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Microbial Genetics

Explore the intricacies of microbial genetics, from DNA replication and gene expression to protein synthesis. Learn about the structure of DNA, bacterial chromosomes, and the processes of replication, transcription, and translation.

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Microbial Genetics

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  1. Microbial Genetics

  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 (A, T, G and C) • 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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