MUNI SCI Bi4025en Molecular Biology Mgr. Jiří Kohoutek, Ph.D. 1 Department of Experimental Biology Lecture 5 • Translation of prokaryotic and eukaryotic mRNAs. 2 Department of Experimental Biology MUNI SCI Sickle eel anemia Substitution of one amino acid in the molecule of Hemoglobin. Loss of function protein/cell/organism 3 Department of Experimental Biology MUNI SCI Sickle eel disease - anemia • In year 1904: • Doctor James Herrick's office in Chicago visited by a patient of African origin with an enlarged heart. • Complains of weakness, fatigue and dizziness. • Herrick diagnoses an anemia. • In the blood sample erythrocytes of unusual sickle-shaped appeared. • For the first time hypothesizes that sickle cell red blood cells are the cause of anemia. Department of Experimental Biology Sickle cell anemia - middle age • In 1927: evidence that the change in shape of normal red blood cells to sickle cell is related to their impaired function - oxygen transport in the body. • In 1949: Linus Pauling, formulates a hypothesis, that the cause of sickle cell anemia could be abnormal form of hemoglobin. • Hypothesis successfully verified by electrophoretic techniques. • In 1954 he was awareded by the Nobel Prize in Chemistry. Linus Pauling (1901 - 1994) 5 Department of Experimental Biology MUNI SCI Sickle cell anemia - middle age • In 1954: Vernon Ingram (1924-2006). • Hemoglobin from healthy (A) and sickle cell (S) cells cleaved by trypsin into fragments that have been divided by electrophoresis and peptide mapping found one different peptide. • Trypsin breaks down proteins after lysine and arginine, unless they are followed by proline. • In the AA sequence of B-chains of hemoglobin the single mismatch was found: valine (hydrophobic) replaced glutamic acid (sixth AA from N-ends). Hydrophobic valine causes clumping of molecules hemoglobin and deformation of red blood cells. 6 Department of Experimental Biology Hemoglobin A (HbA) Hemoglobin S (HbS) f Digest withl % PEPTIDE FRAGMENTS Map .peptides HbA PEPTIDE FRA 1 2 3 4 5 N-©®®@(^) GME 6 IT 7 8 HbS 1 2 3 4 5 MUNI SCI One mutation can severely impact protein function EP im U c A G u uuu fenylalanin UCU serin UAU tyrosin UGU cystein uuc fenylalanin ucc serin UAC tyrosin UGC cystein UUA leucin UCA serin UAA stop UGA stop UUG leucin UCG serin UAG stop UGG tryptofan c CUU leucin ecu prolin CAU histidin CGU arginin cue leucin ccc prolin CAC histidin CGC arginin CUA leucin CCA prolin CAA glutamin CGA arginin CUG leucin CCG prolin CAG glutamin CGG arginin A AUU izoleucin ACU treonin AAU asparagin AGU serin AUC izoleucin ACC treonin AAC asparagin AGC serin AUA izoleucin ACA treonin AAA lysin AGA arginin AUG metionin ACG treonin AAG lysin AGG arginin G GUU valin GCU alanin GAU kys. asparagovä GGU glycin GUC valin GCC alanin GAC GGC glycin GUA valin ■4- GCA alanin gaj \ kys. glutamovä GGA glycin GUG valin GCG alanin GA< GGG glycin H H O I I II threonine — proline-N — C — O-lys J_ CH2 I CH2 glutamic add O II ■c ■ H H O I I II threonine — proline — N — C — O — [ys _L H3C — CH — CH3 valine Normal hemoglobin Sickle cell hemoglobin 7 Department of Experimental Biology https://www.khanacademy.org/science/biology/macromolecules/proteins-and-amino-acids/a/orders-of-protein-structure MUNI SCI Sickle cell disease - pathophysiology Because the glutamic acid-to-valine amino acid change makes the hemoglobin molecules assemble into long fibers. The fibers distort disc-shaped red blood cells into crescent shapes. The sickled cells get stuck as they try to pass through blood vessels, which impairs blood flow leading to serious health problems for patients with sickle cell anemia, including breathlessness, dizziness, headaches, and abdominal pain. 8 Department of Experimental Biology https://www.khanacademy.org/science/biology/macromolecules/proteins-and-amino-acids/a/orders-of-protein-structure Sickle cell disease - genetics 9 Department of Experimental Biology • Why has sickle cell anemia not been eliminated by selection pressure? • Because the selection pressure contributes to the maintenance of the mutant gene in the population against another factor. • Heterozygous individuals, one "healthy" and one "sickle cell" from allele manifest a weaker form of anemia. • At the same time they show a higher degree of resistance to the malaria agent (protozoan Plasmodium) than healthy homozygotes. • The highest incidence of sickle cell anemia is in malarial areas. MUNI SCI Protein structure • Proteins are composed of 20 different essential amino acids. • Accounts for 15% of the native weight of cells. With the exception of water, constitute the main structural component of living organisms. • Participate in the structure and function of their bodies. 10 Department of Experimental Biology MUNI SCI Amino acids H-N—C—C-OH • Proteins consist of polypeptides, polypeptides of amino acids (AA). |^ • In the polypeptide amino acids are joined into H 7 0 strand by covalent bonds. , „ J • There are 20 amino acids; all have a free amino group (NH2) and free carboxyl group (COOH). gjj | c^°uxpyl • Amino acids are distinguished from each other by p lateral groups (R). • The nature of the substituent R varies considerably, it could be a hydrocarbon group, OH, SH, SCH3, COOH, or NH2. • The number of AA combinations in different polypeptides is huge (e.g. for a peptide composed of 7 AK, there are 207 combinations). 11 Department of Experimental Biology Journal of Hygienic Engineering and Design, UDC 577.388:542.949.41]:641.12 MUNI SCI Chemical proterties of lateral groups od AA • Lateral groups of amino acids are source of their structural and functional diversity. 4 types: • hydrophobic (non-polar), • hydrophilic (polar), • acidic (negatively charged), • basic (positively charged). NON-POLAR V p h,n"- C - C H,N*-C-C V ,° H,N*-C-C h,N*-C-c' i h ° H,N*-C-c' CH, "D CH V X X CH, CH, 0 CH, sh 1 1 Cr h,C CH, X / CH, Glycine Alanine Valine Cysteine Proline (Gly ÍG) {Ala / A) {Valí V) (Cys'C) (Pro IP) V M H h,n*-C-C^ V * H,N*-C-c' 3 LV CH, H,N--C- C H,N--C- C CH, '° h,C - CH ř CH, CH, 0 CH /X CH, CH, CH, 1 CH, S I CH, Ó Leucine Iso leucine Methionine Tryptophan Phenylalanine (Leu i L) (IIb/ I) (Met/M) (Trp/W) (Pttef F) POLAR h,n*-C-C' H,N*- C-C L o- H,H--C-C V Z3 H,N*-C-c' H,n4—C— C ch, x° CH; CH, V oh CH ° y X OH CH, 9 oh nh, o Ä nh, 0 Serine Threonine Tyrosine Asparagine Glutamine (Ser/ S) (Thr/T) (TyríY) (Asn/N) (Gin /Q) ____1 1 H.n--C - C Lysine [Lys / K) ARGE H,fJ'-C-C C = nh,* nh, Arginine (Arg/R) h,n'-C-C, Histidine (His/ h) r - CHARGE I H,N*-C-C^ CH, A H,N*-C-C^ CH, CH, I Ax o- o AsparticAcid Glutamic Acid (Asp/D) (Glu/E) 12 Department of Experimental Biology https://ib.bioninja.com.au/standard-level/topic-2-molecular-biology/24-proteins/amino-acids.html MUNI SCI Chemical proterties of lateral groups od AA h3n+ H V Nonpolar side chains Glycine (G) Gly TO" CH3 Alanine (A) Ala T ^° H3N+-C-cf CH V H3C/ NCH3 Valine (V) Val h3n+- I ch h3cx \h3 Leucine (L) Leu H3N+- h3n + Polar side chains s I ch3 Methionine (M) Met ľ ,0 H3N+-C— (T H3n + h -U I ch2 h3n+- Phenylalanine (F) Phe H HjN+-C-C' CHj c-<° 1 V H3C—CH u b. CH3 Isoleucine II) lie H1-í- HjN+- 1 -c—c H3N+— C—C7 1 N0- 1 CH2 n CH2 CH2 H2 fH2 1 NH CH2 C = +NH2 +MH3 NHj Lysine (K) Arginine !R) Lys Arg In addition, there are two AA Selenocysteine - has selenium instead of sulfur. H I HjH—C — COOH í CH2 CYSTEINE I I I H^N —C—COOH CH2 SELENOCYSTEINE Pyrrolysine - derivative of lysine with an attached pyrroline ring. PYRROLYSINE Figure 3-5 Biological Science, 2/e Department of Experimental Biology Histidine (H) His 3 2005 Pearson Prentice Hall, Inc. https://ib.bioninja.com.au/standard-level/topic-2-molecular-biology/24-proteins/amino-acids.html https://kevintbeel.com/2017/04/19/three-acronym-mnemonics-for-remembering-the-amino-acids/ https://www.sciencedirect.com/topics/medicine-and-dentistry/selenocysteine MUNI SCI Peptide bond Amino acids are linked in peptides by covalent peptide bonds. • Peptide bond - also referred to as an amide bond, is formed between the amino group (a-nitrogen atom) of one amino acid and the carboxyl group (carbonyl carbon) of its neighbour, releasing water molecules at the same time. The process is also called - condensation. f^-C-C G-N-C-H H H R? H First amino acid Second amino acid □ipepiide 14 Department of Experimental Biology https://www.sciencedirect.com/topics/engineering/peptide-bond MUNI SCI Levels of protein structure 4 levels of organization of protein structure: • Primary structure: determined by amino acid sequence (encoded by gene). • Secondary structure: refers to local folded structures that form within a polypeptide due to interactions between atoms of the backbone. • Tertiary structure: the assembly of the polypeptide in three-dimensional space. • Quaternary structure: results from the union of two or more polypeptides into a multi-subunit protein. 15 Department of Experimental Biology https://quizizz.com/admin/quiz/606682e1825246001f488abd/dna-structure-function-and-replication-basics MUNI SCI Primary structure of protein Pi ;,■ dt bond The linear sequence of amino acids within a protein is considered the primary structure of the protein. HO 6- 9k HO H 1 R2 I .CH O II ,C. N terminus Rs polypeptide chain Amino -lOtl Amino dCKJ ln the polypeptide chain-protein the left hand amino acid is called the N-terminus, and the right hand amino acid is called the C-terminus. 16 Department of Experimental Biology https://www.nature.com/scitable/topicpage/protein-structure-14122136/ https://socratic.org/questions/57c278efb72cff0d7b97f247 MUNI SCI Secondary structure of protein Secondary structure, refers to local folded structures that form within a polypeptide due to peptide bonds between neighbor amino acis. interactions between atoms of the backbone. The secondary structure does not involve R group atoms. The most common types of protein secondary structures are the a helix and the (3 pleated sheet. Both structures are held in shape by hydrogen bonds, which form between the carbonyl O of one amino acid and the amino H of another. p-pleated sheet o li Y y y li v Y Y 11 ,Č-N'C'»C-Nsc,C-N''CNC-NNC<.C-N''Csč-Ňvc^C 11 I [J I I ' Jí I I I HH R ^HH R rtHH R H H I H H I I H II H H H H .? H I I I -C'^N-Cvc^N-C'^N-Csc^-C-'^N-Cvc^N-C I H H H H H H II O H II^N I R H H II " C N i I R H 17 Department of Experimental Biology https://www.khanacademy.org/science/biology/macromolecules/proteins-and-amino-acids/a/orders-of-protein-structure MUNI SCI Secondary structure of protein - a - helix • a helix - the carbonyl (C=0) of one amino acid is hydrogen bonded to the amino H (N-H) of an amino acid that is four down the chain. (E.g., the carbonyl of amino acid 1 would form a hydrogen bond to the N-H of amino acid 5.) This pattern of bonding pulls the polypeptide chain into a helical structure that resembles a curled ribbon. • Each turn of the helix containing 3.6 amino acids. • The R groups of the amino acids stick outward from the a helix, where they are free to interact. 18 Department of Experimental Biology https://www.khanacademy.org/science/biology/macromolecules/proteins-and-amino-acids/a/orders-of-protein-structure https://cbm.msoe.edu/teachingResources/proteinStructure/secondary.html MUNI SCI Secondary structure of protein - p pleated sheet • (3 pleated sheet - two or more segments of a polypeptide chain line up next to each other, forming a sheet-like structure held together by hydrogen bonds. The hydrogen bonds form between carbonyl and amino groups of backbone, while the R groups extend above and below the plane of the sheet. (a) polypeptide haekhone https://www.mun.ca/biology/scarr/iGen3_03-01 .html 19 Department of Experimental Biology https://www.open.edu/openlearn/science-maths-technology/science/biology/proteins/content- section-1.3.21 MUNI SCI Secondary structure of protein - p pleated sheet • In parallel (3 pleated sheet, polypeptide strands run in the same direction (i.e. from N to C-terminus). • In antiparallel (3 pleated sheet, neighbouring strands extend in opposite directions. (al Aiitiparalkl 20 Department of Experimental Biology https://www.mun.ca/biology/scarr/iGen3_03-01.html MUNI SCI Tertiary structure of protein Tertiary structure is the complex looping and folding that occurs as a result of interactions and bonding between portions, amino acids, of the protein that are farther apart. Tertiary structure is primarily due to interactions between the R groups of the amino acids that make up the protein. Amino acids with nonpolar, hydrophobic R groups cluster together on the inside of the protein, The hydrophilic amino acids cluster on the outside, in order to interact with surrounding water molecules. aortic bond between ionised R-grojps -FT----F*- Disulfide bond between R-groups containing -SH grojps Amino acids with hydrophobic side chains Amino acids wilh hydrophilic side chains 21 Department of Experimental Biology https://www.lecturio.com/concepts/post-translational-protein-processing/ https://slideplayer.com/slide/10879273/ MUNI SCI Tertiary structure of protein • Tertiary structure is mainly stabilized by non-covalent and covalent bonds. • Ionic bond between chemical groups with opposite charges. • Hydrogen bond: between partial electronegative atoms negative charge and hydrogen atoms. • Hydrophobic interactions: between non-polar groups. • Van der Waals interactions: between atoms in close proximity, weak (1/1000 of the covalent bond strength), but important for maintaining conformation. • Metallic - Fe2+. • Single type of covalent bond- disulfide bridges. 22 Department of Experimental Biology Ionic bond Hydrogen bond Hydrophobic bond Disulfide bridge Van der Waals interaction MUNI SCI Quaternary structure of protein HEMOGLOBIN Iron Polypeptide chain Heme Oxygen molecules • Many proteins are made of a single polypeptide chain and don't become any more complex than their tertiary structure. • Some proteins are made up of multiple polypeptide chains, also known as subunits. When these subunits come together, they give the protein its quaternary structure. • One example of a quaternary protein structure is hemoglobin. Hemoglobin is made up of four polypeptide chains, and is specially adapted to bind oxygen in the blood. https://www.khanacademy.orq/scie^ MUNI 23 Department of Experimental Biology acids/a/orders-of-protein-structure SCI https://biologydictionary.net/protein-structure/ Translation • Translation of genetic information from mRNA into amino acid sequence using the genetic code. Components of the translational apparatus: o mRNA o 20 standard amino acids (+ selenocysteine, pyrrolysine) o tRNA o 20 enzymes for amino acid activation (aminoacyl-tRNA synthetase) o Ribosomes o soluble protein series: translational factors IF, EF, RF-ATP, GTP o the translational system represents the dominant part of the metabolic apparatus of the cell. MUNI 24 Department of Experimental Biology r> r» t Translation overwiev 25 Department of Experimental Biology Codon Ribosome Copyright © Pearson Education, lne. publishing as Benjamin Cummings http://uoitbiology12u2014.weebly.com/protein-synthesis-and-genetic-code.html MUNI SCI Genetic code • The genetic code is a set of rules defining how the four-letter code of DNA is translated into the 20-letter code of amino acids, which are the building blocks of proteins. • Each AK is determined by a codon in mRNA. • Each codon is defined by a triplet of nucleotides. • Triplets/codons do not overlap. • 64 possible codons: 61 determine amino acids + 3 termination of translation. • Codons in mRNA are recognized (and temporarily bound on the basis of base pairing) by complementary sequences, anticodons, in tRNA. • Amino acids are carried by specific tRNAs. 26 Department of Experimental Biology https://www.nature.com/scitable/definition/genetic-code-13/ Genetic code • Is degenerate - one amino acid can be encoded by multiple codons (redundance). • These synonymous codons are included in codon families. • Three codons are meaningless (stop codons, termination codons): UAA-ochre, UAG - amber, UGA-opal. • 3 codons are bifunctional: o UGA- opal = meaningless or encodes selenocysteine o UAG - amber = meaningless or encodes pyrrolysine o AUG = initiating or encoding methionine • Is universal - most codons have the same meaning in all living systems (prokaryotes, eukaryotes, viruses). 27 Department of Experimental Biology https://basicmedicalkey.com/s-phase-and-dna-replication/ MUNI SCI Cracking the genetic code Work by Crick and coworkers showed that the genetic code was based on non-overlapping triplets of bases, called codons. H. G. Khorana, R. Holley and M. Nirenberg and others deciphered the encoding the meaning of all codons in 1966. If / H. G. Khorana R. Holley M. Nirenberg In 1968 H.G. Khorana R. Holley M. Nirenberg were awarded by the Nobel Prize in Physiology or Medicine. •" GTGCATCTGACTCCTGAGGAGAAG *" nWA ••• CACGTAGACTGAGGACTCCTCTTC UNM GUGCAUCUGACUCCUGAGGAGAAG (transcription) , RNA 111 II 111 protein --V II L T P C E K 28 Department of Experimental Biology https://www.researchgate.net/publication/282279062_Computational_Statistics_for_the _ldentification_of_Transcriptional_Gene_Regulatory_Networks MUNI SCI Genetic code is formed by triplets of codons Insertion/deletion of one/two base pairs changes the reading frame. dna atg ttt ccc a a a ggg ttt.....ccc ta g '(alela standardního typu) jfá £aa ggg ŤŤf ccc a a a ggg atc inzerce jednoho páru bází í transkripce f 3GG UUU,.....CCC, UAG i translace protein Met - Phe - Pro — Lys - Gly - Phe..... Pro - (konec) A ___včleněný pár bází - mění čtecí rámec dna ATG ATT TCC CAA AGG GTT T.....CC CTA G (mutantníalela) JAC TAA AGG GTT TCC CAA Á GG GAT C transkripce f \GG GUU U.....CC CUA G_ translace 1 protein Met - lle - Ser - Gin — Arg — Val - ..... - Leu - GC i změněná sekvence aminokyselin 29 Department of Experimental Biology fenotyp standardního typu mutantní f* fenotyp MUNI SCI Genetic code is formed by triplets of codons The suppressor mutation cancels or reverts the effect of the original mutation and restores the reading frame. DNA (mutantni alela) supresorova mutace (delece jednoho páru bázi) mRNA protein DNA dvojnásobně mutantni alela revertanta 30 Department of Experimental Biology - včleněný pár bází ~ méni čteci rámec AT G ATT TCC CAA AGG GTT T- jŤÄČ JÄÄ AGG GTT j[ČČí ČÄÄ ^franskrípce AUG, AUU, .UCC, iC_AA AGíj (3UU, ,U_; ^franstece Mel - lie - Ser - Gin - Arg - Val - ■ •CC C TA G G G GAT C •CC CUA G Leu změněná sekvence aminokyselin C delece páru bázi - obrovi původní čteci rámec AT G ATT TCC1 AAA GGG TTT.....CCC TAG TAC TA A AGG TTT CCC AAA GGG ATC ^transkripce mRNA A_UG_ MJjJ ,UC_C AjAA GGG, U_U_UCCC, UAG ^translace protein Met - lle - Ser - Lys - Gly - Phe .....Pro - (konec) změněné aminokyseliny obnovená původní sekvence aminokyselin mutantni fenotyp fenotyp standardního typu M U NI SCI Genetic code is formed by triplets of codons Insertion/Deletion of three bases does not change the reading frame. DNA (gen) AG A T\ C\ /• inzerce tří párů bází ATG ATT GTA CCC AAA G G G TTT.....CCC TAG • • *• mm* #t ** • • o . D #* no «»» ««« *«« •« ■ .»* tif c*t •# t* • • •*• **• *4 » »« TAC TA A CAT G G G TTT CCC AAA G G G AT C transkripce mRNA AUG AUU GUA CCC AAA GGG i^U_U translace protein Met - lle - Val - Pro - Lys - Gly - Phe iA, změněná sekvence s jednou přidanou aminokyselinou 31 Department of Experimental Biology původní sekvence aminokyselin standardního typu CCC UAG > fenotyp standardního typu Pro - (konec) M U NI SCI Suppressor mutations • Some mutations in tRNA genes change the structure of anticodons and therefore lead to a misinterpretation of codons in mRNA. • These mutations, originally found as suppressor mutations, have suppressive effect on other mutations. • Example: tRNA mutations that suppress amber mutations causing termination of string translation in UAG. 32 Department of Experimental Biology MUNI SCI Mutation Amber (UAG) termination of translation mutation (H) Coding sequence of standard gene mRNA 5' aug polypeptide Met transcription translation c ag Gin u a a 3' (end) Coding sequence a t g of mutated gene 3 T AC .11! mutation amber 33 Department of Experimental Biology MUNI SCI Mutation Amber in the absence of suppressor mutations Coding sequence of mutated gene translation in the absence of suppressor tRNA i i i ATG T AC i i i mRNA 5' äug fragment of m polypeptide mutation amber tag ATC. U_l_ transcription T i i i 3' T AA ATT j-1- 5' u a a 3' release factor (end) 34 Department of Experimental Biology MUNI SCI Mutation Amber in the presence of suppressor mutations • Some suppressor mutations alter tRNA anticodons so that mutant tRNA pairs to termination codon. • It leads to incorporation of an amino acid into the polypeptide (instead of termination of translation). translation in the presence of suppressor tRNA 35 Department of Experimental Biology MUNI SCI Standard genetic code Second Letter uuu -UUC - Phe ucu -ucc UUA -UUG - Leu UCA UCG - cuu - ecu - cue CUA Leu CCC CCA CUG- CCG - AUU - ACU - AUC lie ACC AUA - ACA AUG Met LStart ACG - GUU - GCU - GUC GUA Val GCC GCA GUG- GCG - Ser Pro Thr Ala ] UAU -UAC - UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG UGU - Tyr UGC - Cys Stop UGA Stop Stop UCG Trp CGU - His CGC Arg CGA Gin CGG — AGU -| Asn Ser AGC J AGA - Lys AGG- Arg GGU - Asp GGC Gly GGA Glu GGG — UGA (opal) - Selenocysteine UAG (amber) - pyrrolysine Department of Experimental Biology https://open.lib.umn.edu/evolutionbiology/chapter/5-8-using-the-genetic-code-2/ MUNI SCI Nonstandard amino acids - selenocysteine • Selenium = an important rare element necessary for catalytic activity of oxidoreductase, with the amino acid selenocysteine (Sec). • Selenocysteine is 21st amino acid and is located in the active sites of enzymes that participate in oxidation-reduction reactions (glutathione peroxidases, thioredoxin reductases, formate dehydrogenases, some hydrogenases). Selenoproteins are rare (1%o proteins). • Selenocysteine-tRNA is initially charged with serine. Then the attached serine is enzymatically modified to form selenocysteine. OH OH OH Serine (Ser) Cysteine (Cys) Selenocysteine (Sec) 37 Department of Experimental Biology TOXICOLOGY COMMUNICATIONS, 2017VOL. 1, NO. 1, 24-28 MUNI SCI Nonstandard amino acids - selenocysteine • Selenocysteine is not produced by posttranslational modification, but by translation. Condition - alternative use of stop codon UGA. • The gene in question must contain a specific signaling sequence SECIS (selenocysteine insertion sequence), which is transcribed into mRNA, but is not translated into the protein. • SECIS element contains auto-complementary sequences, forming a hairpin structure with a loop. • Special proteins associate with the hairpin and ensures recruitment of tRNA recognizing UGA codon carrying the selenocysteine to the ribosome. 38 Department of Experimental Biology eEFsec Selenocysteine / Stem-loop structure Selenoprotein Synthesis MUNI SCI Nonstandard amino acids - selenocysteine • The use of the codon is not accidental, but depends on the context of the given mRNA. • In bacteria, the tRNA carrying selenocysteine (Sec) first binds to SelB and the complex then binds to a stem and loop in the mRNA. This aligns the tRNA-Sec with a UGA codon within the coding sequence on the mRNA. Selenocysteine is then inserted as part of the growing polypeptide. • In mammals, the protein that binds the stem and loop and the tRNA-Sec is called eEFsec. In addition, the stem and loop are more distant, being found after the stop codon. BACTERIA 70S B H-r-ili tRNA-Sec -i and stem loop GfCwng proton 39 Department of Experimental Biology https://www.sciencedirect.com/topics/medicine-and-dentistry/selenocysteine MUNI SCI Nonstandard amino acids - pyrrolysine • Pyrrolysine (Pyl) is 22nd genetically encoded amino acid, it is a derivative of lysine with an attached pyrroline ring. • Occurrence is limited to archaebacteria Methanosarcinaceae and on the bacterium Desulfitobacterium hafniense. • The involvement of pyrroliethylene in them is associated with an exceptional ability use methylamines as an energy source using methylamine methyl-transferase. • For the synthesis of these enzymes, one UAG termination triplet must be used for the inclusion of pyrrolysine. • Molecular mechanism of alternative interpretation of stop codon is not yet fully known. 40 Department of Experimental Biology https://www.sciencedirect.com/topics/medicine-and-dentistry/selenocysteine Nonstandard amino acids - pyrrolysine OH • Pyrrolysine is made first as a free amino acid and then attached to tRNA-Pyl. • There is no pyrrolysine specific elongation factor. l-Lysine (2) • Moreover, the sequence determinants that specify which UAG codons should be used for pyrrolysine insertion are unclear. • Genome sequencing has found genes homologous to those for the pyrrolysine system in occasional Eubacteria, yet, pyrrolysine itself has not yet been identified directly in these organisms. flUI 41 Department of Experimental Biology https://cen.acs.org/articles/89/web/2011/04/Pyrrolysine-Synthesis-Revealed.html . Transfer RNA - tRNA Transfer ribonucleic acid (tRNA) is a type of RNA molecule that helps decode a messenger RNA (mRNA) sequence into a protein. 70-95 nucleotides. Internal base pairing creates partially ds sections -folding tRNA into a three-dimensional structure. D-loop D G A G 3 A A C UCmJG I I I I GAGC , rn^G 3' A-OH C C 9 A pG „ acceptor s1em U — u G — C G — U A — U U — A U — A U TijiC loop i c u G A C A C m'A I I I I I G m5C U G U G c T if Anticodon loop . C-GAG C — G A —U G — rrfc A — V Cm A U Y G*A A m'G variable loop • Contain anticodon - a sequence of three nucleotides complementary to the codon in mRNA. • The amino acid is covalently (ester) attached to 3'-OH end of tRNA. Dihydrouridine and pseudouridine arms stabilize the secondary structure of the clover leaf. 42 Department of Experimental Biology https://www.nature.com/scitable/definition/trna-transfer-rna-256/ MUNI SCI Transfer RNA - tRNA • Modified nucleotides often appear in tRNA and they help correct folding of tRNA-pseudouridine, methylguanosine, dimethylguanosine, methylinosine, dihydrouridine. 43 Define footer - presentation title / department Biomolecules2017, 7(2), 35; https://doi.org/10.3390/biom7020035 MUNI SCI Structure of tRNA molecule Clover leaf is folded into an L-shaped structure due to hydrogen bonds between different domains. Common ways of illustrating tRNA T loop Anticodon loop Anticodon Anticodon Anticodon 44 Department of Experimental Biology https://www.genome.gov/genetics-glossary/Transfer-RNA MUNI SCI Structure of tRNA molecule • At opposite ends of the L-shaped tRNA structure there are two regions of unpaired nucleotides: •Anticodon composed of three nucleotides complementary to the codon. • Short single-string area at the 3-end, where a specific AK is connected. Ester bond Amino ackJ Intramolecular base-pairing 45 Department of Experimental Biology mRNA 5' https://www.chegg.com/learn/biology/introduction-to-biology/peptidyl-trna-and-transfer-rna MUNI SCI tRNA and redundancy of genetic code • The genetic code is redundant: several codons determine the same amino acid. Possible explanations: • There is more than one tRNA for certain amino acids. • Certain tRNAs can pair with multiple codons. • Both variants are true. 46 Department of Experimental Biology MUNI SCI tRNA and redundancy of genetic code • Precise pairing between codon and anticodoe is required only between nucleotides in the first two positions of the codon, in the third position alternatives are tolerated jittering/"wobble". Codons Amino Acids AGA UUA AGG UUG GCA CGA GGA CUA GCC CGC GGC AUA CUC GCG CGG GAC AAC UGC GAA CAA GGG CAC AUC CUG AAA AGC AGU CCA UCA ACA CCC UCC ACC UUC CCG UCG ACG GUA GUC UAC GUG GCU CGU GAU AAU UGU GAG CAG GGU CAU AUU CUU AAG AUG UUU CCU UCU ACU UGG UAU GUU UAA UAG UGA Ala Arg Asp Asn Cys Glu Gin Gly His lie Leu lys Met Phe Pro Ser Thr Trp Tyr Val stop M W • Thanks to fluctuating base pairing, 20 amino acids and 61 codons are covered only by 31 variants of tRNA molecules. • The exact number of tRNA species is not the same for different species. https://microbenotes.eom/dna-polymerase/#dna-polymerase-iii MUNI 47 Department of Experimental Biology _ _ T FEMS Microbiol Rev. 2012 Nov; 36(6): 1105-1121. CP tRNA and redundancy of genetic code Pairing of the tRNA anticodon with the mRNA codon proceeds from the 5' end of the codon. Once the first two positions are paired, exact base pairing of the third position is less critical The first (5') base of the anticodon can typically pair with either member of the purine or pyrimidine pair in the codon as appropriate: it "wobbles". In this example, the double-ringed G can pair with either a single-ringed U or C. This allows mRNA to be translated with fewer than the 64 tRNAs that would be required without wobble. Some wobble positions can pair with any of the four bases. mRNA 5' ■ ■ ■ Identical leucine tRNAs Normal pairing ■■■3' 5'- Wobble pairing ■■■3' GAG (anticodon) CUcT^ ^CUU Two codons 48 Department of Experimental Biology https://www.mun.ca/biology/scarr/iGen3_06-08.html MUNI SCI Alternative base pairing between anticodon CGI in tRNA and three different (synonymous) codons • I = Inosine __ANH OL HO-i I N H,0 OH °H hurfmlwtär k.JUk/ fs|" N hydrolytic %^*""N p deaminase , Inosine = nucleoside composed of ribose and adenine ino&ine hypoxanthin. In eukaryotes the modification is present in eight different tRNAs. The modification of adenosine to inosine in tRNA anticodons has a profound impact upon codon-anticodon recognition 49 Department of Experimental Biology RNABiol. 2018; 15(4-5): 500-507. MUNI SCI Alternative base pairing between anticodon CGI in tRNA and three different (synonymous) codons It can pairs with uracile, cytosine and adenine. Modified tRNAs with inosine are preferred for the translation of highly repetitive coding sequences. U C A G U C A G kodony mRNA A III 0 GCU GCC GCA CGI CGI CGI alanyl-tRNA Ala1 50 Department of Experimental Biology MUNI SCI Isoacceptor tRNA Isoacceptor tRNAs are tRNAs with different anticodons but incorporating the same amino acid in protein synthesis. Codon UCU UCC UCA UCG AGU AGC tRNA tRNAsen RNASer2 tRNASer3 Antikodon AGG + wobbling AGU + wobbling UCG + wobbling i s c o T) O U Second Codon Letter U c A 6 uuu s UCU ¥ UAU c UGU U u f uuc s UCC Y UAC C UGC c L UUA s UCA Stop UAA Stop UGA A I UUG s UCG Stop v; U6G G L cuu p ecu H CAU R CGU U f L cue p ccc H CAC R CGC C L CUA p CCA Q CAA f» CGA A L CUG p CCG Q CAG CGG G I AUU T ACU N AAU S AGU U A 1 AUC T ACC N AAC S AGC C 1 AUA T ACA K AAA R AGA A M AUG I ACG K AAG « AGG G V GUU A GCU L> GAU 0 ÜÜJ u G V GUC A GCC D GAC G GGC c V GUA A GCA E GAA G GGA A V GUG A GCG - GAG G GGG G 3. 51 Department of Experimental Biology RNA Biol. 2018; 15(4-5): 500-507. MUNI SCI Speficity of tRNA tRNA • Must have the correct anticodon sequence (to respond to the correct codon). • Must be recognized by correct aminoacyl-tRNA synthetases (to carry the correct AA). • Must bind to the correct place of the ribosome (so that it can realize its adapter function). 52 Department of Experimental Biology MUNI SCI Aminoacyl tRNA synthetase Aminoacvl tRNA synthetase • Enzymes that covalently attach specific AA to specific tRNA. • There are 20 AA-tRNA synthetases, one for each AA. • For instance, one AA-tRNA synthetase attaches glycine to all tRNAs, which recognize codons for glycine, etc. • Each AA-tRNA synthetase must recognize: o relevant AA o unpaired nucleotides in the anticodon arm o sequence in AK-tRNA arm. • The exact function of AA-tRNA synthetases is equally important for translation accuracy, as a codon-anticodon bond! 53 Department of Experimental Biology MUNI SCI Two phases of translation • Extra-ribosomal: attachment of an amino acid to its tRNA by activity of aminoacyl-tRNA-synthetase. • Ribosomal: amino acids, bound to appropriate tRNA, are assembled to polypeptide on ribosome based on sequence of codons. The ribosomal phase has three stages: • translation initiation polypeptide chain elongation • translation termination. 54 Department of Experimental Biology MUNI SCI Extra-ribosomal phase of translation 1. Activation of the amino acid 2. Transfer of the aminoacyl group to the tRNA Step 2 ARS(AA-AMP) + tRNA -* AA-tRNA + AMP + ARS • Extra-ribosomal phase is sometimes called "charging" or "loading" the tRNA with an AA. • The synthetase first binds ATP and the corresponding amino acid (or its precursor) to form an aminoacyl-adenylate, releasing inorganic pyrophosphate (PPi). • The adenylate-aa ARS complex then binds the appropriate tRNA molecule's D arm, and the amino acid is transferred from the aa-AMP to either the 2'- or the 3-OH of the last tRNA nucleotide at the 3'-end. • 1. Amino Acid + ATP Aminoacyl-AMP + PPi • 2. Aminoacyl-AMP + tRNA Aminoacyl-tRNA + AMP 55 Department of Experimental Biology International Journal of Biological Macro molecules, Volume 111, May 2018, Pages 400-414 https://en.wikipedia.org/wiki/Aminoacyl_tRNA_synthetase MUNI SCI Ribosomes • Cellular particle made of RNA and protein that serves as the site for protein synthesis in the cell. • The ribosome reads the sequence of the messenger RNA (mRNA) and, using the genetic code, translates the sequence of RNA bases into a sequence of amino acids. • It is the place where codons meet anticodons according to the base pairing rules. • It can assemble onto mRNA, capture and correctly direct tRNA molecules in order to covalently connect amino acids into the polypeptide chain. • Work non-specifically: they synthesize any peptide properly encoded by any mRNA. Department of Experimental Biology https://www.genome.gov/genetics-glossary/Ribosome MU SC Ribosomes • Ribosomes are present in large numbers in all living cells. • Ribosomes occur o free particles in prokaryotic and eukaryotic cells o particles attached to the membranes of the endoplasmic reticulum in eukaryotic cells. • There are about 200,000 ribosomes in the E. coli cell (i.e. 25% of the dry mass of the cell). •Atypical eukaryotic cell in cytoplasm contains millions of ribosomes. Endoplasmic reticulum nuclear envelope nuclear pore smooth endoplasmic reticulum cisternal space cisternae 1)2010 Encyclopedia Britannica, Inc. ribosomes All molecules involved in protein synthesis together forms 1/3 of the dry weight of cells, high energy consumption. 57 Department of Experimental Biology https://www.britannica.com/science/ribosome MUNI SCI Ribosomes Small 30S subunit Large 60S subunit 58 Department of Experimental Biology Ribosomes consist of two major components: the small and large ribosomal subunits. • Each subunit consists of one or more ribosomal RNA (rRNA) molecules and many ribosomal proteins (RPs or r-proteins). • rRNA sequences are very evolutionarily conserved • Ribosome sizes are often expressed according to Svedberg's units "S„ the Svedberg unit, a measure of the rate of sedimentation in centrifugation rather than size. https://commons.wikimedia.org/wiki/File:010_small_subunit-1 FKA.gif https://commons.wikimedia.Org/wiki/File:010_large_subunit-1FFK.gif MUNI SCI Ribosomes Prokaryotes: • Size 70S (20 x 25 nm) • Small subunit 30S: rRNA 16S + 21 proteins • Large subunit 50S: rRNA 5S and 23S + 34 proteins. Eukaryotes: • Usual size 80S (some interspecies variability) • In mitochondria and chloroplasts 60S • Small subunit 40S: rRNA 18S + 33 proteins • Large subunit 60S: rRNA 5S, 5.8S, 28S + 49 proteins 59 Department of Experimental Biology MUNI SCI Ribosomes in prokaryotes and eukaryotes 5S rRNA + 34 polypeptides 23S rRNA 16S rRNA + 21 polypeptides 5S rRNA, 5.8S rRNA •61 + 6.8S rRNA + + 49 polypeptides 28S rRNA 185 rRNA+ > 33 polypeptides Cr. 50S subunit 30S subunit <> 60S subunit 40S subunit Prokaryotic 70S ribosome Eukaryotic 80S ribosome 60 Department of Experimental Biology https://nl.pinterest.com/pin/503418064572288990/?amp_client_id=CLIENT_ID(_)&mwe b_unauth_id={{default.session}}&simplified=true MUNI SCI rRNAs come from the nucleolus • rRNAs are produced by DNA transcription. • In eukaryotes rRNA is produced by RNA-polymerase I in the nucleolus. 61 Department of Experimental Biology https://sciencetrends.com/what-is-the-nucleolus/ MUNI SCI rRNA genes • The rRNA genes are arranged in succession in many copies • Separated by intergene areas (spacebars). • Transcription of these genes is very effective. Department of Experimental Biology https://www.cell.co m/fulltext/S0960-9822(02)01135-1 rRNA genes • The genes for rRNA are transcribed as a longer precursors, which are spliced and modified by post-transcriptional modifications. Pr0kar¥0teS 16S_235 5S Pre-rRNA III I I I-l~l (5.5 kb) 16S_ I_23S__5S I i ' in r I ~~n ii f I I 5S 16S(1.5kb) I 23S (2.9 kb) (CU2kb) Mature rRNAs I 1 * I I I 1 Eukaryotes L8S 5.8S 28S I- I I I I I II I ISS { SM 28S t I ~l III ~ I 5.as !8S(].9kb) \ 0.l6kb Mature rRNAs I ~~l I I 28S (5 kb) 63 Department of Experimental Biology Canadian journal of gastroenterology = Journal canadien de gastroenterologie 14(2):99-110 MUNI SCI Small and Large subunit of ribosome Unique function • Small subunit • correct assembly with respect to mRNA and tRNA. • Large subunit • formation of peptide bonds. • Both subunits are assembled into one complex near the 5 - end of the mRNA, where the translation begins. • mRNA gradually stretches through the ribosome and allows translation one codon at a time. • After completion of translation, the complex disintegrates and the two subunits are separated from each other. 64 Department of Experimental Biology MUNI SCI Structure of the ribosome • Four interacting sites are located at the ribosome: • Binding site for mRNAs. Three binding sites for tRNA, designated A, P and E: • A (aminoacyl) - binds an aminoacyl-tRNA or termination release factors. • P (peptidyl) - binds a peptidyl-tRNA (a tRNA bound to the polypeptide chain), when a stop codon is reached, the peptidyl-tRNA bond of the tRNA located in the P-site is cleaved releasing the newly synthesized protein. • E (exit) - binds tRNA, without AA, before leaving the ribosome. Department of Experimental Biology https://www.sparknotes.com/biology/molecular/translation/section3/ Ribosome is Ribozyme Very complex structure, composition: 2/3 rRNAa 1/3 protein. The three-dimensional structure of the ribosome in 2000 was considered a triumph of the modern biology. Confirmed that rRNA (not proteins) provide the main structural and functional parameters of this complex, including catalysis of protein synthesis! proteins - purple 23S rRNA in orange/white 5S rRNA in burgundy/white A-site tRNA (green) P-site tRNA (red) tRNAH O (P-site) tRNA j 3' direction. • 60S subunit associates with initiation complex when start codon is recognized. • Initiation factors are released when the two ribosomal subunits associate. 80 Department of Experimental biology https://slideplayer.com/slide/5746723/ Met *0 Cap , mRNA Initialion factots l^ + GTP V M*t-tRNAt 405 subunit ♦OS subunit with initiation components Initiation factors 80S initiation compltx inrroduc hon todtntK Anofyui. tenth Edition 0 2012 W. H. t it*i».in and Company MUNI SCI Mimicry of tRNA Molecules 81 Department of Experimental biology CELL, VOLUME 100, ISSUE 3, P311-321, FEBRUARY 04, 2000 MUN SCI Translation - prokaryotes versus eukaryotes prokaryotic eukaryotic function Initiation factor IF1 IF3 IF2 elF3elF4celF6 elF4B elF4F elF2B elF2 elF5 Bind to ribosome submits Bind tomRNA InitiatortRNAdelivery Displacement of other factors Elongation factor EF-Tu EF-Ts EF-g eEF1a eEFIßY eEF2 Aminoacyl tRNA delivery Recycling of EF-Tu or eEF1a Translocation Termination factors RF1.RF2.RF3 eRF Polypeptides Chain release 82 Department of Experimental biology https://slideplayer.com/slide/3330529/ MUNI SCI Termination of translation by tmRNA • Transfer-messenger RNA (tmRNA) is a bifunctional RNA that has properties of a tRNA and an mRNA. • tmRNA uses these two functions to release ribosomes stalled during translation and target the nascent polypeptides for degradation. • This concerted reaction, known as trans-translation, contributes to translational gualitv control and regulation of gene expression in bacteria, when ribosome fails to recognize stop codon or RF can not bind to ribosome. tmRNA is conserved throughout bacteria, and is one of the most abundant RNAs in the cell, suggesting that trans-translation is of fundamental importance for bacterial fitness. 83 Department of Experimental biology Biochimie. 2011 Nov; 93(11): 1993-1997. MUNI SCI Termination of translation by tmRNA tRMA-like domain 111 PK3 if mi níi Ä Í *— - i ; PK4 * * * í íítttl * H J: li * ( 1*— : ANDEN YAiíŤí PK2 Tag reading frame Ajis-r, A/c*-". tRNA-like domain r~-1 Tag reading frame • Longer than tRNA. • Does not have a clover leaf structure. • Alanine bound to the 3-end. • Carries a short sequence encoding 10 AA, followed by a stop codon. StopV 84 Department of Experimental biology Biochimie. 2011 Nov; 93(11): 1993-1997. MUNI SCI Termination of translation by tmRNA • tmRNA*mpB binds the A site of stalled ribosomes and accepts the nascent chain. • The non-stop mRNA is released and preferentially degraded by RNase R. • Translation then resumes using the open reading frame found within tmRNA. • After synthesis of the ssrA peptide, release factors (RF) terminate translation and the ribosome is recycled into large and small subunits. recycled ribosome release of translation of ssrA-tagged chain ssrA peptide . SsrA-tagged chain is degraded by a number of proteases. 85 Department of Experimental biology Adv Protein Chem Struct Biol. 2012; 86: 151-191 MUNI SCI Polysomes 100 nm 100 nm {A) (B) Figure 6-75. Molecular Biology of the Cell, 4-th Edition. • The synthesis of most proteins takes from 20 sec to a few minutes. • Even during this short period of time, multiple ribosomes bind to mRNA. • For intensively translated transcripts, the ribosome mounts on the 5'-end of the mRNA immediately as soon as the previous ribosome frees up enough space for him. • The resulting polysomes contain many ribosomes bound to the same mRNA at a distance of about 80 nucleotides. • High efficiency of the creation of the given Protein. • In bacteria ribosomes to mRNA immediately during its creation by transcription. 86 Department of Experimental biology SCIENCE OF AGING KNOWLEDGE ENVIRONMENT • 26 Feb 2003 • Vol 2003, Issue 8 • p. re3 • DOI: 10.1126/sageke.2003.8.re3 MUNI SCI Translation machinery is often target of antibiotics • Successful protein synthesis is a condition of life. • In the process of translation of prokaryotes and eukaryotes there are subtle differences. • Most antibiotics inhibit the translation of bacteria (not eukaryotes). • Often uses structural differences of ribosomes. • A number of antibiotics have been isolated from fungi that often inhabit the same habitant as bacteria. • In order to withstand evolution, they have developed the ability to produce toxins that kill bacteria that do not harm them. • Fungi are eukaryotic organisms - more closely related to humans than bacteria, their products are useful for protecting humans. 87 Department of Experimental biology https://wou.edu/chemistry/courses/online-chemistry-textbooks/ch450-and-ch451-biochemistry-defining-life-at-the-molecular-level/chapter-9-dna-replication-and-repair-2/ MUNI SCI Translation machinery is often target of antibiotics Growing Protein peptide Tunnel chain synthesis site ^s* SOS \ J (30S Á--3' mRNA Growing polypeptide (a) Three-dimensional detail of the protein synthesis site showing the 30S and SOS subunit portions of the 70S prokaryotic ribosome. Streptomycin Changes shape of 30S portion, causes code on mRNA to be read incorrectly Chloramphenicol Binds to SOS portion and inhibits formation of peptide bond Protein sythesis site Erythromycin Binds to 50S portion, prevents translocation-movement of ribosome along mRNA Tetracyclines Interfere with attachment of tRNA to mRNA-ribosome complex Direction of ribosome movement (b) In the diagram the black arrows indicate the different points at which chloramphenicol, erythromycin, the tetracyclines, and streptomycin exert their activities. Copyright & 2004 Paanon Education. Inc , pubtening as Bcrfamin dimming* 88 Department of Experimental biology MUNI SCI Differences between prokaryotic and eukaryotic translation • In bacteria, the initiation complex is formed directly on the sequences near AUG. • In eukaryotes, 40S first recognizes the 5' end of mRNA and then moves to the initiation point where it connects with the 60S. • elF-4G is bound to polyA-end RNA and to elF-4E bound to cap = only mRNA with full length translated. • N-formlymethinine in prokaryotic 5'end of proteins. 89 Department of Experimental biology MUNI SCI thank you for your attention MéAMWHIU-^ on Mount öl^pia, the New Havert— r This is So casy! I've ComPLĚTCD The tfvÖiCK's CußE ASAiNiM (jNDEfi. T\W£MTY SecomOS! OF COUfiSt! UT mE see. what i wrtve fbg A CLEVřft fei_0vJ LUCE ůC^mftCTr- PANTS ILT'S SEE HovJTttU GET Ot ASSemftLifJCi A *lo*?Ki mooCi- of the i.*fßere SufcVNrT OF THř 90 Department of Experimental biology Trends in Biochemical Sciences, Volume 28, Issue 8, August 2003, Pages 411-418 MUNI SCI