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How to Study Genome 1. Genetic Material 2. Expression Product DNA as Genetic Material DNA encodes all the information in the cell The composition of the DNA is the same in all cells within an organism – Variation among different cells is achieved by reading the DNA differently DNA contains four bases that encode all the information to make an organism’s life DNA DNA Consists of four kinds of bases (A,C,G,T) joined to a sugar phosphate backbone Bases carry the genetic information while the phosphate backbone is structural Two complementary strands of bases (C-G) and (A-T) DNA (Deoxyribonucleic Acid) a Polymer of Deoxyribonucleotide Units Deoxyribonucleotide Deoxy Ribo Nucleotide Deoxy ribo nucleotide Ribose= Five Carbon Sugar Molecule HOCH2 O OH 5´ H 4´ H 3´ HO H 1´ 2´ H OH Ribose (RNA) HOCH2 O OH 5´ H 4´ H 3´ HO H 1´ 2´ H H Deoxyribose (DNA) Backbone Sugar Molecules Nucleotide Phosphate group 5-carbon sugar Nitrogenous base Nucleotide Phosphate Group O O=P-O O 5 CH2 O N C1 C4 Sugar (deoxyribose) C3 C2 Nitrogenous base (A, G, C, or T) DNA Double Helix “Rungs of ladder” Nitrogenous Base (A,T,G or C) “Legs of ladder” Phosphate & Sugar Backbone DNA Double Helix 5 O 3 3 O P 5 O C G 1 P 5 3 2 4 4 2 3 1 P T 5 A P 3 O O P 5 O 3 5 P DNA It is Composed of Four Different Ribonucleotides NH2 C N C N CH 9 C N C N H H Adenine HN O C O C 1 N H C C O C Two Purines N H N C CH 9 C N C N H2 N H Guanine CH3 H Thymine Two Pyrimidines O NH2 C H N C C 1 C N H H Cytosine Nitrogenous Bases PURINES Adenine (A) 2. Guanine (G) 1. A or G PYRIMIDINES 3. Thymine (T) 4. Cytosine (C) T or C BASE-PAIRINGS Purines Pyrimidines Base Pairs Adenine (A) Thymine (T) A=T Guanine (G) Cytosine (C) C G # of H-Bonds 2 3 3 H-bonds G C BASE-PAIRINGS H-bonds G C T A Base Pairing Occurs Through Hydrogen Bonds G-C A-T Chargaff’s Rule Adenine must pair with Thymine Guanine must pair with Cytosine Their amounts in a given DNA molecule will be about the same. T A G C The DNA Backbone is a Deoxyribose Polymer O O P O - Deoxyribose sugars are linked by Phosphodiester Bonds O OH H2 C 5´ O H H 1´ H - O3´ 2´ H H O P O O OH H2 C 5´ O H H 5´-p 1´ H O3´ 2´H O P O O H OH H2 C 5´ O H H 1´ H 3´ HO 2´ H H 5´ 3´-OH 3´ O O P O - 5´ O 5´ 3´ H2 C 5´ O OH H H 1´ H - 3´ 2´ O H H O P O O H2 C 5´ O OH H H 1´ H - O3´ 2´ H H O P O O H2 C 5´ O OH H H 1´ H 3´ 2´ H HO H 3´ 3´ 5´ O O P O O O P O O O H2 C 5´ O OH H H 1´ H 3´ 2´ H O H O P O O H2 C 5´ O OH H H 1´ H 3´ 2´ H O H O P O O OH H2 C 5´ O Base H H 1´ H 3´ 2´ H O H O P O O OH H2 C 5´ O Base H H 1´ H 3´ 2´ H O H O P O O H2 C 5´ O OH H H 1´ OH H2 C 5´ O Base H H 1´ H 3´ 2´ H HO H H 3´ 2´ H HO H DeoxyRibonucleotide NH2 N HC O- NH2 N HC N CH N O O O N P O P O P OCH2 O H H OOOH H HO H CH N N HOCH2 O H H H HO N Deoxyadenosine 5´-triphosphate (dATP) H H DeoxyRibonucleoside Deoxyadenosine O O P O O C CH3 HN C O O C C N H H2 C O H H H T A C G 5´ 3´ H O H NH2 O P O C H N C O O C C N H H2 C O H H H H O H O O P O C CH3 HN C O O C C H H2 C O N H H - H HO H H T A 3´ 5´ G C = A T Double-stranded DNA Forms a Double Helix RNA A polymer composed of nucleotides that contain the sugar ribose and one of the four bases cytosine, adenine, guanine and uracile Polynucleotide containing ribose sugar and uracil instead of thymine Genetic material of some viruses Primary agent for transferring information from the genome to the protein synthetic machinery phosphate group URACIL (U) base with a single-ring structure sugar (ribose) Types of RNA Three types of RNA: a) messenger RNA (mRNA) b) transfer RNA (tRNA) c) ribosome RNA (rRNA) Remember: all produced in the nucleus A. Messenger RNA (mRNA) Carries the information for a specific protein. Made up of 500 to 1000 nucleotides long. Made up of codons (sequence of three bases: AUG methionine). Each codon, is specific for an amino acid. A. Messenger RNA (mRNA) start codon mRNA A U G G G C U C C A U C G G C G C A U A A codon 1 protein methionine codon 2 codon 3 glycine serine codon 4 isoleucine codon 5 codon 6 glycine alanine codon 7 stop codon Primary structure of a protein aa1 aa2 aa3 peptide bonds aa4 aa5 aa6 B. Transfer RNA (tRNA) Made up of 75 to 80 nucleotides long. Picks up the appropriate amino acid floating in the cytoplasm (amino acid activating enzyme) Transports amino acids to the mRNA. Have anticodons that are complementary to mRNA codons. Recognizes the appropriate codons on the mRNA and bonds to them with H-bonds. anticodon codon in mRNA anticodon tRNA molecules amino acid attachment site amino acid amino acid attachment site OH The structure of transfer RNA (tRNA) Transfer RNA (tRNA) amino acid attachment site methionine U A C anticodon amino acid C. Ribosomal RNA (rRNA) Made up of rRNA is 100 to 3000 nucleotides long. Important structural component of a ribosome. Associates with proteins to form ribosomes. Ribosomes Large and small subunits. Composed of rRNA (40%) and proteins (60%). Both units come together and help bind the mRNA and tRNA. Two sites for tRNA a. P site (first and last tRNA will attach) b. A site Ribosomes Origin Cytosol (eukaryotic ribosome) Chloroplasts (prokaryotic ribosome) Complete ribosome 80 S Ribosomal subunit 40 S 60 S rRNA components 18 S 5S 5.8 S 25 S Proteins 70 S 30 S 50 S 16 S 4.5 S 5 S 23 S C. 24 C. 35 30 S 50 S 18 S 5S 26 S C. 33 C. 35 Mitochondrion 78 S (prokaryotic ribosome) C.30 C.50 Ribosomes Large subunit P Site A Site mRNA A U G Small subunit C U A C U U C G Study of Genetic Material Number of chromosomes Banding Number of nucleotides Sequencing Structural genes cloning Non-structural genes Molecular marker Central Dogma of Biology DNA, RNA, and the Flow of Information Replication Transcription Translation Central Dogma (Modifications) (2)Ribozymes Transcription DNA RNA Translation Protein (1) Reverse transcription Replication (2)Self Replication (3)Self Replication DNA Replication 1. Origin of Replication 2. Strand Separation 3. Priming 4. Synthesis of new strand DNA DNA Replication Origins of replication 1. 5’ 3’ Replication Forks: hundreds of Y-shaped regions of replicating DNA molecules where new strands are growing. Parental DNA Molecule 3’ Replication Fork 5’ DNA Replication Origins of replication 2. Replication Bubbles: a.Hundreds of replicating bubbles (Eukaryotes). b.Single replication fork (bacteria). Bubbles Bubbles DNA Replication Strand Separation: 1. Helicase Enzyme which catalyze the unwinding and separation (breaking H-Bonds) of the parental double helix. 2. Single-Strand Binding Proteins Proteins which attach and help keep the separated strands apart. DNA Replication Strand Separation: 3. Topoisomerase enzyme which relieves stress on the DNA molecule by allowing free rotation around a single strand. Enzyme DNA Enzyme DNA Replication Priming 1. RNA primers before new DNA strands can form, there must be small pre-existing primers (RNA) present to start the addition of new nucleotides (DNA Polymerase). 2. Primase enzyme that polymerizes (synthesizes) the RNA Primer. DNA Replication Synthesis of the new DNA Strands 1. DNA Polymerase with a RNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the 5’ to 3’ direction. 5’ 3’ Nucleotide DNA Polymerase RNA Primer 5’ DNA Replication Synthesis of the new DNA Strands 2. Leading Strand synthesized as a single polymer in the 5’ to 3’ direction. 5’ 3’ 5’ Nucleotides DNA Polymerase RNA Primer DNA Replication Synthesis of the new DNA Strands 3. Lagging Strand It also synthesized in the 5’ to 3’ direction, but discontinuously against overall direction of replication. Leading Strand 5 ’ 3’ DNA Polymerase RNA Primer 3’ 5’ 5’ 3’ 3’ 5’ Lagging Strand DNA Replication Synthesis of the new DNA Strands 4. Okazaki Fragment series of short segments on the lagging strand. DNA Polymerase Okazaki Fragment RNA Primer 5’ 3’ Lagging Strand 3’ 5’ DNA Replication Synthesis of the new DNA Strands 5. DNA ligase a linking enzyme that catalyzes the formation of a covalent bond from the 3’ to 5’ end of joining stands. Example: joining two Okazaki fragments together. DNA ligase 5’ 3’ Okazaki Fragment 1 Lagging Strand Okazaki Fragment 2 3’ 5’ DNA Replication Synthesis of the new DNA Strands 6. Proofreading initial base-pairing errors are usually corrected by DNA polymerase. DNA Replication Semiconservative Model Watson and Crick the two strands of the parental molecule separate, and each functions as a template for synthesis of a new complementary strand. DNA Template Parental DNA New DNA DNA Repair Excision repair 1. Damaged segment is excised by a repair enzyme (there are over 50 repair enzymes). 2. DNA polymerase and DNA ligase replace and bond the new nucleotides together. Gene Expression Transcription Translation What is gene expression? Biological processes, such as transcription, and in case of proteins, also translation, that yield a gene product. A gene is expressed when its biological product is present and active. Gene expression is regulated at multiple levels. Expression of Genetic Information Beadle and Tatum (1941) showed in the fungus Neurospora crassa that there is a relationship between a gene and each enzyme needed in a biochemical pathway, resulting in the one geneone enzyme hypothesis (now modified to one gene-one polypeptide, since not all proteins are enzymes and some require more than one polypeptide). Expression of Genetic Information Production of proteins requires two steps: Transcription involves an enzyme (RNA polymerase) making an RNA copy of part of one DNA strand. There are four main classes of RNA: i. Messenger RNAs (mRNA), which specify the amino acid sequence of a protein by using codons of the genetic code. ii. Transfer RNAs (tRNA). iii. Ribosomal RNAs (rRNA). iv. Small nuclear RNAs (snRNA), found only in eukaryotes. Translation converts the information in mRNA into the amino acid sequence of a protein using ribosomes, large complexes of rRNAs and proteins. Steps of gene expression Transcription – DNA is read to make a mRNA in the nucleus of our cells Translation – Reading the mRNA to make a protein in the cytoplasm mRNA Synthesis DNA template: 3’-to-5’ RNA synthesis: 5’-3’; no primer needed 59 Expression of Genetic Information Only some of the genes in a cell are active at any given time, and activity also varies by tissue type and developmental stage. Regulation of gene expression is not completely understood, but it has been shown to involve an array of controlling signals. a. Jacob and Monod (1961) proposed the operon model to explain prokaryotic gene regulation, showing that a genetic switch is used to control production of the enzymes needed to metabolize lactose. Similar systems control many genes in bacteria and their viruses. b. Genetic switches used in eukaryotes are different and more complex, with much remaining to be learned about their function. Prokaryotic gene organization Prokaryotic transcriptional regulatory regions (promoters and operators) lie close to the transcription start site Functionally related genes are frequently located near each other These “operons” are transcribed into a single mRNA with internal translation initiation sites Prokaryotic Gene Expression Promoter Cistron1 Cistron2 CistronN Terminator Transcription RNA Polymerase mRNA 5’ 3’ 1 2 Translation C N N N Ribosome, tRNAs, Protein Factors C N C 1 2 Polypeptides 3 Operons a promoter plus a set of adjacent genes whose gene products function together. usually contain 2 –6 genes, (up to 20 genes) these genes are transcribed as a polycistronic transcript. relatively common in prokaryotes rare in eukaryotes Operon System The lactose (lac) operon Pi I Q3 P Q1 Z Q2 Y • Contains several elements – – – – lacZ gene = β-galactosidase lacY gene = galactosidase permease lacA gene = thiogalactoside transacetylase lacI gene = lac repressor – – – – Pi = promoter for the lacI gene P = promoter for lac-operon Q1 = main operator Q2 and Q3 = secondary operator sites (pseudo-operators) A Regulation of the lac operon Pi I Q3 P Q1 Z Q2 LacZ lacI repressor Y LacY Inducer molecules→ Allolactose: - natural inducer, degradable IPTG (Isopropylthiogalactoside) - synthetic inducer, not metabolized A LacA Eukaryotic gene expression Eukaryotic gene Expression Transcripts begin and end beyond the coding region (5’UTR and 3’UTR) The primary transcript is processed by: a. 5’ capping b. 3’ formation polyA c. splicing Mature transcripts are transported to the cytoplasm for translation Regulation of gene expression Promoter 1. DNA replication Gene (red) with an intron (green) Plasmid single copy vs. multicopy plasmids 2. Transcription 3. Posttranscriptional processing 4. Translation 5. Posttranslational processing Primary transcript mRNA degradation Mature mRNA inactive protein active protein Protein degradation Gene regulation of the transcription Condition 2 1 Chr. I 1 10 Chr. II Chr. III 2 19 “turned “turned “turned off” off” on” on” 4 5 6 7 8 3 11 12 20 21 22 constitutively expressed gene 13 14 15 16 23 induced gene 24 9 17 25 18 26 repressed gene inducible/ repressible genes Gene regulation upregulated gene expression 1 2 10 19 Condition 43 down regulated gene expression 3 4 11 12 20 21 22 constitutively expressed gene 5 7 8 13 14 15 16 17 23 6 24 25 9 18 26 Definitions Constitutively expressed genes Genes that are actively transcribed (and translated) under all experimental conditions, at essentially all developmental stages, or in virtually all cells. Inducible genes Genes that are transcribed and translated at higher levels in response to an inducing factor Repressible genes Genes whose transcription and translation decreases in response to a repressing signal Housekeeping genes –genes for enzymes of central metabolic pathways (e.g. TCA cycle) –these genes are constitutively expressed –the level of gene expression may vary Modulators of transcription Modulators: (1) specificity factors, (2) repressors, (3) activators 1. Specificity factors: Alter the specificity of RNA polymerase Examples: σ-factors (s70, s32 ) s70 Standard promoter s32 Housekeeping gene Heat shock promoter Heat shock gene Modulators of transcription 2. Repressors: mediate negative gene regulation may impede access of RNA polymerase to the promoter actively block transcription bind to specific “operator” sequences (repressor binding sites) Repressor binding is modulated by specific effectors Effector (e.g. endproduct) Repressor Operator Promoter Coding sequence Negative regulation Repressor Effector Example: lac operon RESULT: Transcription occurs when the gene is derepressed Negative regulation Repressor Effector (= co-repressor) Example: pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism Modulators of transcription 3. Activators: mediate positive gene regulation bind to specific regulatory DNA sequences (e.g. enhancers) enhance the RNA polymerase -promoter interaction and actively stimulate transcription common in eukaryotes Activator RNA pol. promoter Coding sequence Positive regulation Activator RNA polymerase Positive regulation Activator Effector RNA polymerase Post-Transcriptional Modification in Eukaryotes primary transcript formed first then processed (3 steps) to form mature mRNA then transported to cytoplasm Step 1: 7- methyl-guanosine “5’-cap” added to 5’ end Step 2: introns spliced out; exons link up Step 3: Poly-A tail added to 3’ end mature mRNA 5’-cap- exons -3’ PolyA tail 81 Intron Splicing in Eukaryotes • Exons : coding regions • Introns : noncoding regions • Introns are removed by “splicing” GU at 5’ end of intron AG at 3’ end of intron 82 Splicesomes Roles in Splicing out Intron RNA splicing occurs in small nuclear ribonucleoprotein particles (snRNPS) in spliceosomes 83 Splicesomes Roles in Splicing out Intron 5’ exon then moves to the 3’ splice acceptor site where a second cut is made by the spliceosome Exon termini are joined and sealed 1 2 1 2 1 2 84 Translation Three parts: 1. initiation: start codon (AUG) 2. elongation: 3. termination: stop codon (UAG) Translation Large subunit P Site A Site mRNA A U G Small subunit C U A C U U C G Initiation aa1 aa2 2-tRNA 1-tRNA anticodon hydrogen bonds U A C A U G codon G A U C U A C U U C G A mRNA peptide bond aa3 aa1 aa2 3-tRNA 1-tRNA anticodon hydrogen bonds U A C A U G codon 2-tRNA G A A G A U C U A C U U C G A mRNA aa1 peptide bond aa3 aa2 1-tRNA 3-tRNA U A C (leaves) 2-tRNA A U G G A A G A U C U A C U U C G A mRNA Ribosomes move over one codon aa1 peptide bonds aa4 aa2 aa3 4-tRNA 2-tRNA A U G 3-tRNA G C U G A U G A A C U A C U U C G A A C U mRNA aa1 peptide bonds aa4 aa2 aa3 2-tRNA 4-tRNA G A U (leaves) 3-tRNA A U G G C U G A A C U A C U U C G A A C U mRNA Ribosomes move over one codon aa1 peptide bonds aa5 aa2 aa3 aa4 5-tRNA U G A 3-tRNA 4-tRNA G A A G C U G C U A C U U C G A A C U mRNA peptide bonds aa1 aa5 aa2 aa3 aa4 5-tRNA U G A 3-tRNA G A A 4-tRNA G C U G C U A C U U C G A A C U mRNA Ribosomes move over one codon aa4 aa5 Termination aa199 aa3 primary structure of a protein aa2 aa200 aa1 200-tRNA A C U mRNA terminator or stop codon C A U G U U U A G Translation Ribosome Amino Acids forming Peptide chain P Site Met His Tyr A Site Val Pro 3’ E Site tRNA anti-codon 5’ codon AUG CAU GGA UAC GUA CCU mRNA strand Translation The difference • Eukaryotic and prokaryotic translation can react differently to certain antibiotics Puromycin an analog tRNA and a general inhibitor of protein synthesis Cycloheximide only inhibits protein synthesis by eukaryotic ribosomes Chloramphenicol, Tetracycline, Streptomycin inhibit protein synthesis by prokaryotic ribosome End Product The end products of protein synthesis is a primary structure of a protein. A sequence of amino acid bonded together by peptide bonds. aa2 aa1 aa3 aa4 aa5 aa199 aa200 Polyribosome • Groups of ribosomes reading same mRNA simultaneously producing many proteins (polypeptides). incoming large subunit 1 incoming small subunit 2 3 4 polypeptide 5 6 7 mRNA TYPES OF PROTEINS Enzymes (Helicase) Carrier (Haemoglobine) Immunoglobulin (Antibodies) Hormones (Steroids) Structural (Muscle) Ionic (K+,Na+) Coupled transcription and translation in bacteria original base triplet in a DNA strand As DNA is replicated, proofreading enzymes detect the mistake and make a substitution for it: a base substitution within the triplet (red) POSSIBLE OUTCOMES: OR One DNA molecule carries the original, unmutated sequence VALINE PROLINE The other DNA molecule carries a gene mutation THREONINE VALINE LEUCINE HISTIDINE GLUTAMATE A summary of transcription and translation in a eukaryotic cell