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How to Study DNA 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 Meischer (1860’s) DNA slightly acidic and composed of large amounts of phosphorous and nitrogen 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 (CG) and (A-T) DNA primary structure DNA (Deoxyribonucleic Acid) a Polymer of Deoxyribonucleotide Units Deoxyribonucleotide Deoxy Ribo Nucleotide 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 DeoxyRibonucleotide Phosphate Group O O=P-O O 5 CH2 O N C1 C4 Sugar (Deoxyribose) C3 C2 Nitrogenous base (A, G, C, or T) DeoxyRibonucleotide 5-carbon sugar (Deoxy ribose) Nitrogenous base Phosphate group 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 NITROGEN BASES It is composed of four different nitrogen bases 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 More of Chargaff’s Work In any sample of DNA the following is true: – Amount of Cytosine = Amount of Guanine – Amount of Thymine = Amount of Adenine Polymerization of Nucleotides 3’ carbon 5’ carbon Polynucleotides (Nucleic Acids) 5’ end 3’ end 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 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 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 RIBO NUCLEIC ACID 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 uracile 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. Codon There are 20 different possible amino acids to make from different codons. 3 possible stop codon 1 start codon TAC on DNA AUG on RNA Codon Chart Start Codon Codon Chart 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 70 S 30 S 50 S Mitochondrion 78 S (prokaryotic ribosome) 30 S 50 S rRNA components 18 S 5S 5.8 S 25 S 16 S 4.5 S 5 S 23 S Proteins 18 S 5S 26 S C. 33 C. 35 C.30 C.50 C. 24 C. 35 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. Replication Forks: Hundreds of Y-shaped regions of replicating DNA molecules where new strands are growing. 3’ 5’ 3’ Parental DNA Molecule Replication Fork 5’ DNA Replication Origins of replication 2. Replication Bubbles: Hundreds of replicating bubbles (eukaryotes). Bubbles Bubbles DNA Replication Strand Separation: Unwinding and separation of the parental double helix DNA 1. Helicase Enzyme which catalyze the breaking H-Bonds between 2 nitrogen bases from different strand. 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: The attachment of complementary primer on the single stranded DNA 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: The additional of nucleotide on RNA primer 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. What is gene expression? The activation of a gene that results in a protein. 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 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). 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 cells Translation – Reading the mRNA to make a protein in the cytoplasm Three (3) regulatory elements of transcription Coding region: DNA that code for a specific polypeptide (protein) Promoter : DNA segment that recognizes RNA polymerase Operator : Element that serves as a binding site for an inhibitor protein (modulator) that controls transcription Promoter Region on DNA Upstream from transcription start site Initial binding site of RNA polymerase and initiation factors (IFs) Promoter recognition: a prerequisite for initiation Prokaryotic promoter regions -35 site = TTGACA -10 site: “TATA” box 66 Promoter Region on DNA (TATA box) Pol II Eukaryotic Promoter Elements GC box ~200 bp CCAAT box ~100 bp TATA box ~30 bp Transcription start site (TSS) Pol II Eukaryotic Promoter Elements Cap Region/Signal: nCAGTnG TATA box: (~ 25 bp upstream) TATAAAnGCCC CCAAT box: (~100 bp upstream) TAGCCAATG GC box: (~200 bp upstream) A T A G G C G nGA Prokaryotic and eukaryotic 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 Expression mainly by controlling transcription 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 Genes that work together are located together A promoter plus a set of adjacent genes whose gene products function together. They are controlled as a unit They usually contain 2 –6 genes (up to 20 genes) These genes are transcribed as a polycistronic transcript. It is relatively common in prokaryotes It is 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 The lac operon: model for gene expression Includes three protein synthesis coding region-sometimes called "genes" as well as region of chromosome that controls transcription of genes Genes for proteins involved in the catabolism or breakdown of lactose When lactose is absent, no transcription of gene since no need for these proteins When lactose is present, transcription of genes takes place so proteins are available to catalyze breakdown of lactose Eukaryotic gene Eukaryotic gene Expression 1.Transcripts begin and end beyond the coding region 2.The primary transcript is processed by: 5’ capping 3’ formation / polyA splicing 3.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 Regulation of gene expression Gene expression is regulated—not all genes are constantly active and having their protein produced The regulation or feedback on gene expression is how the cell’s metabolism is controlled. This regulation can happen in different ways: 1. Transcriptional control (in nucleus): e.g. chromatin density and transcription factors 2. Posttranscriptional control (nucleus) e.g. mRNA processing 3. Translational control (cytoplasm) e.g. Differential ability of mRNA to bind ribosomes 4. Posttranslational control (cytoplasm) e.g. changes to the protein to make it functional When regulation of gene expression goes wrong—cancer! 1. Transcription control Eukaryotic gene expression 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 2. 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 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 88 Splicesomes Roles in Splicing out Intron RNA splicing occurs in small nuclear ribonucleoprotein particles (snRNPS) in spliceosomes 89 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 90 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 Prokaryotes vs eukaryotes: key points Prokaryotes Eukaryotes Operons (functional grouping) Monocistronic RNAs (One mRNA, one protein) Polycistronic mRNAs (single mRNA, multiple ORFs) Ribosome scanning No splicing Regulatory sequences lie near (~100 bp) the start site Translation is concurrent with transcription Often spliced Regulatory sequences can be far (>1 kb) from the start site RNA processing is concurrent with transcription; translation occurs in a separate compartment 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