inJbcriubk atiiÍ tfKcific Jl-třitLWii in cr:ll tAructuit uai - ■-■--- Jíl luil be nfMTimMluLJy- iílJufltJ áml iPř Hfttúdudbk UůdíL" bell i-if-finL-rj jin^ ii.íqii ulily canlralled ■rri-cirtJLincES ir, ihe truufarmitiíH] nÉ apeciac iypn <4 Jatvivxňiifub, Thi* pli»ii«rawn w** flnt dwíflb*J by Gnftfth (1) vrbo HJCCKLlrd n imruíocituji^ mi i■ r.iII ■.-•A nDa-nci}»uiirni (K) vilUl Acivpl frera flc* ipefiůc tjrpc inlů Eij^y tflca;pwiLp.Jcd -Hnit virufcnl (5() urllůufa hjelůtůfc#ouůflpMlůt vařičiy oJ Irii^:-(ycUůHíjyiiH (tai 3J* po&iifck ^iťtbrio. Llic ILmJ4ů a4 Ihia bacut-i^Lůpcctta. Nuclein Nuclein - acidic substance rich in nitrogen and phosphorus J. F. Miescher -1864 Isolated from blood of wounded patients and cleaved by pepsin (proteolytic enzyme) Courtwy of Herrn Courvofeler Pornalt-Sammlung, University of Base) Noncommercial, educational UK only MB 2 2023 Roles of genetic material Genotype - storage of genetic information and its transition to the offspring Phenotype - expression of genetic information to particular properties of an individual Evolutionary role - adaptation of an organism/species to the environment through the changes in genetic information MB 2 2023 4 Terminology Gene - several "defi nit ions" depending on the point of view: classic genetics (Mendel) - elementary unit of hereditary genetic information molecular genetics - part of DNA coding for RNA (and as a consequence coding for some property of the individual) structural genes coding for mRNA/protein (+ regulatory regions) genes coding for functional RNA (miRNA,...) strict - structural gene - part of DNA that codes for protein sequence Allele - particular variant of the respective gene Genome - complete DNA of organism (molecular) x complete genetic information of organism x sum of genes (classic) Genotype - the combination of particular alleles of all genes in individual Phenotype - the sum of actual individual properties (as a result of expression of particular genotype in the respective environment) Genophore - the carrier of genetic information, usually a molecule of DNA (often used for bacteria) MB 2 2023 5 Information biopolymers Deoxyribonucleic acid (DNA) • linear heteropolymer composed from 2-deoxyribonucleotides connected by phosphodiester bonds • usually as a stable and resistant double helix • serves as a storage of genetic information, as a template for its reproduction (replication) and as a template for the expression of genetic information to the phenotype (transcription) Ribonucleic acid (RNA) • linear heteropolymer composed from ribonucleotides connected by phosphodiester bonds • usually as a single-stranded structure of variable length, structure and reactivity • many functions depending on type of RNA Protein • linear heteropolymer composed from 20 (21) amino acids connected by peptide bonds • highly variable structures, properties and functions MB 2 2023 6 Central dogma of molecular biology Nucleic acids DNA n Replication Transcription! Reverse transcription (retroviruses, ...) RN/p Replication Translation 1 Protein MB 2 2023 Gene P0U5F1 POU class 5 homeobox 1 [ Homo sapiens (human) ] Gere ID: 5460, updated or 17-Sep-201E * Summary * 7 Official Symbol POU5F1 pro rfedbyHGHC Official Full Name POU class 5 homeobox 1 : ; =s= v hghc Primary source HGNC:HGNC:9221 See related Ensembl:ENSG00000204531 HPRD:012S2: MIM:1G4177: Veoa:QTTHIJMGQ00QQ031206 Gene type protein coding RefSeq status REVIEWED Organism Homo sapiens Lineage Eu^aivota l,-1etaica Chcrdata Craniata Vertebrata Euteleostami Mammalia: Eutheria: Also known as OCT3; OCT4; OTF3; OTF4; QTF-3; Oct-3; Oct-4 Summary This gene encodes a transcription factor containing a POU homeo;:c "ain that clays a N A T U R K 7S7 equipment, and to |)r. (i. K. U. Ifrwom and lim oftj.lnni nud Iři of K.K.8. Discovery 11 for llmir pnrt ut making the ol we r vat ions. •V. it c. B. ť■•nu J. II . Aii.l Jrv ■ « im li II ulf ľi[>. • tJnwin. *• « .Irti'a.Jíaŕ .Irfaw i iMimM M. Wilkins E. Chargaff MOLECULAR STRUCTURE OF NUCLEIC ACIDS A Structure for Deoxyribose Nucleic Acid WE wish to suggest a structure for lho mI) nf deoxyril»o*e nucleic acid i' clwiin structure lias alwi been aug 'raaor mi tlm press). Tn his model HM lire mi the outside anil tint Ikvho* on tlm si togotlmr by hydrogen lunula. This i describ I m rather illHleflned, and for thin rva*oii we shall not comment on it. Wo wish 111 put f«irwun 1 a radically dinVren* structure for tlm nail of uVoxyrtlwi*' nucleic acid. Thiw structure luw two helical chain* each coiled ronn«l the taunt- i»Xi* (see-diagram). We have made the usual chemical assiimption*, namely, that each rlmin consist* of pliospliato chewier _■'!]■■ joining 3-n-deoxy-nbofuraniMn reajditos with 3'..V huk»K>*. Tim two chaiiiH (hut not writ bases) are related by a dyad |mrpcndicular to the fibre axis. !'■ 'I. ' '.-i na follow right-liaitditl lichees, bill owing t<> (he dyad tlm sequence* of the atoms in tlm two chain* mn in »{i]KMito directions. Each (limn |iwi*ely nf t the other a pynmidine hydrogen Inrnda nre mm I to psii. : i, positi' p>*rimidme A. If it ta (Mrtiirrmd that •trucIure in the most itlLtt*. ia. with the kcto figuration*) it ia found bMM can bond logetlm. Franklin's X-ray photograph shows I purine) with thyrnine t tparjnt) with c\tóaine | DNA's "B'-form (1952) In other wortu, if an i.*,..UM>- mM1 ,w „m..,m u. a jiftir, on eitlmr eliam. limn on the** aatuunptionn Ihe OllMf member mtut 1m ll^-mino; itirailarly for L'n.iriii. ■ and cytokine. The teigiinnce of baaeN on a nf. K m. I - not ap|Mtar to lm roatrioted in any way. However, if only apooifte pain, of base* nan ú-fonnetl. it follow* thai if tlm aoqm-iioc of batten on one cliain ia given, then tlm nrqiieiiee oil th" otlmr chain w automatieally detenninml. It haa been found exp-rnnontally*'- tliat tlm ratio of tlie omotinta of lulenum tn thymine, anjl the ratio • if guanine turytotme, are always. \-er>- clove to unity for deoxynbuao nucleic acid. It ia probably nh;...,.':,!. to build this atntcture with a nlxMe sugiu- in jilaee of tlm deoxyrilxne, ae tlie extra oxygen Atom wtmtd make |qq cIimo a van dor Wiuila contact. Tlie provioualy pubtialied X-ray '. Wilkin- 1 • ľ. K. V- i-.l.t. i anil tlmir .workers at MB 2 2023 21 DNA structure - Pauling model Linus Pauling A PROPOSED STRUCTURE FOR THE NUCLEIC ACIDS By Linus Pauling and Rohrrt B. Corby Gates and Ckhllin Laboratories of CHKmsrRV,* California Institute of Tbciinology Comtnuiik-ateu' l)ecemlM.>r 31, 1962 The nucleic acids, as constituents of living organisms, are comparable in importance to the proteins. There is evidence that they are involved in the processes of cell division and growth, thai they participate in the transmission of hereditary characters, and that they are ini|x>rtant constituents of viruses. An understanding of the molecular structure of the nucleic acids should be of value in the effort to understand the fundamental phc- 92 CHEMISTRY: PAUUXG AMD COREY Proo N. A. S. which are involved in ester linkages. This distortion of the phosphate group from the regular tetrahedral configuration is not supported by direct experimental evidence; unfortunately no precise structure determinations have been made of any phosphate di-esters. The distortion, which corresponds to a larger amount of double bond character for the inner oxygen atoms than for the oxygen atoms involved in the ester linkages, is a reason- \ FIGURE fi Plan of the nucleic Kid structure, showing several nucleotide residues. able one, and the assumed distances are those indicated by the observed values for somewhat similar substances, es|>ecially the ring compound SsOj, in which each sulfur atom is surrounded by a tetrahedron of four oxygen atoms, two of which are shared with adjacent tetrahedra, and two unshared. The 0—0 distances within the phosphate tetrahedron are 2.:i2 A (between the two inner oxygen atoms), 2.4») A, 2.55 A, and 2.60 A. The MB 2 2023 22 Various types of double helix B-DNA DNA in water/salt soulutions A-DNA Z-DNA • DNA in crowding solutions • CpG sequences in crowding conditions • RNA Left handed Zig-zag step MB 2 2023 23 Reversed Watson-Crick pairing Base protonation N H H ^ Adeiioiine -M H I 1+Vh pKl=l.<5 H H H CT N I N *|An I H Guano&inc N V H N I H N N N H pKi = 9.2 H Uridine 0 N 1 H H H H o n t- H Thymidine 0 N 1 R CH, H •base protonation might alter the base reactivity • free bases have pK far from physiological •pK of bases in DNA might be closer to pH 7.4 • cytosine in Cn sequences has pK~7 - cytosine i-motif H 1 + 1 ^ R Cylidiric N H I RMB 2 2023 25 DNA double helix x ions / water • phosphates in DNA backbone are negatively charged - repulsion • this is compensated by interaction with ions (Na+, K+, Mg2+,...) or water (H-H bonds) 26 Stability of DNA double helix T 7"m = melting temperature hydrogen bonds AT = 2 x GC = 3 Tm increases with GC and length base stacking various Tm increases with length and ions repulsion of backbone phosphates Mg2+>Na+ Tm increases with ions MB 2 2023 27 Base-pair parameters in double helix Shear(Sx) Buckle (k) Shift (Ox) Tilt(r) Stretch (Sy) Stagger (Sz) Propeller (it) Opening [a) Lu et al., 2003, NAR Rise (Dz) MB 2 2023 Twist (>•) + Coordinate frame 3' Types of nucleic acids • linear (human chromosome) x circular (bacterial genome) • single-stranded (most RNAs) x double-stranded (human DNA) MB 2 2023 29 Superhelicity Overwound topological domains form compact large scale chromatin structures Supercoiling influences higher levels of chromatin organisation Underwound topological domains have a decompacted large-scale structure and 'ij»t»'"«»■•£•:■•;•♦ :v;/^^.Vi .' ;•[*>•:«•••':;V;-*:.:v> .-•■•"•../•.v: -\ Superhelicity happen mostly as a result of transition of polymerase complex and unwinding of DNA (helicase,...) during replication and transcription. Topoisomerases • Enzymes that relax the superhelicity • Topo I - works on 1 DNA strand • Topo II - works on 2-strand DNA MB 2 2023 30 Reactivity of bases with amino acids Double-stranded NA: Interaction of Hoogsteen side with amino acid in major groove. n Mparagine (or glubumi nt) Serine (or threonine) Aiginine Arginine Figure 2-16 [nleractions involving two hydrogen bonds between amino acids and bases that can occur through the major groove of a double helix. MB 2 2023 31 Reactivity of bases with amino acids Single-stranded NA: Interaction of Watson-Crick side with amino acid. II 0 0-... H c; h Axpartic (or glutamic) 0 1 R H R Asparagine (or glutaminc) ,CHa ^H-"-0*"'rJ'",H I Asparagine (or glutaminej H Hi I cT C H I H Asparaginic (or giiiiamine) CH, H 1 '■■ H'' .....H I N H^C CH; H HSCT I I C H. © I Asparagine (or glutatnine) Asparagine (or glutamic) Arginine Figure 2-17 [nieracticirii involving two hydrogen bonds between amino acids and bases that take the place of Watson-Crick base pairing. MB 2 2023 32 Genome composition percentage 0 10 20 i-1-r 30 T 40 T 50 T 60 T 70 T 80 90 T 100 LINEs SINEs retroviral-like elements —1 DNA-only transposon 'fossils' TRANSPOSONS simple sequence repeats — segmental duplications JL introns protein-coding regions — I_ GENES non-repetitive DNA that is neither in introns nor codons J REPEATED SEQUENCES UNIQUE SEQUENCES Molecular Biology of the Cell (© Garland Science 2008) MB 2 2023 33 Repetitive sequences - repeats Some sequences in genome are unique, usually the genomic sequences (both coding and non-coding). In contrast, other sequences exist in many copies - repetitive sequences (repeats). The length of repeat (microsatelites 2-6 bp x LINE 6-7000 bp), as well as the number of copies (several - 1.5M SINE in human) is highly variable. Structure: • direct repeats 5'...AGTC. 3'...TCAG. .AGTC .TCAG .3' .5' Position: • Tandem repeats 5'...AGTC...CTGA...3' 3'...TCAG...GACT...5' • inverted repeats + palindromes 5'...AGTC...GACT...3' 3'...TCAG...CTGA...5' Interspersed repeats MB 2 2023 34 Inverted repeats Hairpin 5'- Palindrome AGTCGACT 3' 5- c- T-G-A- -G -A -C -T Hairpin with loop 5'- Inverted repeat AGTCTGAGCTGACT 3' 5- A G G C C-T-G-A- -G -A -C -T MB 2 2023 35 Inverted repeats Cruciform A G G C Inverted repeat 5-AGTCTGAGCTGACT 3-TCAGACTCGACTGA 3' 5' 5-3- c- T-G-A-T-C-A-G- -G -A -C -T --A --G -T -C. A ■3' ■5' T C MB 2 2023 36 Special types of repetitions - transposons Interspersed repetitions with various lengths and number of copies. LTR - long terminal repeat -100 bp - 5 kbp - variant of retrotransposons LINE - long interspersed nuclear elements - up to 6 kbp - human> 500k copies - 3 types (LI, L2, L3) - only some LI are able to transpose SINE - (Alu,...) short interspersed nuclear elements - up to 500 bp - human ~ 1,5M copies MB 2 2023 37 Loops and hairpins in RNA 16S RNA 7W. u*C* no a*ugc aucuggag0 accc„ug caaSu acgagc uguug ugcucg ugccgaucuggaccuuaaca|j _ cc cgc Vaaurcc -g^ a g . _C^ i/qo ccuua uccuuug uuccc CGG uc ggggu agg a a ac a.GG GCCcc Ö oc Ca., IK|UC tRNA (Lys) ......?*, D-loop \ 5' pG-C • G -G -A ■ U ■ u ■ u 3' A-OH C c A ■ C • c ■ u u • A ■ A mm R6:1.301-1.54; mm RS: 1.051-1.300 mm R4: 751-1.050 R3:501-750 mm R2:251-500 mm Rl:1-250 Anticodon loop „ V A C U C m2G I I I I G AG C , v mJG C C A G A Cm U acceptor stem TipC loop I c u G A C A C I I I I I G m5C U G U G c C T,tl U 7^ A , — G G — G — U — nrC — >P A Y variable loop G™A a MB 2 2023 Nature Reviews | Microbiology Functional types of RNA hnRNA mRNA Translation; codes for protein sequence pre-rRNA rRNA Translation; part of ribosome DNA -> pre-tRNA tRNA Translation; amino acid carrier snRNA, snoRNA,... Splicing/modification of RNA SÍRNA, miRNA,... Gene expression regulation MB 2 2023 39 Hoogsteen pairing - triplexes Hoogsteen pairing-G-quadruplexes Guanine quadruplexes GGGNnGGGNnGGGNnGGG • gene expression regulation • telomere structure transcriptional activation altered transcription (Huppert J.L, Chem Soc Rev, 2008) IV V (Biffi G., et al., Nat Chem, 2013) 8' VEGF-A (Brooks T. A., et al., FEBS J, 2010) 5' -UTR Exon 1 Intron 1 Exon 2 20% £ o 15% O = >C 5% Intron 2 Exon 3 3' -UTR -If- Regions 25,747 13,909 28,239 26,321 24.963 21,838 20,613 20.882 SUrV6yed l-TSS .Poly-A MB_2_2023 (Maizels N. and Gray L.T., PloS Genet., 2013) 250 bp 42 Base reactivity Hydrofobic bases with high ability to form hydrogen bonds are reluctant to be freely expressed into water environment around - if there is any chance to avoid this and lower the base exposition to the environment by any type of base pairing or base stacking, the bases tend to form a structure. Even the "single-stranded" RNA or DNA forms, in fact, compact structure with number of base pairs. MB 2 2023 43 Packing of DNA into chromosome CP- T At the simpl is a double-structure of simplest level, chromatin stranded helical DNA. ONA double helix DNA is complexed with histones to form nucleosomes. Each nucleosome consists of eight histone proteins around which the DNA wraps 1.65 times. Nucleosome core of eight histone molecules ... that forms loops averaging 300 nm in length. A chromatosome consists of a nucleosome plus the HI histone. Chromatosome The chromatosomes fold up to produce a 30-nm fiber... 30 nm . The 300-nm fibers are compressed and folded to produce a 250-nm-wide fiber. MB 2 2023 I Tight coiling of the 250-nm fiber produces the chromatid of a chromosome. 1400 nm 44 (Nature Education, 2013) Binding of DNA to a histone octamer linker DNA I-1 core histones of nucleosome \ "beads-on-a-string" nucleosome includes form of chromatin ~200 nucleotide pairs of DNA NUCLEASE DIGESTS LINKER DNA I released nucleosome core particle Vi nm DISSOCIATION WITH HIGH CONCENTRATION OF SALT octamenc histone core 147-nucleotide-pair DNA double helix J* DISSOCIATION 1 \ 50 nm H2A H2B H3 H4 Molecular Biology of the Cell (© Garland Science 2008) MB 2 2 Folding of nucleosomes into 30 nm fiber MB_2_2023 Molecular Biology of the Cell (© Garland Science 2008) 46 30 nm fiber binds to protein scaffold folded 30-nm fiber looped domain high-level expression of genes in loop histone modifying enzymes chromatin remodeling complexes RNA polymerase proteins forming chromosome scaffold Molecular Biology of the Cell (© Garland Science 2008) MB 2 2023 47 Chromosome One Chromosome (two identical Chromatides) O i Short Arm (p) Long Arm (q) Centromere - here are the chromosomes connected to the system of cellular microtubules - important for chromosome segregation during cell division Telomere - terminal part of chromatides that protect the end from being recognised as a double-strand break by a DNA repair machinery Molecular Biology of the Cell (© Garland Science 2008) MB 2 2023 Nature Reviews | Cancer 48 Chromosome Fully condensed chromosomes are present only during the cell division, otherwise they are more or less decondensed to a lower levels of structure, especially in transcriptionally active sites (euchromatin). Transcriptionally inactive parts of DNA, as well as repetitive regions or telomeres are much more condensed (heterochromatin). Various types of chromatin differ in epigenetic markers of both DNA (5-methyl cytosine) and histones (methylation a acetylation). MB 2 2023 49 Mycoplasma BACTERIA I ANDARCHAEA 10; I I I I I III 106 PROTISTS Arabidopsis PLANTS I Drosophila INSECTS I MOLLUSKS ■■■■■ shark CARTILAGINOUS FISH | Fugu zebrafish BONY FISH AMPHIBIANS REPTILES BIRDS human MAMMALS I I IIIIII I I IIIIII 107 108 I I I Mill 109 I I I Mill 10 10 I I I Mill 10 ii I I I II III 10 12 number of nucleotide pairs per haploid genome MB_2_2023 Figure 1-37 Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008) 50 Table 1-1 Some Genomes That Have Been Completely SPECIES ARCHAEH Sequenced HABITAT GENOME SIZE (1000s OF NUCLEOTIDE PAIRS PER ESTIMATED NUMBER OF GENES CODING FOR HAPLOID GENOME) PROTEINS Methanococcus jannaschii lithotrophic, anaerobic, hydrothermal vents 1664 1750 methane-producing Archaeoglobus fulgidus lithotrophic or organotrophic, hydrothermal vents 2178 2493 anaerobic, sulfate-reducing Nanoarchaeum equitans smallest known archaean; hydrothermal and 491 552 anaerobic; parasitic on another, volcanic hot vents larger archaean EUCARYOTES Saccharomyces cerevisiae minimal model eucaryote grape skins, beer 12,069 -6300 (budding yeast) Arabidopsis thaliana model organism for flowering soil and air -142,000 -26,000 (Thale cress) plants Caenorhabditis elegans simple animal with perfectly soil -97,000 -20,000 (nematode worm) predictable development Drosophila melanogaster key to the genetics of animal rotting fruit -137,000 -14,000 (fruit fly) development Homo sapiens (human) most intensively studied mammal houses -3,200,000 -24,000 Genome size and gene number vary between strains of a single species, especially for bacteria and archaea. The table shows data for particular strains that have been sequenced. For eucaryotes, many genes can give rise to several alternative variant proteins, so that the total number of proteins specified by the genome is substantially greater than the number of genes. MB_2_2023 Table 1-1 (part 2 of 2) Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008) 51 Levels of structure of biopolymers DNA RNA Protein Primary Secondary 43 .0 a—u a-u g-c , ,.ga, u gguau" u. I I I I I * u A O A A c u a 5> HEART INSTITUTE, NATIONAL INSTITUTES OF HEALTH Communicated t>ji Richard B. Roberts, October I, 1964 Previous studies utilizing randomly ordered synthetic polynucleotides to direct amino acid incorporation into protein in E. coti extracts indicated that RNA codewords corresponding to valine, leucine, and cysteine contain the bases (UUG).1-' The activity of chemically denned trinucleotides in stimulating the binding of a specific C"-aminoacyl-sRNA to ribosomes, prior to peptide bond formation,1 provided a means of investigating base sequence of RNA codewords and showed that the sequence of a valine RNA codeword is GpUpU.' 53 Properties of genetic code • genetic code is based on triplets - one aminoacid in protein is coded by a sequence of three nucleotides in DNA (RNA] Triplet = Codon x anticodon = complementary sequence on particular tRNA that carries the mRNA CGUGGUACGAUUGGAUGUL _i ■ . ■ i_._11_._11_ Protein Arg Gly Thr Me Gly CyS respective aminoacid • genetic code is universal - individual triplets code for the same aminoacid in almost all organisms (x mitochondria) CGU = Arginine CGU = Arginine CGU = Arginine • genetic code is degenerated - one aminoacid might be coded by several different triplets (but the opposite is not true) CGC Arginine MB 2 2023 AGA 54 Genetic code Second nt First nt U C A G Third nt Phe Ser Tyr Cys U U Phe Ser Tyr Cys C Leu Ser STOP STOP/Sel A Leu Ser STOP Trp G Leu Pro His Arg U C Leu Pro His Arg C Leu Pro Gin Arg A Leu Pro Gin Arg G Thr Asn Ser U A lie Thr Asn Ser C Thr Lys Arg A Met/START Thr Lys Arg G Val Ala Asp Gly U G Val Ala Asp Gly C Val Ala MB 2 Ala Glu 2023 Glu Gly A Val Gly G Reading frames Genetic code is based on triplets - three possible ways of reading (reading frames), but only one is correct. mRNA CGUGGUACGAUUGGAUGU 1—i—1 ■—i—■■—i—"—i—> >—i—"—i—> Proteini Arg Gly Thr Me Gly Cys mRNA CGUGGUACGAUUGGAUGU '—i—>'—i—m—i—"—i—-'—i—"—i— Protein2 Val Val Arg Leu Asp mRNA CGUGGUACGAUUGGAUGU •—i—• ■—i—n—i—"—i—1'—i——i Protein3 Trp Tyr Asp Trp Met MB 2 2023 56 Genetic code Although the genetic code is universal, the usage of particular codons, as well as the amount of particular tRNAs and aminoacyl transferases differ Modification of synthetic genes for recombinant protein production according to the expression system used (Bacteria, human,...) might be highly beneficial. MB 2 2023 57