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Molecular Biology Informational Macromolecules DNA/RNA/PROTEINS Cells Chemical Machines Coding devices I. The Blueprint of Life: Structure of the Bacterial Genome • 4.1 Macromolecules and Genes • 4.2 The Double Helix • 4.3 Genetic Elements: Chromosomes and Plasmids 4.1 Macromolecules and Genes • Functional unit of genetic information is the gene • Genes are in cells and are composed of DNA 4.1 Macromolecules and Genes • Three informational macromolecules in cell – DNA – RNA – Protein 4.1 Macromolecules and Genes • Genetic information flow can be divided into three stages – Replication: DNA is duplicated (Figure 4.3) – Transcription: information from DNA is transferred to RNA • mRNA (messenger RNA): encodes polypeptides • tRNA (transfer RNA): plays role in protein synthesis • rRNA (ribosomal RNA): plays role in protein synthesis – Translation: information in RNA is used to build polypeptides Replication Transcription C A G T T A DNA Dark green strand is template for RNA synthesis. C C G T G A G C T T DNA polymerase 5′ T A 3′ C A C T G G A G G C G A T G G C G A C U U U C U C C T G A G C A A G G A G A G C C C T T C G C G T C A C G 3′ G C 5′ G mRNA 5′ A A C Protein T G C T C G U RNA polymerase A Translation Messenger RNA is template for protein synthesis. tRNA mRNA G A 5′ U G C G A C U U G G A G C C U G A U U G G A G G A C C C 3′ Ribosome Figure 4.3 Microbial Genetics Structure of INFORMATIONAL MOLECULES STRUCTURE OF GENOME WHY DNA? MUTATIONS, WHY CHANGE? PROOF DNA KEY MOLECULE CENTRAL DOGMA DNA MAKES RNA MAKES PROTEIN FEATURES OF DNA 1. STORES GENETIC INFO IN ITS BASE SEQUENCES 2. GREAT PHYSICAL & CHEMICAL STABILITY 3. DNA TRANSFER GENETIC INFO TO PROGENY DNA USING OLD STAND AS TEMPLATE FOR NEW STRAND 4. GENETIC CHANGE CAN OCCUR!! MUTATIONS, WITHOUT LOST OF PARENT INFO ONE DAUGHTER CELL IDENTICAL TO PARENT ONE DAUGHTER CELL SLIGHTLY DIFFERENT 5. MUTATION: CHEMICAL ALTERATION REPLICATION ERROR HYDROPHOBIC !. WATER FREE KEEPS RATE LOW 2. REPAIR SYSTEM Proof DNA Information Molecule 1.1928 Fred Griffth Streptococcus pneumoniae TRANSFORMING PRINCIPLE 2. 1944 Oswald Avery, Colin Macloed & Maclyn McCarty PURIFIED Cell extract PROVED TO BE DNA 3. Blender Experiment: Alfred Herchey & Martha Chase E. coli and Phage T2 Transforming Principle Streptococcus pneumoniae Smooth colonies (S) Capsulated Virulence Factor Rough colonies (R) Non-capsulated Non-virluent 1. Heat Killed (S) non-lethal in mice 2. Mixed Live (R) bacteria & Dead (S) Bacteria DEATH (MICE) 3. DEAD MICE-ISOLATED (S) BACTERIA (ALSO OCCURRED ON NUTRIENT MEDIA & CELL-FREE EXTRACT) 4. S-CELL EXTRACT = ‘TRANSFORMING PRINCIPLE” UNKNOWN! Avery, Macloed, & Mccarty Purified S “cell Extract” Proved to be DNA 1. Isolated DNA from cell Extract of S cells 2. Added DNA to live R cells= 1/104 was S colony 3. S cells and R cells remained after growth media 4. Polysaccharide Capsule Material + R cells- R cells 1. Chemical Test proved it was DNA 2. Physical Test proved it was DNA 3. Transforming Activity NOT LOST! proteoases Ribonucleases 4. Transforming Activity LOST DNAase Blender Experiment E. Coli Phage T2 1. Radiolabelled DNA 32 P Protein 35S Amino Acids Cysteine/methionine 2. Mixed T2 Phage with culture of E. coli cells 3. Blended mixed after short period of ATTACHMENT 4.BLENDED MIXTURE & CENTRFUGED 5. Progeny 32 P in pellet material and 35S in supernatant ROSALAND FRANKLIN 1951 X-RAY DIFFRACTION DNA CRYSTALS HELICAL STRUCTURE CLOSELY PACKAGED/ NITROGEN SIDE CHAINS PO4 GROUPS EYWIN CHARGAFF 1950 BIOCHEMICAL ANALYSES COMPLIMENARY COMPOSITION A=T G=C WATSON/CRICK/WILKINS BUILT MODEL OF DNA DOUBLE HELIX ANTIPARALLEL STRANDS 10 BP/HELIX TURN EQUAL WIDTH 34A° (10-7) ANGSTRAM MAJOR GROUVES/MINOR GROUVES (PROTEIN BINDING) Semiconservative Melelson & Stahl 15N Medium 14N 21 14N % 15N 22 4.1 Macromolecules and Genes • Genetic information flow can be divided into three stages – Replication: DNA is duplicated (Figure 4.3) – Transcription: information from DNA is transferred to RNA • mRNA (messenger RNA): encodes polypeptides • tRNA (transfer RNA): plays role in protein synthesis • rRNA (ribosomal RNA): plays role in protein synthesis – Translation: information in RNA is used to build polypeptides Polysystronic 4.1 Macromolecules and Genes • Central dogma of molecular biology – DNA to RNA to protein • Eukaryotes: each gene is transcribed individually • Prokaryotes: multiple genes may be transcribed together 4.3 Genetic Elements: Chromosomes and Plasmids • Genome: entire complement of genes in cell or virus • Chromosome: main genetic element in prokaryotes • Other genetic elements include virus genomes, plasmids, organellar genomes, and transposable elements 4.3 Genetic Elements: Chromosomes and Plasmids • Viruses contain either RNA or DNA genomes – Can be linear or circular – Can be single- or double-stranded • Plasmids: replicate separately from chromosome – Great majority are double-stranded – Most are circular – Generally beneficial for the cell (e.g., antibiotic resistance) – NOT extracellular, unlike viruses 4.3 Genetic Elements: Chromosomes and Plasmids • Chromosome is a genetic element with "housekeeping" genes – Presence of essential genes is necessary for a genetic element to be called a chromosome • Plasmid is a genetic element that is expendable and rarely contains genes for growth under all conditions 4.3 Genetic Elements: Chromosomes and Plasmids • Transposable elements – Segment of DNA that can move from one site to another site on the same or a different DNA molecule – Inserted into other DNA molecules – Three main types: • Insertion sequences • Transposons • Special viruses Ways DNA GETS INTO CELLS TRANSFORMATION: Naked DNA or Plasmids incorporated into cell genome Competent Host Cells 1. Chemical used to open “Porins” cell transport channels 2. Electroporation- Pulsed electrical field- electric shotgun 3. Markers used to test if cell are Transformed Antibiotic resistantPGLO fluorscent green protein 4. Plate cells on selective growth media view colonies /UV light Transduction/ Viruses Need host to grow DNA 0r RNA NOT Both Ss or ds Structure Capsid coat or shell- PROTEIN/ ICOSAHEDRON Genetic Molecule DNA or RNA Host Specific: T2 phage/E. coli Type of Infection Lytic- 30 min 5000/1000 progeny cell lyses BURST Lysogenic Phage- linear DNA incorporated cell DNA CONJUGATION CELL SEX CELL TO CELL CONTACT-EXCHANGE BODY FLUIDS (DNA) SEX PILI F+-------F+ CONFERES RESISTANCE (ANTIBIOTIC) DEGRADATION CAPACITY Plasmids 4.2 The Double Helix • Four nucelotides are found in DNA (Figure 4.1): – Adenine (A) – Guanine (G) – Cytosine (C) – Thymine (T) • Backbone of DNA chain is alternating phosphates and the pentose sugar deoxyribose • Phosphates connect 3′-carbon of one sugar to 5′-carbon of the adjacent sugar Complementary DNA strands 3′-Hydroxyl 5′-Phosphate Hydrogen bonds Phosphodiester bond 5′-Phosphate 3′-Hydroxyl Figure 4.4 E coli Nucloid/Genome Single genome Circular Double stranded Complimentary binding Anti parallel Super coiled Major/minor grooves 4.6 X 106 bp 2000/3000 genes 4.2 The Double Helix • Size of DNA molecule is expressed in base pairs • 1,000 base pairs = 1 kilobase pair = 1 kbp • 1 million base pairs = 1 megabase pair = 1Mbp • E. coli genome = 4.64 Mbp • Each base pair takes up 0.34 nm of length along the helix • 10 base pairs make up 1 turn of the helix 4.2 The Double Helix • Supercoiled DNA: DNA is further twisted to save space (Figure 4.6) – Negative supercoiling: double helix is underwound – Positive supercoiling: double helix is overwound • Relaxed DNA: DNA has number of turns predicted by number of base pairs • Negative supercoiling is predominantly found in nature • DNA gyrase: introduces supercoils into DNA (Figure 4.7) Relaxed circular DNA Nucleoid Break one strand. Nick Proteins Relaxed nicked circular DNA Supercoiled circular DNA Rotate one end of broken strand around helix and seal. Supercoiled domain Chromosomal DNA with supercoiled domains Figure 4.6 DNA gyrase makes double-strand break One part of circle is laid over the other. Relaxed circle Helix makes contact in two places. Unbroken helix is passed through the break. Double-strand break resealed behind unbroken helix Following DNA gyrase activity, two negative supercoils result. Supercoiled DNA Figure 4.7 DNA REPLICATION 1. Parent to offspring: Semi- conservative 2. Enormous supply of ATP to unwind strands 3. ssDNA Template using bp (sense Strand) 4. N.T. (bp) added 1 X 1 to end of growing chain POLYMERASE ENZYME 5. Sequence of growing chain: daughter Cell COMPLEMENTARY to base sequence in PARENT Strand (Sense Strand) e.g. AT DNA Enzymes Polymerase catalyzes addition of N.T. synthesizes DNA 5’-------------3’ de NOVO NEEDS 3’0H for synthesis Primase- RNA short strand (15nt) COMPLEMENATORY TO TEMPLATE DNA REPLICATION FORK- Bidirectional Theta Structure ORI- ORIGIN OF REPLICATION LEADING STRAND- CONTINOUS (1 PRIMER) DISCONTINOUS- PRIMED MANY TIMES Helicases SSB DNA Topoisomerases Gyrase DNA polymerase I DNA polymerase III 5’---3’ DNA primase DNA ligase REPLISOME COMPLEX TOPOISOMERASES T1 NICK 1 STRAND T2 NICKS 2 STRANDS GYRASE- REMOVES SUPERCOILING HELICASE- UNWINDS DNA STRANDS PRIMASE- SYNTHESIS OF RNA PRIMER SSB- SINGLE STRANDED DNA BINDING PROTEINS Figure 4.9 4.3 Genetic Elements: Chromosomes and Plasmids • The Escherichia coli chromosome • Escherichia coli is a useful model organism for the study of biochemistry, genetics, and bacterial physiology • The E. coli chromosome from strain MG1655 has been mapped using conjugation, transduction, molecular cloning, and sequencing (Figure 4.8) Figure 4.8 4.3 Genetic Elements: Chromosomes and Plasmids • Some features of the E. coli chromosome – Many genes encoding enzymes of a single biochemical pathway are clustered into operons – Operons are equally distributed on both strands – ~5 Mbp in size – ~40% of predicted proteins are of unknown function – Average protein contains ~300 amino acids – Insertion sequences (IS elements) II. Transmission of Genetic Information: DNA Replication • 4.4 Templates and Enzymes • 4.5 The Replication Fork • 4.6 Bidirectional Replication and the Replisome 5′ 3′ 5′ 5′ 3′ 3′ Parental strand Semiconservative replication + Daughter strand Figure 4.11 Growing point DNA polymerase activity. PPi cleaved off Deoxyribonucleoside triphosphate Figure 4.12 4.4 Templates and Enzymes • DNA polymerases catalyze the addition of dNTPs • Five different DNA polymerases in E. coli – DNA polymerase III is primary enzyme replicating chromosomal DNA • DNA polymerases require a primer (Figure 4.13) – Primer made from RNA by primase RNA primer 5′ DNA 3′–OH G UC U U A C U G A T C A GG T T C A T C GG A CG T A T C C AG AA T G A C T AG T CC AA G T AG C C T G C A T A G AG CC T T A CG A T C AGG C A G T 3′ 5′ Figure 4.13 4.5 The Replication Fork • DNA synthesis begins at the origin of replication in prokaryotes • Replication fork: zone of unwound DNA where replication occurs (Figure 4.14) • DNA helicase unwinds the DNA • Extension of DNA (Figure 4.15) – Occurs continuously on the leading strand – Discontinuously on the lagging strand • Okazaki fragments are on lagging strand 3′ Replication fork Helicase 5′ ATP 3′ ADP + Pi Helicase direction 5′ Figure 4.14 3′ 5′ RNA primer Single-strand binding protein 5′ Leading strand DNA polymerase III 3′ Lagging strand Helicase Primase RNA primer 5′ 3′ Figure 4.15 4.5 The Replication Fork • Connecting DNA fragments on the lagging strand (Figure 4.16) – DNA synthesis on lagging strand continues until it reaches previously synthesized DNA – DNA polymerase I removes the RNA primer and replaces it with DNA – DNA ligase seals nicks in the DNA Origin of replication Replication forks Newly synthesized DNA Theta structure Movement 3′ Origin (DnaA binding site) 5′ Lagging 3′ Leading 3′ 5′ 5′ Replication fork Movement 5′ 5′ Lagging 3′ Leading 3′ 5′ Origin 3′ Replication fork Figure 4.17 5′ 3′ DNA polymerase III 3′-OH RNA primer 3′ 5′ 5′-P 5′ 3′ 3′ 5′ DNA polymerase I 5′ 3′ Excised RNA primer 3′ 5′ DNA ligase 3′-OH 5′-P 5′ 3′ 3′ 5′ 5′ 3′ 3′ 5′ Figure 4.16 Helicase Direction of new synthesis Newly synthesized strand DNA polymerase III 5′ 3′ RNA primer Leading strand template DNA helicase DNA gyrase 5′ 3′ Tau Parental DNA RNA primer DNA polymerase III 3′ DNA primase 5′ 5′ 5′ Lagging strand template Newly synthesized strand Direction of new synthesis Single-strand DNA-binding proteins Figure 4.18 4.6 Bidirectional Replication and the Replisome • DNA replication is extremely accurate – Proofreading helps to ensure high fidelity • Mutation rates in cells are 10–8 to 10–11 errors per base inserted • Polymerase can detect mismatch through incorrect hydrogen bonding • Proofreading occurs in prokaryotes, eukaryotes, and viral DNA replication systems Termination DETAILS NOT KNOWN Link as a CHAIN UNLINKED BY TOPOISOMERASES Coupled with cell wall synthesis DNA PARTITIONED EQUALLY INTO DAUGHTER CELLS CELL DIVISION PROTEIN “ORCHESTRATES” FtsZ POLYMERIZATION Precursors dATP, dCTP, dGTP, dTTP Ribonucleotide Reductase Polymerase : DNA Kinase 5’---------3’ Ori Theta formation Primosomes- Primase, and Polymerases Helicase Seperates DNA Strands Gyrase- unwinds DNA Topoisomerase I & II Ligase – Reseals DNA stands