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Microbial Genetics

Microbial Genetics. and biotechnology. Define Terms. Genetics Genome / Genomics Chromosomes Gene Genotype Phenotype Recombination. Control of Gene Expression. DNA Structure. Double stranded Nucleotide Nitrogen Bases Sugar Phosphate Base Pairs Hydrogen Bonds A-T C-G Alpha helix

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Microbial Genetics

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  1. Microbial Genetics and biotechnology

  2. Define Terms • Genetics • Genome / Genomics • Chromosomes • Gene • Genotype • Phenotype • Recombination

  3. Control of Gene Expression

  4. DNA Structure • Double stranded • Nucleotide • Nitrogen Bases • Sugar • Phosphate • Base Pairs • Hydrogen Bonds • A-T • C-G • Alpha helix • 5’ – phosphate group • 3’ – hydroxyl group

  5. RNA Structure • Single strand • Nucleotide • Nitrogen base • Sugar • Phosphate • Base Pairs • A-U • C-G • Three types • mRNA • rRNA • tRNA

  6. Prokaryotic Chromosomes • Location • Nucleoid region • No membrane • Number • Most have 1 • Some species have 2, the second linear • Appearance • Circular • Ds • Loops and coils

  7. E. coli genome / chromosome

  8. DNA Replication • Semiconservative • Replication fork • Single origin • Bidirectional • 2 Leading strands • 2 Lagging strands • Enzymes • Helicases • DNA polymerases 5’ to 3’ • I for leading strand • II digest RNA primer • III for lagging strand • DNA ligase • DNA gyrase • Hydrogen bonds broken and reformed • Methylation of adenine bases • Initiation sites for replication • Turn on or Turn off gene expression • Protect against viral infections • DNA repair Polymerase I & II

  9. DNA Replication Enzymes • DNA Helicase • DNA Primase • DNA Polymerase • DNA Gyrase • Topoisomerases • DNA Ligase

  10. Leading/Lagging Strands

  11. DNA Replication Overview

  12. Binary Fission

  13. Binary Fission • Asexual reproduction • DNA replicated • FTs proteins • Divisome apparatus • Peptidoglycan • Plasma membrane • Double numbers

  14. Plasmids

  15. Plasmids • 2% of genetic information (5-100 genes) • ds, circular extra chromosomal DNA • Independent replication • Cellular Traits • F-Fertility • R-Resistance : inactivate AB, toxins, heavy metals • Dissimilation: catabolism of unusual substances • Bacteriocins • Virulence : enzymes, toxins, attachment

  16. Rolling Method for DNA replication and F-Plasmid • Rolling Method • One strand remains in loop • Second strand breaks away and rolls of loop • Both strands serve as templates for daughter strand • Occurs during conjugation

  17. Plasmid Integration (Episome)

  18. Transcription • DNA  RNA • mRNA • rRNA • tRNA • Initiation • Sigma factor on RNA polymerase • binds to promoter sequence on DNA • Will be release after 10 nucleotides • RNA polymerase • unzips, unwinds DNA • Lacks proof reading ability • Elongation • 5’ to 3’, slower • Ribonucleotide sequences • Base pairs : A-U [instead of Thymine] C-G • Termination • Self • Terminator sequence • G-C rich area • Protein-dependant • Terminator protein • Separates DNA and RNA polymerase

  19. Sigma Factors for RNA polymerase Where RNA polymerase binds to DNA

  20. Prokaryotic RNA • Transcription = RNA  Polypeptides • RNA • mRNA • Code for several polypeptides along strand • Each code has codons: Start and Stop • tRNA • Acceptor stem • Anticodon • Wobble • rRNA • 70S Ribosomes • 50S: 23S + 5S rRNA and 33 proteins • 30S: 16S rRNA and 21 proteins • Binding Sites on Ribosomes • A: accepts tRNA with AA • P: holds tRNA for base pairing anticodon to mRNA codon for polypeptide • E: release [Exit] for tRNA

  21. Translation Steps • Initiation • 30S • tRNA @ P site • 50S • GTP used • Elongation • New tRNA @ A site • Ribozyme in 50S forms peptide bond • GTP used • Termination • Release factor proteins • Stop codon on mRNA

  22. Importance of rRNA structures

  23. Regulation of Gene Expression • Constitutive • Not regulated • Always “on” at fixed rate • Transcription • Translation • 60-80% • Polypeptides need in large amounts • Regulated • Only when needed • Control synthesis of genes for enzymes • Induction • Repression • Control enzyme activity: feedback • Noncompetitive inhibition • Competitive inhibition

  24. Enzyme Reguation

  25. Operon Parts • Operator • Controls access • On/off • Promoter • RNA polymerase binds • All or none • Regulator • Genes at distant site control transcription • Repressor binds to operator to block • Structural genes • Code for enzymes

  26. Operon regions on DNA

  27. Operation of Operon Gene expression

  28. Operator Always “on” unless switched off by repressor

  29. Promoter Region

  30. Regulation of Operator Genes • Negative Control • Repressor • On/Off • Interacts with operon • Types • Inducible • Repressible • Positive Control • Activator protein • Determines rate • Directly interacts with genome • Facilitates transcription

  31. General Regulatory Control animation

  32. “Negative” Genetic Control of Enzyme synthesis and formation • Operon Model • Operator (O) • Promoter (P) • Structural genes • Regulatory genes • Makes repressor • Active binds to Operator • Inactive unable to bind to Operator • Types • Inducible Operons • Repressor Active • Operon Off • Inducer needed • Catabolic Pathways • Repressible Operons • Repressor inactive • Operon On • Corepressor needed • Anabolic pathways

  33. Repressor Proteins • Regulatory • Control Gene expression • Binds to operator • Cues from metabolites • Active form = blocks • Inactive form = allows transcription

  34. Active Repressor Proteins

  35. Inactive Repressor Proteins

  36. Inducible Operon • Repressor • Active • Bound to operon • Operon off • Need Inducer to inactivate repressor • Inducer • metabolite that can bind to repressor • Inactivates repressor • Operon “induced” on

  37. Lac Operon: Inducible

  38. Use of Lactose

  39. Repressible Operon • Repressor inactive • Operon on • Need co-repressor • Metabolite • Binds to repressor • Activates repressor

  40. TRP Operon: Repressible

  41. “Negative” Gene Control

  42. Which Regulatory Gene Operon is this?

  43. The “other” one  • Positive Control of Gene Expression • Catabolite Activator Protein (CAP) • Binds to promoter region • Enhance affinity for RNA polymerase • Stimulate gene expression

  44. CAP (Catabolite Activator Protein) • cAMP binds and activates CAP (crp) • cAMP-CAP bind to promoter • Increase RNA polymerase affinity • Allow efficient transcription • Determines rate No cAMP, no CAP binding, rate slows

  45. Mutations • Define • Types • Silent • Point • Mis-sense • Non-sense • Sense [aka silent] • Substitution • Transition: purine for purine • Transversion: purine for pyrimadine • Frameshift • Insertions • Deletions • Causes • Spontaneous • Induced • Chemical • Physical • Conditional • Adaptive • Transposons Inversion (transposons) Thymine dimer (radiation)

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