Molecular Biology II Department of Molecular Pharmacy Mgr. Marie Brázdová, Ph.D. (FAF MUNI, BFU AVČR) Mgr. Marek Petr (FAF MUNI, BFU AVČR) Mgr. Daniel Renciuk, Ph.D., (BFU AVČR) PharmĎr. Jakub Treml, Ph.D. (FAF) brazdovam@pharm.muni.cz, petrmpharm.muni.cz. The structure and function of biopolymers during the transitions of genetic information Daniel Renciuk 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 rough nonpathogenic mutant bacterium O o o o live R strain cells grown in presence of either heat-killed S strain cells or cell-free extract of S strain cells TRANSFORMATION i «•I S strain Some R strain cells are transformed to S strain cells, whose daughters are pathogenic and cause pneumonia CONCLUSION: Molecules that can carry heritable information are present in S strain cells. (A) S strain cells i r fractionation of cell-free extract into classes of purified molecules 1 1 RNA protein DNA lipid carbohydrate 1 1 1 1 1 1 1 molecules tested for transformation of R strain cells o° wo R R strain strain s strain °o R strain R strain CONCLUSION: The molecule that carries the heritable information is DNA. (B) Molecular Biology of the Cell (© Garland Science 2008) MB 2 2022 0. Avery C. MacLeod M.McCarty STUDIES ON THE CHEMICAL NATURE OF THE SUBSTANCE INDUCING TRANSFORMATION OF PNEUMOCOCCAL TYPES ItTDCcnoN or Tsanstoimatton >y a Desoxwbomucxek: Acid Fraction I So lai ED FltOH PSBUMOCOCCUB TvtT HI By OSWALD T. AVKRY, M.DW COLIN M. MacUOD, MP,. a*d MACLYK McCAKTY,' M-D. {Frtm tkc Htnfiui of The Rx\«» - — «1 ■ktffaSNm POUdmltHHl ptiMniMKa i-fvf fJLM inm «X mi iMUJG iau>m mm cadaaa iUi ■ Tilt f https://www.ncbi.nlm.nih.gov/gene/5460 Go to nucleotide: Graphics FASTA GenBank NC_000006.12: 31M..31M (S.3Kbp] C " | Find: 131,171,500 131,171 K 131,170,503 131,170 K I X Tools T I íi Trads - 31,185,500 31,189 K 131,188,500 131,188 K 131,187,500 131,187 K 131,188,500 131,188 K 131,185,500 131,185 K 131,184,500 131,184 K ISiJi Senes, NCEI Heme sapiens Ännctaticn Release 108, 2016-06-07 M1.002701.S NP_B02692.2: P0U A 5' UTR 4 Regulatory regions Promoter + enhancers UU L±U P0UEF1 N 11.001173531.2 NM_203289.E W1_001285987.1 WL001285986.1 Introns = noncoding regions Exons = coding regions 5837.1: t 2: tran >)P_08107l8979.1: t HPJ013E 5: ■IP J090Í 8.2 HPJ01167002.1: POU.. NP.976034.4: POU d... NP.001272916.1: POU.. NP_B01272915.1: POU.. Genes Most eukaryotic genes contain introns, that are transcribed into primary RNA transcript and introns are consecutively removed by splicing process on spliceosome to form final mRNA. Intron = noncoding region Exon = coding region Prokaryotic genes do not contain introns and they are directly transcribed into mRNA that serves as a template for translation into protein. Exon = coding region MB 2 2022 10 Nucleic acids NUCLEOTIDE,-' V NUCLEOSIDE Phosphate 5\carbon sugaf NH. j Nitrogenous base M6-2-20M- 11 5-carbon sugar - pentose (3-D-2-deoxyribose MB 2 2022 (3-D-ribose 12 Base N 2^. 6 7 5 8 3 \ H Purine Adenine Guanine Pyrimidine H o N Chi H Cytosine Thymine MB 2 2022 O H. N O^N H Uracil 13 Nucleic acid nomenclature Base Nucleoside Nucleotide NTP G Guanine Guanosine Guanosine triphosphate A Adenine Adenosine Adenosine triphosphate T Thymine Thymidine Tymidine triphosphate C Cytosine Cytidine Cytidine triphosphate U Uracil Uridine Uridine triphosphate MB 2 2022 14 Modified bases tRNA o x HN^NH O O pseudouracil HN I R dihydrouracil Oxidative damage NH O 8-oxo adenine 8-oxo guanine Metabolism Epigenetics xanthine < hypoxanthine o NH, N N I R CH, NH "N 2 yOH CH, inosine MB_2_2025-methyl cytosine 5-hydroxymethyl cytosineis Conformation of N-glycosidic bond oh nh, ■ n oh oh OP ISO" 0° 120" ISO" Figure 2-8 The glycosidic torsion angle ^ is defined by 04- C1-N9-C4 for purines and 04'-Cl'-Nl-C2 for pynmidines When ^ = (T the 04'-Cl' is eclipsed by the N9-C4 bond in purines and by the N L-C2 bond in pyrimidines. The syn conformations correspond to 0° ± 90"; anti conformations correspond to 180" ± 90". In nucleotides steric hindrance limits the conformations actually found to a much narrower range of angles thai depend on sugar pucker and base. The syn MB ^nl^j^tions are usually found with x — 45° ± 45*; anti ^oTllbrmations are usually found with X — -135^ ± 45°. Formation of sugar-phosphate backbone 5' end (phosphate)fho—p—o—\ O^ "N' o N NH2 NH2 Phosphodiester bond 0 o II II HO-P-O-P-OH 1 l OH OH 000 ii HO—P—O—P—O—P—O—i 1 i i OH OH OH REPLICATION DNA-de pen dent DNA polymerase TRANSCRIPTION DNA-dependent RNA polymerase REVERSE TRANSCRIPTION RNA-dependent DNA polymerase NHc ,0. OH MB 2 2022 17 Base reactivity - hydrogen bonds Hydrogen bond - weak electrostatic interaction of two polar groups - one covalently binds hydrogen (DONOR - usually -N-H a -0-H); the second (ACCEPTOR) is usually N or 0 Length: ~ 2.8 A (2 - 3,4 A) Energy: < 1 kcal/mol both depend on particular atoms and on Watson-Crick base pairing TEST 2 hydrogen bonds hydrogen bonds Chargaff rules: = XT = ZC Pu = I Py MB 2 2022 19 DNA double helix MB 2 2022 DNA double helix • two molecules (strands) of DNA • the helix is right-handed • the strands are antiparallel - their 5'-3' direction is opposite in the context of double helix direction • similar content of purines and pyrimidines; content of A = T, G = C (Chargaff rules) • result - the strands are complementary - i.e. according to the Watson-Crick base pairing rules we can predict the sequence of one strand according to the sequence of the other • on average the double helix contains 10,5 base pairs per turn of the helix, which is about 3,4 nm in length MB 2 2022 21 DNA structure -Watson and Crick model F. Crick J.D. Watson M. Wilkins „.. April 2">. na N \ I r H K W •t, trnul l*i I--. i. K It |*m»m» mml %tm mi m m «t NJUi. ^■■in> // n.f ,.»<*- . ». MMNl M . MM W »W IN m i. Mi* r*v»t» r»r*. ■ n i |M».M » »-- MM f*—) «■■!<>■■ ft.llM.MJ >IECULAR STRUCTURE OF NUCLEIC ACIDS StfwLtiKe for Droxyriboa* Nuclm Anil i 1» iiik • m>mi aW Mm mJi •><•>.••» MMa-» m«( iD.VAl. Tkta i m..H btWlIM »liirll a~ .if analpnU. TW Mi .« ftaatjal 7» III* null laaua m*r> J 4 V m il» »-lir>-♦« i«i. mkihI •■■ ««■» w »r Imm •f M Ike mmm liiliili •> IM 1W U MW 10 will inai 11 n*+x I !■ lkal TIm iImimpw m! • jihMfrfniriM miwi TI- mi^Ui.. liVtr V** j Ilr, KaulM,, 'Vfi'iilimT mTTTni ■«» »ic » i>.n- • ~» «l~ ~— «• rw.t >hi im» nai tn Im m»W ill. » M Ml MV HIIImAi Mai ill*! mmm) MB iJM IÍIMmI UMBflM* l*y fcllpWMMI UwVM illM M m rww i»*ai«"L «1 (.» I KM WMill •* iImH M4 "MIMMlW Ma at. w- »'M% In MU i ♦i»»rl • nwl»«H| .llll.~r alM|ftlU« t<1 Franklin's X-ray photograph shows Jlak ^.m-Tf DNA's B'-form (1952) • MMt, *-u «i*)t*it *.1mml. it***. \m tUm»* mmmjm*ii* n* lit* *^H*>r wwinbir nun U> ifiyinm** | •uttiUrtr Jur ptnuu Ma*! iyOi—f, Tím mmjmwmm 4 hmmm Ml if — . • •( [• v- . J I.I • !I (I HM (<»MR <*•*•*•! i< U*** (kr MM* Hl faltÉtta* *•.*»*•« W» 1" MMI? ■ itl M I It. lit* ml MntthlM. lit. II t*l~ ritr I i llwi **v M*lW ImIImtnc* .-T tlv ilt-MtU M* •WMl «JRM IlkMNrt i II Hi I **> W**(«M IM*M m i - i •»* )***•• •mm.i^U*..! ir*-f**»!.••••)> i gMMjJi >*XT "*«V '***" llMMMU ti*t I flti MMM* MIMMiml i'.l'l ilnlJU III tf(* *>«niltHM, UM'l>*>1l«kf ti*» •**«• dá MM mwii I U iNi.l.lltia it, IriaHTMT Will. • M* |a*f aaa>«J*M|m*laM« ll*» lllft Wad ImV ||alWWWn ^IIMWIMIW ill I NtMt WW MT IM. M II I Wt!.lt>*>. I**- II II rTMtklu. *M*I ll«*M# *♦*■*»»ili...*> Ml 2 2022 22 DNA structure - Pauling model am..uisritY. r.ituvi. i\r> ciwf.i r*« n a a »»1Kb are inwiheet in eater bnluiKra. Thu iliitnrtxwi t| the !.hi/.|ihan (roup Im the regular I. trabr.tr.I (■••iitiguratifii rt not aunported by direct rxpmmrnul rvjdelicr. unfortunately no |urw«e ■tructure detcrtmiiatioii* have brni mailt of any |Jvi»phat«- di-caten. The dtaturtuti, which rur nrapond* to a Urgcr auiuunt u4 double bund rh.ir^ti i for the murr oxygen I than for the oxygen atortu involved in thr i-»trr linkage*, i* a rrawm Vi -I I Jx Linus Pauling A PROPOSED STRICTURE FOR THE NUCLEIC ACIDS By Linus Pauling and Roiiert B. Corby Gatbs anu Ckkli.in ! in .k v.. ih-.i -. iif CiiHMisrav,* Caliporma Instititb nr Tbchnolouv CfMiiiuuiik-atL-il Dt$a*nhtf 31, 1952 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 ami growth, that they participate in the transmission of hereditary characters, and that they are important constituents of viruses. An understanding of the molecular structure of the nucleic acids should be of value in the effort to understand the fundamental plic- Han «4 tW aurkV MM .Irwrtiirr. «ho«*«f «•>««! «ikwMan able our. and thr ..wuuird .balance* are tho*r indicated »»>■ the value* loe somewhat aimiUr lulwUnrrv raprcrallt the ring SaUt, in winch rarh aulfur atom ta aurnanvlrd bv a tetrahedron ol (our oivr.ru iit..mv two iA whvh are ibaml with adjacent ttlruhedra, and two uittbarrd The i) < i drttance* within the phmphate telrahedroei an 2.:C A (between the two inner oxygn atom*). 2.4« A, '.* &\ A. and 2.U0 A. The MB 2 2022 23 Various types of double helix B-DNA A-DNA Z-DNA • DNA in water/salt soulutions • DNA in crowding solutions • CpG sequences in crowding conditions • RNA MB 2 2022 24 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 2022 26 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) 27 Stability of DNA double helix T ^m = melting temperature hydrogen bonds AT = 2 x GC = 3 base stacking various repulsion of backbone phosphates Mg2+>Na+ Tm increases with GC and length Tm increases with length and ions Tm increases with ions MB 2 2022 28 Base-pair parameters in double helix Shear(Sx) Shift (Dx) Tilt(r) Stretch (Sy) Propeller (ir) Slide (Dy) Roll 0») Stagger (Si) Opening [a) Lu et al., 2003, NAR Rise (Dz) MB 2 2022 Twist (u.) 3' 5' 5' I 3' Coordinate frame Types of nucleic acids • linear (human chromosome) x circular (bacterial genome) • single-stranded (most RNAs) x double-stranded (human DNA) MB 2 2022 30 Superhelicity Overwound topological domains tomn compact large scale Chromat»! structures Underwound topological domains have a decompacted large-scale structure 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 2022 31 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 2022 32 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 Q h r Asparagine (or glutaminc) ,CHa ^H-"-0*"'rJ'",H I Asparagine (or glutaminej H Hi I c; 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 2022 33 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 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 2022 34 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 2022 35 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 2022 36 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 2022 37 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 2022 38 Loops and hairpins in RNA 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 2022 40 Hoogsteen pairing - triplexes • gene expression regulation ^u ^u ^Y ^Y ^u ^Y R / Hoogsteen pairing-G-quadruplexes Guanine quadruplexes GGGNnGGGNnGGGNnGGG • gene expression regulation • telomere structure promoter gene H 1 1 l \ 9 0 l V \ c_y transcriptional activation altered transcription (Huppert J.L, Chem Soc Rev, 2008) IV V (Biffi G., et al., Nat Chem, 2013) 4 ýv-/x . - • - - (Brooks T. A., et al., FEBS J, 2010) 5" -UTR Exon 1 Intron 1 Exon 2 E Intron 2 Exon 3 3' -UTR TL Regions 25,747 13.909 surveyed 28,239 26,321 fTSS 24.963 21,838 Poly-A MB_2_2022 (Maizels N. and Gray L.T., PloS Genet., 2013) 20.882 250 bp 43 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 2022 44 Packing of DNA into chromosome At the simplest level, chromatin is a double-stranded helical structure of DNA. ONA double helix v1 ,V 1 ,/||r I Each nucleosome consists of eight histone proteins around which the DNA wraps 1.65 times. A chromatosome consists of a nucleosome plus the HI histone. The chromatosomes fold up to produce a 30-nm fiber... The 300-nm fibers are compressed and folded to produce a 250-nm-wide fiber. MB 2 2022 Tight coiling of the 250-nm fiber produces the chromatid of a chromosome. 1400 nm 45 (Nature Education, 2013) Binding of DNA to a histone octamer linker DNA I-1 core histones of nucleosome \ "beads on a string" form of chromatin NUCLEASE DIGESTS LINKER DNA nucleosome includes -200 nucleotide pairs of DNA I released nucleosome core particl Ti ? e 11 nm « Jl i DISSOCIATION WITH HIGH CONCENTRATION OF SALT octameric histone core 147-nucleotide-pair DNA double helix DISSOCIATION 50 nm ( 1 I \ w *r H2A H2B H3 H4 Molecular Biology of the Cell (© Garland Science 2008) MB 2 2' Folding of nucleosomes into 30 nm fiber MB_2_2022 Molecular Biology of the Cell (© Garland Science 2008) 47 30 nm fiber binds to protein scaffold MB_2_2022 Molecular Biology of the Cell (© Garland Science 2008) 48 Chromosome One Chromosome (two identical Chromatides) Molecular Biology of the Cell (© Garland Science 2008) 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 Nature Reviews | Cancer MB 2 2022 49 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 2022 50 Amoeba PROTISTS ^■■■HHHHHHHHtH Arabidopsis bean lily fern number of nucleotide pairs per haploid genome MB_2_2022 Figure 1-37 Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008) 51 Table 1-1 Some Genomes That Have Been Completely Sequenced SPECIES SPECIAL FEATURES HABITAT GENOME SIZE ESTIMATED (1000s OF NUMBER NUCLEOTIDE OF GENES PAIRS PER CODING FOR HAPLOID GENOME) PROTEINS ARCHAEA Methanococcus jan nasch ii 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_2022 Table 1-1 (part 2 of 2) Molecular Biology of the Cell, Fifth Edition (© Garland Science 2008) 52 Levels of structure of biopolymers DNA RNA Protein Primary Secondary 43 .0 A-ll /> u A—j M lf~cuau*u<> w4 C- -0« 5>uM B. ftoWu. iw*r I, IMi PrcvwuR ttudira uliliiuur. randomly ordered «ynlhcUe pnlvniiclnntida* to direct unixw mad incorporation into protein in E. ccti extracts indicated that RNA codeword- correeounduis to valine, leucine, and cysteine contain the hasra (I'UG).'-* The activity of ehemieajly dennod tnnucWollde* in atiimilatin* the binding of a tprcinc i' •-aiiiinnaryUKNA to riTKurwiiKA, pfkif to peptide bond formation,' provided a mean* of investigating baas aaooanca of RN'A codrwordR and •tviuol that tbe sequence of a valine RNA codeword ii GpUptJ.* 54 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 2022 AGA 55 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 2022 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 2022 57 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 2022 58