1. Copyright © 2011 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell, University of Tennessee, Knoxville M I C R O B I O L O G Y WITH DISEASES BY TAXONOMY, THIRD EDITION Chapter 7 Microbial Genetics

2. Copyright © 2011 Pearson Education Inc. The Structure and Replication of Genomes Genetics – Study of inheritance and inheritable traits as expressed in an organism’s genetic material Genome – The entire genetic complement of an organism – Includes its genes and nucleotide sequences  Gene - Segment of DNA:  Gene codes for a functional product (usually a protein or regulation site)

3. Copyright © 2011 Pearson Education Inc. The Location of Genes Chromosome – Genes essential for survival Plasmid – Extra chromosomal DNA that replicate independently. Not essential for normal bacterial metabolism, growth, or reproduction. Can confer survival advantages

4. Copyright © 2011 Pearson Education Inc. E. coli genome Figure 7.2 1 single looped chromosome made of 4,300 genes 1.6 millimeters in length (800 times the length of the cell)

5. Copyright © 2011 Pearson Education Inc. The structure of nucleic acids Figure 7.1  Nucleic acid is made of repeating units of nucleotides  Nucleotides: 5 carbon sugar phosphate nitrogen base

6. Copyright © 2011 Pearson Education Inc. The structure of nucleic acids Figure 7.1

7. Copyright © 2011 Pearson Education Inc. Figure 8.2 The Transfer of Genetic Information Horizontal Gene Transfer DNA Replication Protein Synthesis

8. Copyright © 2011 Pearson Education Inc. DNA Replication Figure 7.4 Bacteria must replicate their DNA as the first step in binary fission Each strand of nucleotides serves as a template for a complimentary new strand. The process is semiconservative because each new double helix is composed of an old strand of nucleotides from the parent molecule and one newly-formed strand.

9. Copyright © 2011 Pearson Education Inc. DNA Replication

10. Copyright © 2011 Pearson Education Inc. DNA Replication

11. Copyright © 2011 Pearson Education Inc. DNA replicates only in a 5′ to 3′ direction Since strands are antiparallel, new strands are synthesized differently – Leading strand synthesized continuously – Lagging strand synthesized discontinuously DNA Replication

12. Copyright © 2011 Pearson Education Inc. Fig. 8.3 DNA Replication

13. Copyright © 2011 Pearson Education Inc. Leading Strand Synthesis 1. Helicase unwinds DNA and creates replication fork 2. Stabilizing Proteins bind to prevent reannealing 3. RNA polymerase (Primase) synthesizes short RNA sequences called primers, which serve as starting points for DNA synthesis 4. DNA polymerase III binds and adds nucleotides to hydroxyl group at 3′ end of nucleic acid 5. DNA polymerase I replaces RNA primer with DNA

14. Copyright © 2011 Pearson Education Inc. Lagging Strand Synthesis 1. RNA polymerase (Primase) synthesizes short RNA sequences called primers, which serve as starting points for DNA synthesis 2. DNA polymerase III binds and adds nucleotides to hydroxyl group at 3′ end of nucleic acid 3. Okazaki Fragments short, newly synthesized DNA fragments 4. DNA polymerase I replaces RNA primer with DNA 5. Ligase seals gap between Okazaki Fragments

15. Copyright © 2011 Pearson Education Inc. Lagging Strand Synthesis http://highered.mcgraw- hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/00724373 16/120076/micro04.swf::DNA%20Replication%20Fork

16. Copyright © 2011 Pearson Education Inc. Figure 8.2 The Transfer of Genetic Information Horizontal Gene Transfer DNA Replication Protein Synthesis

17. Copyright © 2011 Pearson Education Inc. Gene Expression Gene expression occurs when gene activity leads to a protein product in the cell ( Protein Synthesis ). A gene does not directly control protein synthesis; instead, it passes its genetic information on to RNA, which is more directly involved in protein synthesis. Difference between DNA and RNA RNA is single-stranded; is composed of the sugar ribose; substitutes uracil for thymine

18. Copyright © 2011 Pearson Education Inc.

19. Three types of RNA are involved in gene expression: – messenger RNA ( mRNA ) carries genetic information to the ribosomes – ribosomal RNA ( rRNA ) is found in the ribosomes – transfer RNA ( tRNA ) transfers amino acids to the ribosomes, where the protein product is synthesized RNA Types

20. Copyright © 2011 Pearson Education Inc. Protein Synthesis Central dogma of genetics – explains of the flow of genetic information Steps: 1.Transcription = DNA transcribed to RNA 2.Translation = RNA translated to form polypeptides (proteins) Transcription Translation DNA triplets RNA codons Protein (genotype) (phenotype)

21. Copyright © 2011 Pearson Education Inc. Transcription Process of making mRNA from a DNA template by complementary base pairing. Only the section of DNA that codes for a needed protein is copied. Coding strand : strand of DNA that codes for a protein; the strand that is the same as mRNA except for the substitution of bases - uracil for thymine. Template strand : complimentary to coding strand; this is the strand that is transcribed to make mRNA. RNA polymerase attaches to a site called the promoter. This is what determines which strand of DNA is copied.

22. Copyright © 2011 Pearson Education Inc. Transcription Coding strand = Coding Strand A – T – G – T – T – G – A – C Template Strand T – A – C – A – A – C – T – G mRNA A – U – G –U – U – G – A – C

23. Copyright © 2011 Pearson Education Inc. Transcription Figure 7.8a Promoter– specific nucleotide sequence at beginning of a gene; tells the RNA polymerase where to start transcription RNA Polymerase binds to promoter by sigma factor that unwinds DNA begins reading the DNA sequence on the template strand from 3’ to 5’; makes mRNA in 5’ to 3’ direction http://www.youtube.com/watch?v=ztPkv7wc3yU

24. Copyright © 2011 Pearson Education Inc. Prokaryotic mRNA can code for several polypeptides Figure 7.12

25. Copyright © 2011 Pearson Education Inc. Translation Process of translating the DNA code from mRNA into an enzyme or other protein. Occurs on the ribosomes. Ribosomes move along the mRNA, reading the genetic code three bases at a time which is a codon Each mRNA codon codes for a specific amino acid

26. Copyright © 2011 Pearson Education Inc. Genetic Code Figure 8.9 Universal Code 64 mRNA Codons code for amino acid 20 Amino Acids ( wobble effect) 1 Start Codon 3 Stop Codons

27. Copyright © 2011 Pearson Education Inc. Transfer RNA Figure 7.13a-b  Short (about 75 nucleotides) piece of folded RNA containing 3 loops  tRNA anticodon is found on the bottom loop of the molecule and is complementary to the mRNA codon  tRNA has an acceptor stem specific for an amino acid

28. Copyright © 2011 Pearson Education Inc. Ribosomal structures Figure 7.14

29. Copyright © 2011 Pearson Education Inc. Assembled ribosome and its tRNA-binding sites Figure 7.15

30. Copyright © 2011 Pearson Education Inc. Gene Function Translation – Three stages of translation – Initiation – Elongation – Termination – All stages require additional protein factors – Initiation and elongation require energy (GTP)

31. Copyright © 2011 Pearson Education Inc. The initiation of translation in prokaryotes Figure 7.16

32. Copyright © 2011 Pearson Education Inc. The elongation stages of translation Figure 7.17

33. Copyright © 2011 Pearson Education Inc. One prokaryotic mRNA, many ribosomes and polypeptides Figure 7.18

34. Copyright © 2011 Pearson Education Inc. Gene Function Translation – Stages of translation – Termination – Release factors recognize stop codons – Modify ribosome to activate ribozymes – Ribosome dissociates into subunits – Polypeptides released at termination may function alone or together

35. Copyright © 2011 Pearson Education Inc. Translation http://highered.mcgraw-hill.com/olc/dl/120077/micro06.swf

36. Copyright © 2011 Pearson Education Inc.

37. CLASS EXERCISE

38. Copyright © 2011 Pearson Education Inc. 5’ AATTATGGGACTTTAATGA (DNA)

39. Copyright © 2011 Pearson Education Inc. 75% of genes are expressed constantly 25% of genes are regulated by inhibiting translation or transcription to conserve energy Operons – a group of genes that work together – Inducible operons – gene usually not transcribed (off); must be turned on by a substance – Repressible operons – gene always transcribed (on); must be turned off Regulation of “Gene Expression”

40. Copyright © 2011 Pearson Education Inc. Operon Model Regulatory Gene codes for a repressor protein that controls process of transcription Promoter DNA segment where RNA polymerase attaches to begin transcription Operator DNA segment where the repressor protein binds to prevent the attachment of the promoter gene Structural genes code for specific proteins

41. Copyright © 2011 Pearson Education Inc. The lac operon Inducible Operon Figure 7.19a Inducer - substances that initiates transcription, i.e. turns on gene Inducible enzymes – enzymes synthesized in the presence of an inducer ( ex. B-galactosidase, permease, transacetylase )

42. Copyright © 2011 Pearson Education Inc. The lac operon Figure 7.19b Inducer -Allolactose which is formed when cell takes in lactose Inducible Operon http://youtu.be/h5p05aFzWdA

43. Copyright © 2011 Pearson Education Inc. The lac operon, an inducible operon Figure 7.20

44. Copyright © 2011 Pearson Education Inc. The tryp operon Figure 7.20a Repressors - block the ability of RNA polymerase to initiate transcription; usually inactive until activator is present. Used when enzymes are not needed – (ex.enzymes produced to synthesize amino acid tryptophan) Tryptophan in environment represses gene expression of enzymes Repressible Operon

45. Copyright © 2011 Pearson Education Inc. The tryp operon Figure 7.20b Activated Repressor binds to Tryptophan Repressible Operon http://youtu.be/1arC3WkQNVQ