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Microbial Genetics

Microbial Genetics. Chapter 7 Dr. Ackman. Definitions. Genetics the study of heredity, genes and the mechanisms that they carry this information Replication Expression Genome Complete genetic information of the cell. Definitions. Chromosome

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Microbial Genetics

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  1. Microbial Genetics Chapter 7 Dr. Ackman

  2. Definitions • Genetics • the study of heredity, genes and the mechanisms that they carry this information • Replication • Expression • Genome • Complete genetic information of the cell

  3. Definitions • Chromosome • The structures that are composed of DNA that carry the hereditary information • Gene • Segments of the chromosome that code for a specific product (usually a protein) • Genomics • Sequencing and molecular characterization of genomes

  4. Definitions • DNA (deoxyribose nucleic acid) • Nucleotides • 3 components • Phosphate • Deoxyribose sugar • Nitrogenous base • Adenine, thiamine, cytosine or guanine • Double helix (complementary strands) • Base pairs • A-T • C-G • A-U (RNA) • Hydrogen bonds

  5. DNA • Base sequence codes for protein • 4 letter alphabet • Genetic code • Determines how nucleotide sequence is converted into amino acid sequences • Complementary strand allow precise duplication

  6. DNA to proteins • Gene on DNA • Converted to mRNA • mRNA on ribosome • tRNA brings amino acids to ribosome for protein synthesis

  7. Definitions • Genotype • Genetic information of the organism • Information that codes for characteristics of the organism • Phenotype • The expressed or physical characteristics of the organism • The expression of the genotype

  8. Bacterial Chromosome (DNA) • Bacterial chromosome • Single • Circular • Attached one or many sites to plasma membrane

  9. Bacteria chromosome • Escherichiacoli • 4.6 million base pairs • 4300 genes • 1mm long • 1,000 X length of cell • Supercoiled • Topoisomerase II • DNA gyrase

  10. Bacterial chromosome • Genetic map • Mapped in minutes • Based on time for chromosome exchanged between two cells

  11. DNA replication • Parental strand • Two new “daughter strands” • Each strand acts as template for new strands • Semiconservative replication

  12. DNA Replication • Carbons in nucleotide numbered 1`-5` • Complementary sugars are upside down to one another • Strands run 5`3` on each side

  13. DNA Replication • Steps in replication • DNA unwinds • DNA polymerase • Adds nucleotides to 3` end • Replication fork forms • Leading strand forms towards the fork • 5`3`

  14. DNA Replication • DNA replication • Lagging strand • Needs RNA primer • Removed by DNA polymerase • Synthesized discontinuously • Moves away from fork • Okazaki fragments • 1000 nucleotides • DNA ligase fuses segments

  15. Bacterial DNA Replication • Bacterial DNA replication • E. coli • Occurs bidirectionally • Two replication forks • Continues until forks meet

  16. RNA Synthesis • Transcription • Process of taking DNA code and converting to RNA code • Translation • Converting RNA (mRNA) with tRNA to form amino acid sequences and proteins • Occurs at ribosome

  17. Protein Synthesis • Three types of RNA • mRNA - messenger • tRNA - transfer • rRNA – ribosomal • DNA unzips at gene

  18. Transcription • RNA polymerase binds to DNA at promoter • Only coding strand of DNA is template • 5`3` direction • RNA polymerase assembles RNA nucleotides

  19. Transcription • RNA chain grows • RNA stops growing at terminator site • mRNA strand released from DNA • DNA zips up • mRNA intermediate between DNA and translation

  20. Translation • Bacterial translation • Protein synthesis • Decoding mRNA to amino acids and proteins • Codons • Groups of 3 nucleotides • Sequence of codons determines amino acid sequence • Several codons for a single amino acid • Degeneracy • Allows for mutations

  21. Translation • 64 codons • (43) • Sense codons • Code for amino acids • 61 codons • Nonsense codons • Stop codons • UAG, UAA, UGA • Signal end of protein synthesis • AUG • Start codon • Formylmethionine • Usually removed from protein

  22. Translation • tRNA • Transfer RNA • Anticodon • Complementary to codon • Amino acid attached • Brings amino acid to ribosome

  23. Translation • 1 – components needed come together • Ribosome • tRNA • mRNA • 2 – tRNA carries first amino acid ( ?) to ribosome and mRNA

  24. Translation • 3 – second amino acid brought to ribosome • P – site • Site of first amino acid • A – site • Site of second amino acid • Peptide bond forms

  25. Translation • 4 – after peptide bond first tRNA is released to find amino acid

  26. Translation • 5 – ribosome moves along mRNA until tRNA is in P site • Process continues down mRNA

  27. Translation • 6 – ribosome continues down mRNA • Peptide chain elongates

  28. Translation • 7- polypeptide (protein) released • Ribosome moves down mRNA until stop codon • UAG, UAA, UGA • Polypeptide released

  29. Translation • 8 – tRNA is released and ribosome disassembles • tRNA, mRNA, and ribosome can be used again

  30. Review

  31. Other points • Ribosome moves 5`3` direction • Additional ribosome may attach and begin synthesizing protein • Prokaryotes can start translation before transcription is complete

  32. Eukaryotic differences • Transcription takes place in nucleus • mRNA completed prior to entry in cytoplasm • Exons – Expressed DNA, code for protein • Introns – intervening DNA, do not code for protein • Removed by ribozymes

  33. Regulation of Bacterial Gene Expression • All metabolic reactions are catalyzed by enzymes (proteins) • Feedback inhibition stops a cell from performing unneeded chemical reactions • Stops enzymes that are already synthesized • What prevents synthesis of enzymes that are not needed?

  34. Regulation of Bacterial Gene Expression • Protein synthesis requires tremendous energy • Cell does not waste energy • Regulating protein synthesis economizes cells energy

  35. Regulation of Bacterial Gene Expression • Genes • 60-80% are constitutive • Not regulated • Products produced at fixed rate • Genes turned on all the time • Code for enzymes essential to major life processes • Enzymes needed for glycolysis

  36. Regulation of Bacterial Gene Expression • Genes • Inducible genes • Production of enzymes is regulated • Inducible enzymes • Present only when needed • Trypanosoma • Surface glycoproteins • Produces one glycoprotein at a time • Eludes immune system

  37. Regulation of Bacterial Gene Expression • Regulation of transcription • Repression • Decreases gene expression • Decrease enzyme synthesis • Response to overabundance of an end product • Regulatory proteins called repressors • Block RNA polymerase

  38. Regulation of Bacterial Gene Expression • Regulation of transcription • Induction • Turns on genes • Substance that turns on gene • Inducer • Inducible enzymes

  39. Regulation of Bacterial Gene Expression • Induction enzymes • β-galactosidase (E. coli) • Cleaves lactose • Medium without lactose = little to no β-galactosidase • Lactose added to medium large amounts of β-galactosidase produced • Lactose is converted to allolactose • Allolactose is the inducer • Enzyme reduction

  40. Operon Model • Three genes for lactose utilization • Located next to each other on bacterial chromosome • Regulated together • Called structural genes • lac structural enzymes are transcribed and translated • lac for lactose

  41. Operon Model • Operon model • lac operon • Promoter region • Region of DNA where RNA polymerase initiates transcription • Operator region • Go or stop signal for transcription of the structural genes • Structural genes • Genes for metabolism of lactose

  42. Operon Model • Inducible operon • Near lac operon is regulatory gene • I gene • Codes for repressor protein

  43. Operon Model • Lactose is absent • Repressor binds to operator site • RNA polymerase is inhibited • No transcription of structural genes • No mRNA • No enzymes are synthesized

  44. Operon Model • Lactose is present • Converted to allolactose • Inducer • Inducer binds to receptor protein • Receptor protein altered • Does not fit into operator site • RNA polymerase is not inhibited • Structural genes are transcribed to mRNA then translated into enzymes • An inducible operon

  45. Operon Model • Repressible operon • Tryptophan synthesis • EDCBA structural genes • Also has promoter and operator region

  46. Operon Model • Repressible operon • Structural genes transcribed and translated • Tryptophan is synthesized

  47. Operon Model • Repressible operon • Excessive tryptophan accumulates • Tryptophan acts as corepressor • Corepressor binds to repressor protein • Repressor protein binds operator and structural genes no longer transcribed

  48. Lactose regulation • Lactose operon • Depends on level of glucose in medium • Enzymes for glucose metabolism are constitutive • When glucose is absent cAMP (cyclic AMP) accumulates in cell • cAMP binds to cAMP receptor protein (CRP) • This binds to lac promoter • Initiates transcription by allowing mRNA polymerase to bind to the promoter • Transcription of lac operon requires • Presence of lactose • Absence of glucose • cAMP is an alarmone • Chemical alarm signal the cell uses to respond to environmental or nutritional stress

  49. lac operon

  50. Lac operon • Catabolite repression • Inhibition of the metabolism of other carbon sources by glucose • Glucose effect

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