Nucleic acid and protein Flow of genetic information
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1 Nucleic acid and protein Flow of genetic information References: Glick, BR and JJ Pasternak, 2003, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington DC, pages and Groves MJ, 2006, Pharmaceutical Biotechnology, 2nd ed., CRC, Taylor & Francis, pages Genetic information flow 1
2 Nucleic acids and proteins DEBBIE S. RETNONINGRUM SCHOOL OF PHARMACY INSTITUT TEKNOLOGI BANDUNG Genetic information flow 2
3 NUCLEIC ACIDS DNA AND RNA Polymer/Polynucleotides Monomer : nucleotide Components : sugar, phosphate, base Genetic information flow 3
4 Structure of nucleotides and polynucleotides mabrybio211/chapter05/ch5.htm Genetic information flow 4
5 Genetic information flow 5
6 DNA Backbone : Phosphate-sugar base in the anti parallel strands Polarity: 5 3 vs 3 5 Complement : Base pairs (C=O and N-H bases between polinucleotides Phosphodiester bond (between nucleotides) Genetic information flow 6
7 CHROMOSOM ROMOSOMES VS PLASMID Genetic information flow 7
8 CELL DNA : TO STORE GENETIC INFORMATION RNA : INTERMEDIATE OF GENE EXPRESSION Proteins : PRODUCT OF GENE EXPRESSION SPECIFIC FUNCTION Genetic information flow 8
9 The Central Dogma of Molecular Biology Genetic information flow 9
10 Genetic information flow 10
11 DNA vs RNA Sugar: deoxyribose Base: AGCT Double strand Prokaryote: cytosol Eukaryote: nucleus Storage of genetic information Sugar: ribose Base: AGCU Single strand Prokaryote: cytosol Eukaryote: nucleus and cytosol Product of transcription Genetic information flow 11
12 POLYPEPTIDE PROTEIN Polymer Monomer : amino acids (20 aa) Polarity : N - C Peptide bond Genetic information flow 12
13 Amino acids Genetic information flow 13
14 Amino acid One letter symbol Three letter symbol alanine A Ala arginine R Arg asparagine N Asn aspartic acid D Asp cysteine C Cys glutamic acid E Glu glutamine Q Gln glycine G Gly histidine H His isoleucine I Ile leucine L Leu lysine K Lys methionine M Met phenylalanine F Phe proline P Pro serine S Ser threonine T Thr tryptophan W Trp tyrosine Y Tyr valine V Val Genetic information flow 14
15 Amino acid sequences of human insulin Genetic information flow 15
16 Amino acid sequences of interferon α 2a MCDLPQTHSLGSRRTLMLLAQMLRISLFSCLKDRH DFGFPQEEFGNQFQKAETIPVLHEM IQQIFNLFST KDSSAAWDET LLDKFYTELY QQ LNDLEACVIQGV GVTETPLMKEDSILAV RKY FQRITLYLKEKKYSPCA WEVVRAEIMRSFS LSTNLQESLRSKE Length is 165 amino acids Genetic information flow 16
17 The Four Levels of Protein Structure Genetic information flow 17
18 The primary structure of a protein Genetic information flow 18
19 The secondary structure of a protein Heliks α: Ala, Phe, Leu (often) Arg, Glu, Pro (rare) β-turn: Pro, gly β-sheet: Gly, Ala, Ser Pro: distrupts Genetic information flow 19
20 Secondary structure of protein Collagen helix: left handed helix Sequence tends to form collagen helix: Gly X Y X= often Pro Y= unusual amino acid derivative Genetic information flow 20
21 Secondary vs tertiary structure Genetic information flow 21
22 TERTIARY STRUCTURE : α-heliks β-sheet β-bend Non-covalent interactions: Van der waals interaction Electrostatic interaction Hydrogen bonds Hydrophobic interaction Disulphide bond Genetic information flow 22
23 The quarternary structure of a protein Genetic information flow 23
24 QUARTENER STRUCTURE Association of two or more polypeptide strands Can be the same or different polypeptides (homodimer vs heterodimer) Consists of subunits / monomers Weak non covalent interaction: Hydrophobic interaction Hydrogen bond Van der waals interaction Disulfide bond Genetic information flow 24
25 PROTEIN FUNCTION Genetic information flow 25
26 Location of Protein in Bacterial Cell Gram positive Gram negative Genetic information flow 26
27 Nucleic acids vs protein Nucleotides Polarity 5 3 Phosphodiester bond Storage of genetic information Amino acids Polarity N C Peptide bonds Product of gene expression Genetic information flow 27
28 PROTEIN: Therapeutic agents INSULIN, INTERFERON, STREPTOKINASE, ERYTHROPOEITIN, MONOCLONAL ANTIBODI, HBsAg DNA: GENE THERAPY, DNA ANTISENSE, OLIGONUCLEOTIDES, DNA VACCINE Genetic information flow 28
29 FLOW OF GENETIC INFORMATION
30 PRODUCTS OF PHARMACEUTICAL BIOTECHNOLOGY NUCLEIC ACIDS (DNA AND RNA) PROTEINS (DRUG, VACCINES, DETECTION KITS): PRODUCTS OF TRANSCRIPTION AND TRANSLATION OF GENETIC MATERIAL PEPTIDES (SHORTER THAN PROTEINS) Genetic information flow 30
31 The Central Dogma of Molecular Biology Genetic information flow 31
32 WHY IS UNDERSTANDING TRANSCRIPTION IMPORTANT? PROTEIN IS NEEDED IN LARGE AMOUNTS ONE STRATEGY IS TO INCREASE TRANSCRIPTION EFFIENCY Genetic information flow 32
33 TRANSCRIP RIPTION RNA SYNTHESIS (mrna, rrna, trna) USING INFORMATION STORED IN DNA RNA POLYMERASE (PROTEIN) PROKARYOTE (CYTOSOL); EUKARYOTE (NUCLEUS) PROMOTOR (STRONGvsWEAK) TERMINATOR (STRONG vs WEAK) OPERATOR (REGULATION) ACTIVATOR SITES (REGULATION) Genetic information flow 33
34 TRANSCRIPTION mrna Genetic information flow 34
35 TRANSCRIP RIPTION AND BIOTECH CHNOLOGY STRONG PROMOTOR AND TERMINATOR PROMOTOR AND TERMINATOR RECOGNIZED BY HOST RNA POLYMERASE Genetic information flow 35
36 Genetic information flow 36
37 Genetic information flow 37
38 REVERSE TRANSCRIP RIPTION RNA DNA (cdna) REVERSE TRANSCRIPTASE VIRUS (RETROVIRUS, HIV) AND Hepatitis Bvirus (HBV) Genetic information flow 38
39 Genetic information flow 39
40 TRANSLATION SYNTHESIS OF POLYPEPTIDES/PROTEINS USING INFORMATION IN mrna mrna IS USED AS TEMPLATE (trna AND rrna ARE NOT TRANSLATED, BUT THEY FUNCTION DIRECTLY LOCATION IN RIBOSOME COMPONENTS THAT ARE REQUIRED mrna RIBOSOME BINDING SITE (RBS) FOR PROKARYOTE AUG (START CODON) UAA, UGA, UAG (STOP CODONS) Genetic information flow 40
41 Genetic information flow 41
42 Stop codons Start codon Genetic information flow 42
43 ORGANIZATION ON OF PROKARYOTIC GENE Coding sequence 5 3 Promoter RBS Inisiation of Start codon transcription Stop codon 3 5 terminator Components of transcription: promotor, terminator, site of transcription initiation Components of translation: RBS, start codon, stop codons Genetic information flow 43
44 Genetic information flow 44
45 Eukaryote vs Prokaryote Genetic information flow 45
46 Genetic information flow 46
47 ORGANIZATION ON OF EUKARYOTIC GENE Genetic information flow 47
48 TRANSCRIP RIPTION (EUKARYOT OTE) DIRECT PRODUCT OF TRANSCRIPTION: PRIMARY RNA (INTRON AND EXON) ADDITION OF CAP (5 END) AND POLY A TAIL (3 END) REMOVAL OF INTRON (SPLICING) MATURE mrna IS TRANSPORTED TO CYTOSOL Genetic information flow 48
49 WHY IS KNOWLEDGE OF TRANSCRIPTION IN EUKARYOTE IMPORTANT? MANY PROTEINS FROM EUKARYOTE ARE USED AS THERAPEUTIC PROTEINS INFORMATION IN PROTEIN MOLECULES IS LOCATED IN MATURE mrna WITHOUT INTRON TO ISOLATE DNA ENCODING FOR PROTEIN IS STARTED BY ISOLATING MATURE mrna THEN CONVERTED TO DNA (mrna DNA) Genetic information flow 49
50 REVERSE TRANSCRIPTION PROBLEM IN EUKARYOTE IS THAT GENE CONTAINS INTRON DAN EXON SYNTHESIS OF DNA USING mrna AS TEMPLATE THE RESULT OF THE DNA IS CALLED cdna, complementary DNA (DOES NOT CONTAIN INTRON) CATALYZED BY REVERSE TRANSCRIPTASE Genetic information flow 50
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