2. Translation – synthesis of proteins Which cells: all cells having nuclear DNA Where in the cell: ribosoms (free or attached to ER) mitochondrias Differences between prokaryotes and eukaryotes: prokaryotes: transcription, processing of transcript and translation are not separated by space eukaryotes: translation starts after the mRNA synthetized in nucleus is transported to cytoplasma

3. Which molecules are necessary for synthesis of proteins ? Amino acids Many enzymes Protein factors ATP and GTP Mg 2+ , K + ions

4. Genetic code Proteins – 20 different AA RNA – 4 bases Each amino acid is characterized by a triplet of bases in mRNA – codon Codons consisting of three nucleotide can provide 64 codons -> 61 of them code for 20 amino acids 3 codons are STOP codons (UAA,UAG, UGA) Nierenberg (1961) – poly(U) sequence of mRNA produced polyphenylalanine by translation  sequence UUU is the codon for phenylalanine

5. Genetic codon G Gly Glu Ala Val A Gly Glu Ala Val C Gly Asp Ala Val U Gly Asp Ala Val G G Arg Lys Thr Met A Arg Lys Thr Ile C Ser Asn Thr Ile A U Ser Asn Thr Ile G Arg Gln Pro Leu A Arg Gln Pro Leu C C Arg His Pro Leu U Arg His Pro Leu G Trp Stop Ser Leu A Stop Stop Ser Leu U C Cys Tyr Ser Phe U Cys Tyr Ser Phe 3´ G A C U 5´ Third base Second base First base

7. Degeneracy of the genetic code

9. The reading frames Only one of the three possible frames is the right one, it begins at the start codon AUG recognized by the Met-tRNA Met

10. Efect of mutations Mutations are structural alterations that result from damage of DNA molecules or unrepaired errors during replication They can be transcribed into mRNA By translation of altered base an abnomal sequence of amino acids can appear in the protein

12. Types of mutation (cont.) 2. insertion – one or more nucleotides are added to DNA 3. deletion – one or more nucleotides are removed from DNA The damage of the protein depends on the number of deleted or inserted nucleotides If three nucleotides (or more triplets) are inserted/deleted without a change of the reading frame, polypeptides with inserted/deleted amino acyl residues will be synthesized. If one or two nucleotides are inserted/deleted, the result is a "frame-shift mutation„ that gives nonsense codons, distinct primary structure of proteins, etc.

13. Example of point mutation Point mutations in the genes for hemoglobin: There is known about 800 of structural variants of human hemoglobin Most of them results from point mutations and are not harmfull. Some of them cause diseases. Methemoglobinemia – e.g. replacement of single histidine by tyrosine in  -chain  protein is unattackable for the treatment of methemoglobin reductase, the methemoglobin in blood rises Sickle cell anemia – missense mutation, GTG replaces GAG  replacement of glutamate by valine in position 6 of  -chain  the chain become less soluble and precipitate when deoxygenated

15. General structure of tRNA molecules The cloverleaf structure

17. Aminoacyl-AMP (mixed anhydride) + ATP C O C O H N H 2 R H 2Pi + 1. Activation of AA C C O H N H 2 R N N N N N H 2 O O H O H O P O O O

18. 2. Transfer of activated AA to 3´-end of tRNA Cyt t-RNA 3´- end of t-RNA Ester bond between -COOH of amino acid and 3´-OH of ribose Amino acid C O C N H 2 R N N N H 2 P O O - N N C H 2 O O H O O P O O O - C H 2 O O H O O

19. (at least 20 distinct enzymes in cells) exhibit the very high degree of specifity for amino acids. The enzyme molecule recognizes both a specific amino acid and a specific tRNA. These enzymes discriminate accurately, the overall rate of occurence of errors in translating mRNA is less than 1 in 10 000. This high specifity is oft called the 2 nd genetic code . It depends on specific location of some bases in tRNA molecules, not on the sole anticodon . Aminoacyl - tRNA synthetases

21. Ribosomes Large ribonucleoprotein particles – composed of proteins and RNA Free floating in the cytoplasma or attached to the membranes Ribosomes are workbenches for protein synthesis

22. Ribosomes Ribosomes consist of a large and a small subunit Inactive ribosomes exist as loose subunits that aggregate into comlete particles when they get ready for protein synthesis. Large ribosomal subunit has three binding sites for molecules of tRNA – P, A, E P-peptidyl-tRNA A-aminoacyl-tRNA E-free tRNA (exit) E P A

23. Prokaryotic x eukaryotic ribosomes 80S 40S 60S 50% 18S 5S 5,8S 28S Free floating in cytoplazma or attached to ER membrane 70S 30S 50S 65% 16S 5S 23S Free floating in cytoplazma or attached to plasmatic membrane Sedimentation constants: complete ribosome small subunit large subunit RNA content RNA-small subunit RNA-large subunit Placement in the cell Human Bacterial Property

24. Initiation Ribosome has to associate with mRNA and the initiator tRNA Formation of initiation complex. Soluble cytoplasmic factors helps in initiation – initiation factors Also GTP, ATP are involved Differences between eukaryonic and prokaryonic cells

25. Initiation u eukaryotes Small ribosomal subunit Large ribosomal subunit Guanine cap mRNA Iniciation factors It involves formation of a complex composed of methionyl t-RNA met , mRNA and a ribosome. antikodon Aminocyl-tRNA tRNA 5´-P-P-P-5´- 5´ G E P A G

26. 40S Binding of factors eIF3 and eIF1A to a small subunit eIF3 eIF1A

27. eIF2 GTP AMP 3´ Binding of activated Met to tRNA Met Binding of GTP to eIF2 G Met

28. Met Factor eIF2 recognizes the initiation aminoacyl-tRNA 3´ UAC Binding of complex GTP-eIF2 to t-RNA Met G GTP eIF2

29. m-RNA 5´-P-P-P-5´- 5´ G CBP (cap binding protein) binds to the cap on 5´end of mRNA CPB (eIF-4F) is composed of a number of other initiation factors (eIFs) A C A U G U U G C C…...

30. Met Complex Met-tRNA Met , eIFs and GTP binds to the snaller ribosomal subunit Preiniciation complex G GTP eIF2 UAC

31. m-RNA Binding of m-RNA to preiniciation complex Reaction requires ATP for unwinding a hairpin loop in the mRNA (helicase activity of an eIF subunit) The complex scans mRNA from 5 ´end until it locates the AUG start codon G GTP eIF2 UAC A C C G U A A C A U G U U G C C G 5´-P-P-P-5´-

33. Differences in between prokaryotes and eukaryotes 70s (30s a 50s) 80s (40s a 60s) ribosomes 3 12 and more Iniciation factors formylmethionine methionine First AA No cap, Shine-Dalgarno sequence in mRNA about 10 nucleotides upstream of the AUG start codon is attached to a complementary sequence in 16S RNA Cap on the 5´end of mRNA binds IFs and 40S subunit containing t-RNA met . mRNA is scanned until AUG Binding of mRNA to smaller ribosomal subunit prokaryotes eukaryotes

36. Antikodon is AAC Amino acids is leucin Which amino acid will be added? The next codon is UUG UAC A P E A C C G U A A C A U G U U G C C G 5´-P-P-P-5´-

38. UAC A P E A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- AAC Met Leu Leu-tRNA binds to the site A A P E GTP is hydrolyzed to GDP + Pi, complex GDP-EF1  is released GDP-EF1  Pi Proces elongace je u prokaryotů a eukaryontů velmi podobný (odlišné kofaktory elongace)

39. UAC A P E A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- AAC Met Leu A P E Binding site on tRNA Met is empty Peptidyltransferasa is rRNA. It is a component of 28S RNA subunit 60S – ribozyme activity Formation of peptide bond (transpeptidation) Synthesis of proteins starts with N-terminal Peptidyltransferase catalyzes the release of methionine from tRNA and its transfer to leucine. A peptide bond between carboxyl group of methionine and amino group of leucine is formed

40. UAC A P E A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- AAC Met Leu A P E Movement of met-tRNA to the site E 1. EF2 and GTP bind to ribosome EF2 + GTP 2. tRNA Met moves to the site E, site P become free G

41. A P E A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- AAC Met Leu A P E Result of animation of page 42 UAC Movement of met-tRNA to the site E G

42. A P E A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- AAC Met Leu A P E Translocation and release of Met-tRNA GDP-EF2 + Pi Movement of ribosome with respect to mRNA and its base-paired tRNAs  peptidyl-tRNA moves into the P site Site A is empty GTP is hydrolyzed, EF2 will release Direction of movement

43. Which further steps will follow? Next step of elongation Further tRNA charged with amino acid (proline) binds to the site A

45. Energy consumption Aaminoacyl-tRNA formation ATP  AMP + 2 Pi 2 Binding of aminacyl-tRNA to the site A GTP  GDP + Pi 1 Translocation of peptidyl-tRNA to the site P GTP  GDP + Pi 1 Equivalent of ATP 4 ATP 4 ATP are required for synthesis of one peptide bond. Further energy is required for initiation and synthesis of nukleotides. Proteosynthesis rate Prokaryotes ~ 100 peptide bond s Eukaryotes ~ 100 peptide bonds/min

46. Stimulation of proteosynthesis by insulin CBP (cap binding protein complex) has subunit eIF4E This subunit is blocked by protein 4E-BP Insulin triggers phosphorylation of 4E-BP Phosphorylated 4E-BP looses its afinity to eIF4E  eIF4E become free for participation in protein synthesis Insulin is anabolic hormone, it stimulates proteosynthesis. It affects the synthesis through the interaction with CBP.

47. Polysomes NH 2 NH 2 NH 2 NH 2 As one ribosome moves along the mRNA produsing a polypeptide chain, a second ribosome can bind to the vacant 5´-end of mRNA. Many ribosomes can simultaneously translate a single mRNA, forming a complex known as polysome. A single ribosome covers approximately 80 nucleotides of mRNA. Therefore, ribosomes are positioned on mRNA at intervals of approximately 100 nucleotides. Simultaneous translation of mRNA on more ribosomes

49. Effects of antibiotics on prokaryotic proteosynthesis the differences in proteosynthetic procedure between eukaryotes and bacterias are exploited for clinical purposes some antibiotics inhibits specifically proteins of bacterial ribosomes Binds to A-site on ribosome and trigers premature termination Puromycine Binds to 50S ribosomal subunit and inhibits translocation Erytromycine Binds to 50S ribosomal subunit and inhibits peptidyltransferase Chloramfenikol Binds to 30S ribosomal subunit and inhibits binding of aminoacyl-tRNA to site A Tetracycline Binds to 30S ribosomal subunit, inhibits formation of initiation complex.Triggers errors in reading frame of mRNA. Streptomycine Effect Antibiotics

53. Targeting of proteins to subcelular and extracellular locations Synthesis of proteins on polysomes in cytosol Proteins remain in the cytosol or enters the organeles (nucleus, mitochondria). They contain amino acid sequence ( targeting sequence) that facilitate their transport into a certain organelle Synthesis of proteins on ribosom bound to RER Transport to lysosomes, ER, Golgi complex, cellular membranes or secretion

54. Transport of proteins syntesized on RER RER membrane Signal peptide (ussualy 15-30 hydrophobic amino acids on N-terminl end) Signal recognition particle (SRP) SRP-receptor Signal peptidase Cleaved signal peptide 1. 2. 3 4 5 6

56. Lyzosomes Secretory vesicles Cis Golgi Trans Golgi http://vcell.ndsu.nodak.edu/animations/proteintrafficking/first.htm Transport of proteins syntesized on RER

58. Principles of intracellular sorting Example 1: Proteins determined for lysosomes are marked by N-bonded oligosacharides terminated by mannose-6-P Prot-oligosaccharide-mannose-6-P „ address“ is recognised by specific membrane receptors in Golgi complex that embedds the protein into the clathrine coated vesicles

59. Example 2: Proteins destinated for ER have sequence Lys-Asp-Glu-Leu on their carboxy terminal lys-asp-glu-leu Proteins are transported back from Golgi complex to ER Principles of intracellular sorting

60. Glycosylation of proteins Glycoproteins N -linked carbohydrate chain Involving the amide nitrogen of asparagine They differ in composition of saccharides and and the way of synthesis O-linked carbohydrate chain Involving hydroxyl side chain of serine or threonine

61. Synthesis of N-glycoproteins The oligosaccharide chain is first assembled on the dolichodiphosphate backbone Dolichol is isoprene (see the synthesis of cholesterol) n=18-20 Dolicholdiphosphate is bonded to membrane of ER. Activated monosaccharides are gradually attached to the terminal phosphate. The oligosaccharide chain is then transferred en bloc to suitable Asn residues of apoglycoproteins during their synthesis on membrane-bound polyribosomes H- [CH 2 -C=CH-CH 2 ] n -CH 2 -CH-CH 2 -CH 2 OH CH 3 CH 3

62. Synthesis of oligosaccharide precursor Transfer to protein Dolichol-P Dolichol-P-P ← GlcNAc ← GlcNAc Dolichol-P-P ← ( GlcNAc ) 2 ← ( Man ) 5 Dolichol-P-P ← ( GlcNAc ) 2 ( Man ) 9 Dolichol-P-P ← ( GlcNAc ) 2 ( Man ) 9 ( Glc ) 3 2 UDP -GlcNAc 5 GDP -Man 4 Dolichol-P-Man 3 Dolichol-P-Glc

63. Trimming and final processing of N -linked glycoproteins: ( Glc ) 3 ( Man ) 9 ( GlcNAc ) 2 -protein – 3 Glc ( Man ) 9 ( GlcNAc ) 2 -protein ( Man ) 5 ( GlcNAc ) 2 -protein ( Man ) 3 ( GlcNAc ) 2 -protein pentasaccharide core region High-mannose N -linked glycoproteins up tp 4 Man removed – 2 Man glycosylation Hybride types Complex type N - linked glycoproteins glycosylation Late phase processing Trimming

64. Complex type (triantennary chain) Hybride type (one antenna) High-mannose type (before trimming to core region) Examples of plasma - type ( N - linked ) oligosaccharides The boxed area encloses the pentasaccharide core region common to all N -linked glycoproteins.

65. Synthesis of O-glycoproteins is posttranslational process that takes place exclusively in the Golgi complex and which is direct – glycosyls from nucleotide sugars (NuDP-glycoses) are transferred to side chains of Ser or Thr residues and elongated by other nucleotide sugars (Thr) (Thr) ……… Ser……………. ……… Ser……………. OH UDP-monosach. UDP O-monosacharide

66. Regulation of gene expression Gene expression – formation of proteins or RNA products Generally only small fraction of genes in a cell are expressed at any time Gene expression is regulated differently in prokaryotes and eukaryotes

68. Regulation of gene expression in prokaryotes Regulation is less complex tham the multicellular eukaruotes Gene expression is regulated mainly by controlling the initiation of gene transcription. The most extensive studied is bacterium E.coli. Its genom includes 4x10 6 base pairs  E.coli should be able of making several thousands of proteins Under normal conditions they synthesize only about 600-800 different proteins. Many genes are inactive and only those genes are expressed that generate the proteins required for growth in that particular enviroment

69. Housekeeping genes and inducible genes Expression of some genes is constitutive – they are expressed at a reasonably constant rate and are not the subject of regulation - housekeeping genes Some genes are inducible - their expression requires an inducer or activator

70. Operons theory Structural gens of bacterias are grouped into units called operons DNA 3´ mRNA promotor 1 3 2 Structural genes operon AUG AUG UAA AUG UGA UAG 5´ Prot 1 Prot 1 Prot 1

72. Regulation of RNA polymerase binding by repressors – negative control Regulatory gene DNA mRNA repressor A B C operator structural genes repressor je encoded by regulatory gene Its product, the repressor protein, difuses to the promoter and binds in the region of the operon called operator Operator is located within the promoter or near of its 3´-end Repressor blocks the binding of RNA-polymerase to the promotor Synhesis of mRNA does not occur

73. Repressor is controlled by two mechanisms Induction Corepression Inductor is a small molecul that binds to repressor, changes its conformation and triggers its release from the operator transcription can start Inductors: small molecules of nutrients or their metabolites repressor is not active until corepressor is bonded to it. The complex repressor-corepressor binds to operator preventing binding of RNA polymerase transcription stops Corepressors: small molecules of nutrients or their metabolites

74. Example of induction Induction of lac operon in E.coli by lactose Enzymes for metabolizing glucose by glycolysis are produced constitutively If the milk sugar lactose is available, the cell adapt and begin to produce three additional enzymes required for lactose metabolism These enzymes are encoded by lac operon A metabolite of lactose ( allolactose - isomer of lactose that is formed spontaneously) serves as an inducer, binds to the repressor and inactivates it. RNA polymerase can bind to promotor and transcribe the structural genes of the lac operon (  -galactosidase, permease and transacetylase) Glucose can prevent activation of the lac operon (see fig.80)

75. Example of corepression Corepression of trp operon (synthesis of tryptophane at E.coli) genes for enzymes of tryptophane synthesis (5 enzymes) are located in trp operon Tryptophan ise corepresssor, it binds toinactive repressor, binds its conformation. The complex tryptophan-repressor inhibits transcription of operon. http://www.biology.ualberta.ca/facilities/multimedia/uploads/genetics/trpoperon2.swf

76. Stimulation of RNA polymerase binding - positive control Regulatory proteins binds to promoter and stimulate the binding of RNA-polymerase Regulatory protein is activated on the base of presence/or absence of small molecule of nutrient of its metabolite in the cell Catabolite repression

77. Example of positive control Transcription of lac operon at E.coli transcription of lac operon is affected by allolactose only when the glucose is absent Decrease of glucose level results in increase of cAMP (it is not known why) cAMP binds to its receptor in the cell (cAMP-receptor protein -> CRP) Complex cAMP-CRP binds to the regulatory site of lac operon, stimulates the binding of RNA polymerase to promotor and transcription of genes for metabolism of lactose -> cells metabolize lactose, only when it does not have adequate supply of glucose

78. Atenuation of transcription Some operons are regulated by process that interrups (attenuates) transcription after it has been initiated The cause is the change of secondary structure of mRNA Attenuator („retarder“ ) is a sequence included in operon Processes of translation occurs at the same time with transcription The rate of transcription affects the formation of hair-pin loops on mRNA

79. Atenuation of transcription – trp operon E.coli 1 2 3 4 1 2 3 4 1 2 Low conc. of Trp High conc. of Trp STOP Ribosom is attenuated Quickly moving ribosome Sequence 1 contains codones for Trp 5´ mRNA

83. Regulation of availability of genes for transcription Chromatin in the nucleus: Condensed (heterochromatin ) – genes are inactive Euchromatin- genes produce mRNA In cells of differentiated tissues only the genes that have some role in the cell are active Long-term changes in the activity of genes occur during development as chromatin goes from a diffuse to a condensed state and vice versa.

89. B) Regulation at the level of transcription Basal regulation of transcription (common for all genes) Regulation by the components of „basal transcription complex“ (RNA polymerase binding the TATA box, TATA binding proteins and further basal transcription factors binding on promoter or RNA-polymerase) Genes regulated only in this way: constitutively expressed genes Specific regulation of gene expression: Gene specific transcription factors bind to the specific regulatory sequences . See the lecture 13

90. Regulation of transcription factors Down(up)-regulation Modulation by binding of stimulatory and inhibitory ligands Mutual cooperation of transcription factors Phosphorylation/ dephosphorylation of transcription factors regulated growth actors, cytokines, peptide hormones atd.

91. C) Postranscriptional regulation Alternative splicing Alternative splicing and variation of the site of polyadenylation cause that one gene can produce various proteins (see the lecture 13) RNA editing In some instances, RNA is „edited“ after transcription. Primary transcript and the sequence of gene are the same, but bases are altered or nucleotides are added or deleted after the transcript is synthesized.

92. RNA editing synthesis of apoB in hepatocytes and enterocytes CAA Gen apoB transcript CAA hepatocyte enterocyte CAA mRNA U AA Stop! translation Liver apoB -4563 AA Intestinal apoB – 2152 AK

93. Synthesis apoB in hepatocytes and enterocytes (apo B is a component of chylomicrons and VLDL) Gen apoB produces protein containing 4563 amino acids in liver The same gene produces apoB containing only 2152 amino acids in enterocytes Conversion of C(cytosin) to U (uracil) by deamination of mRNA transcript generates the stop-codon in intestinal mRNA. Thus the protein formed in the enterocyte has only 48% of lenght in comparision with apoB in liver

94. D) Regulation at the level of translation Regulation ussualy involves the initiation of protein synthesis by eIFs (eukaryotic initiation factors) See Fig. 34,46