Microbial Genetics

The Structure and Replication of Genomes LEARNING OBJECTIVE Compare and contrast the genome of prokaryotes and eukaryotes 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 2

Figure 7.1 The structure of nucleic acids-overview

The Structure and Replication of Genomes The Structure of Prokaryotic Genomes Prokaryotic chromosomes Main portion of DNA, along with associated proteins and RNA Prokaryotic cells are haploid (single chromosome copy) Typical chromosome is circular molecule of DNA in nucleoid 4

Figure 7.2 Bacterial genome-overview

The Structure and Replication of Genomes LEARNING OBJECTIVE Describe the structure and function of Plasmids The Structure of Prokaryotic Genomes Plasmids Small molecules of DNA that replicate independently Not essential for normal metabolism, growth, or reproduction Can confer survival advantages Many types of plasmids Fertility factors Resistance factors Bacteriocin factors Virulence plasmids 6

The Structure and Replication of Genomes LEARNING OBJECTIVE Compare and contrast the genome of prokaryotes and eukaryotes The Structure of Eukaryotic Genomes Nuclear chromosomes Typically have more than one chromosome per cell Chromosomes are linear and sequestered within nucleus Eukaryotic cells are often diploid (two chromosome copies) 7

Figure 7.3 Eukaryotic nuclear chromosomal packaging-overview

The Structure and Replication of Genomes The Structure of Eukaryotic Genomes Extranuclear DNA of eukaryotes DNA molecules of mitochondria and chloroplasts Resemble chromosomes of prokaryotes Only code for about 5% of RNA and proteins Some fungi and protozoa carry plasmids 9

The Structure and Replication of Genomes LEARNING OBJECTIVE Describe the replication of DNA as a semicooservative process. Compare and contrast the synthesis of leading and lagging stands in DNA replication . DNA Replication Anabolic polymerization process that requires monomers and energy Triphosphate deoxyribonucleotides serve both functions Key to replication is complementary structure of the two strands Replication is semiconservative New DNA composed of one original and one daughter strand 10

Figure 7.4 Triphosphate deoxyribonucleotides as building blocks and energy sources in DNA synthesis-overview

Figure 7.5 Semiconservative model of DNA replication Original DNA First replication Second replication Original strand New strands

The Structure and Replication of Genomes DNA Replication Initial processes in replication Bacterial DNA replication begins at the origin DNA polymerase replicates DNA only 5 to 3 Because strands are antiparallel, new strands are synthesized differently Leading strand synthesized continuously Lagging strand synthesized discontinuously 13

Figure 7.6a DNA replication: initial processes Chromosomal proteins (histones in eukaryotes and archaea) removed DNA polymerase III 3´ Replication fork 5´ DNA helicase Stabilizing proteins Initial processes

Figure 7.6b DNA replication: synthesis of leading strand Primase 3´ Replication fork 5´ Leading strand P + P Triphosphate nucleotide RNA primer Synthesis of leading strand

Figure 7.6c DNA replication: synthesis of lagging strand Replication fork Triphosphate nucleotide RNA primer Okazaki fragment Lagging strand 5´ 3´ 5´ DNA ligase Primase DNA polymerase III DNA polymerase I Synthesis of lagging strand

The Structure and Replication of Genomes DNA Replication Other characteristics of bacterial DNA replication Bidirectional Topoisomerases remove supercoils in DNA molecule DNA is methylated Control of genetic expression Initiation of DNA replication Protection against viral infection Repair of DNA 17

Figure 7.7 The bidirectionality of DNA replication Origin Parental strand Replication forks Daughter strand Replication proceeds in both directions Termination of replication

The Structure and Replication of Genomes DNA Replication Replication of eukaryotic DNA Similar to bacterial replication Some differences Uses four DNA polymerases Thousands of replication origins Shorter Okazaki fragments Plant and animal cells methylate only cytosine bases 19

The Relationship Between Genotype and Phenotype Gene Function LEARNING OBJECTIVE Explain how the genotype of an organism determine its phenotype. The Relationship Between Genotype and Phenotype Genotype Set of genes in the genome Phenotype Physical features and functional traits of the organism 20

The Transfer of Genetic Information Gene Function LEARNING OBJECTIVE State the central dogma of genetic, and explain the roles of DNA and RNA in polypeptide synthesis. The Transfer of Genetic Information Transcription Information in DNA is copied as RNA Translation Polypeptides synthesized from RNA Central dogma of genetics DNA transcribed to RNA RNA translated to form polypeptides 21

Figure 7.8 The central dogma of genetics 5´ 3´ DNA (genotype) 3´ 5´ Transcription mRNA 5´ 3´ Translation by ribosomes NH2 Methionine Arginine Tyrosine Leucine Polypeptide Phenotype

The Events in Transcription Gene Function LEARNING OBJECTIVE Describe three steps in RNA transcription , mentioning the following: DNA , RNA polymerase , promoter, 5’ to 3’ direction and terminator . The Events in Transcription Four types of RNA transcribed from DNA RNA primers mRNA rRNA tRNA Occur in nucleoid of prokaryotes Three steps Initiation Elongation Termination 23

Figure 7.9 Transcription-overview RNA polymerase attaches nonspecifically to DNA and travels down its length until it recognizes a promoter sequence. Sigma factor enhances promoter recognition in bacteria. RNA polymerase 5´ 3´ DNA 3´ 5´ Promoter Sigma factor Terminator Attachment of RNA polymerase Upon recognition of the promoter, RNA polymerase unzips the DNA molecule beginning at the promoter. “Bubble” 5´ 3´ 3´ 5´ Template DNA strand Unzipping of DNA, movement of RNA polymerase Initiation of transcription “Bubble” Triphosphate ribonucleotides align with their DNA complements and RNA polymerase links them together, synthesizing RNA. No primer is needed. The triphosphate ribonucleotides also provide the energy required for RNA synthesis. 5´ 3´ 3´ 3´ 5´ Growing RNA molecule (transcript) 5´ 5´ 3´ Template DNA strand 5´ 3´ Elongation of the RNA transcript 5´ 3´ 3´ 5´ Promoter 5´ 3´ Terminator RNA transcript released Self-termination: transcription of DNA terminator sequences causes the RNA to fold, loosening the grip of polymerase on DNA. Enzyme-dependent termination: Rho pushes between polymerase and DNA, releasing polymerase, RNA transcript and Rho. RNA polymerase Rho termination protein Rho protein moves along RNA C-G rich hairpin loop 3´ Template strand Termination of transcription

Figure 7.10 Concurrent RNA transcription RNA polymerases Promoter 5´ 5´ 5´ 3´ 3´ 3´ 3´ 3´ 3´ 3´ 3´ 5´ 5´ Sigma factor Template DNA strand RNA 5´ 5´ 5´

The Events in Transcription Gene Function The Events in Transcription Transcriptional differences in eukaryotes RNA transcription occurs in the nucleus Transcription also occurs in mitochondria and chloroplasts Three types of RNA polymerases Numerous transcription factors mRNA processed before translation Capping Polyadenylation Splicing 26

Figure 7.11 Eukaryotic mRNA Exons (polypeptide coding regions) Template DNA strand 3´ 5´ Introns (noncoding regions) Transcription Exon 1 Exon 2 Exon 3 Pre-mRNA 5´ cap Intron 1 Intron 2 Intron 3 Poly-A tail Intron 1 Processing Spliceosomes 5´ 3´ mRNA splicing Exon 1 Exon 3 Exon 2 mRNA (codes for one polypeptide) 5´ 3´ Nuclear envelope Nucleoplasm Nuclear pore Cytosol mRNA

Gene Function Translation LEARNING OBJECTIVE Describe the genetic code in general, and identify the relationship between codons and amino acids. Describe the translation of polypeptides. Identifying the rol of the three type of RNA. Translation Process where ribosomes use genetic information of nucleotide sequences to synthesize polypeptides 28

Figure 7.12 The genetic code

Gene Function Translation Participants in translation Messenger RNA Transfer RNA Ribosomes and ribosomal RNA 30

Figure 7.13 Prokaryotic mRNA Promoter Gene 1 Gene 2 Gene 3 Terminator 3´ 5´ Template DNA strand Transcription Start codon AUG Start codon AUG Start codon AUG UAA UAG UAA 5´ 3´ mRNA 3´ mRNA Ribosome binding site (RBS) Stop codon RBS Stop codon RBS Stop codon Untranslated mRNA Translation Polypeptide 1 Polypeptide 2 Polypeptide 3

Figure 7.14 Transfer RNA-overview

Figure 7.15 Prokaryotic ribosomes-overview

Figure 7.16 Transfer RNA binding sites in a ribosome tRNA- binding sites Large subunit Large subunit E site P site A site Nucleotide bases mRNA 5´ 3´ Small subunit Small subunit mRNA Prokaryotic ribosome (angled view) attached to mRNA Prokaryotic ribosome (schematic view) showing tRNA-binding sites

Gene Function Translation Three stages of translation Initiation Elongation Termination All stages require additional protein factors Initiation and elongation require energy (GTP) 35

Figure 7.17 The initiation of translation in prokaryotes Initiator tRNA Large ribosomal subunit tRNAfMet Anticodon mRNA Start codon E 5´ 3´ P A P A P A Small ribosomal subunit Initiation complex

Figure 7.18 The elongation stage of translation-overview Peptide bond E E 5´ 3´ 5´ 3´ P A P A Movement of ribosome one codon toward 3´ end 5´ 3´ E P A E 5´ 3´ P A E 5´ 3´ P A Two more cycles Growing polypeptide E 5´ 3´ P A

Figure 7.19 A polyribosome in a prokaryotic cell-overview

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 39

Gene Function Translation Translation differences in eukaryotes Initiation occurs when ribosomal subunit binds to 5 guanine cap First amino acid is methionine rather than f-methionine 40

Regulation of Genetic Expression Gene Function LEARNING OBJECTIVE Describe the use of ribswitches and short interference RAN in genetic control. Explain the operon model of transcriptional control in prokaryotes. Contrast the regulation of an inducible operon with that of a repressible operon ,and give an exaple of each. Regulation of Genetic Expression 75% of genes are expressed at all times Other genes transcribed and translated when cells need them Allows cell to conserve energy Regulation of protein synthesis Typically halts transcription Can stop translation directly 41

Regulation of Genetic Expression Gene Function Regulation of Genetic Expression Control of translation Genetic expression can be regulated at level of translation Riboswitch mRNA molecule that blocks translation of the polypeptide it encodes Short interference RNA (siRNA) RNA molecule complementary to a portion of mRNA, tRNA, or a gene that binds and renders the target inactive 42

Regulation of Genetic Expression Gene Function Regulation of Genetic Expression Nature of prokaryotic operons An operon consists of a promoter and a series of genes Some operons are controlled by a regulatory element called an operator 43

Figure 7.20 An operon Operon Promoter Operator Structural genes Regulatory gene 1 2 3 4 3´ 5´ Template DNA strand

Regulation of Genetic Expression Gene Function Regulation of Genetic Expression Nature of prokaryotic operons Inducible operons must be activated by inducers Lactose operon Repressible operons are transcribed continually until deactivated by repressors Tryptophan operon 45

Figure 7.21 The lac operon-overview

Figure 7.22 CAP-cAMP enhances lac transcription cAMP bound to CAP RNA polymerase Transcription proceeds CAP binding site Promoter Operator lac genes

Figure 7.23 The trp operon-overview

Mutations of Genes Mutation LEARNING OBJECTIVE Define mutation. Mutation Change in the nucleotide base sequence of a genome Rare event Almost always deleterious Rarely leads to a protein that improves ability of organism to survive 49

Mutations of Genes Types of Mutations Point mutations LEARNING OBJECTIVE Define point mutation and describe three types. Types of Mutations Point mutations Most common One base pair is affected Insertions, deletions, and substitutions Frameshift mutations Nucleotide triplets after the mutation are displaced Insertions and deletions 50

Figure 7.24 Effects of the various types of point mutations-overview

Mutations of Genes Mutagens Radiation Chemical mutagens LEARNING OBJECTIVE Discuss how different types of radiation cause mutation in a genome. Describe three kind of chemical mutagens and their effects. Mutagens Radiation Ionizing radiation Nonionizing radiation Chemical mutagens Nucleotide analogs Disrupt DNA and RNA replication Nucleotide-altering chemicals Result in base-pair substitutions and missense mutations Frameshift mutagens Result in nonsense mutations 52

Figure 7.25 A pyrimidine (thymine) dimer Ultraviolet light Thymine dimer

Figure 7.26 The structure and effects of nucleotide analogs-overview

Figure 7.27 The action of a frameshift mutagen Normal DNA Acridine Replication Deletion Insertion Daughter DNA

Mutations of Genes Frequency of Mutation Mutations are rare events LEARNING OBJECTIVE Discuss the relative frequency of deleterious and useful mutations. Frequency of Mutation Mutations are rare events Otherwise organisms could not effectively reproduce Mutagens increase the mutation rate by a factor of 10 to 1000 times 56

Figure 7.28 DNA repair mechanisms-overview

Identifying Mutants, Mutagens, and Carcinogens Mutations of Genes LEARNING OBJECTIVE Contrast the positive and negative selection techniques for isolating mutants. Describe the Ames test , and discuss its use in discovering carcinogens. Identifying Mutants, Mutagens, and Carcinogens Mutants Descendants of a cell that does not repair a mutation Wild types Cells normally found in nature Methods to recognize mutants Positive selection Negative (indirect) selection Ames test 58

Figure 7.29 Positive selection of mutants-overview

Figure 7.30 Negative (indirect) selection-overview Inoculate bacteria onto complete medium containing tryptophan. Mutagen Bacterial suspension Incubation Bacterial colonies grow. A few may be tryptophan auxotrophs. Most are wild types. Stamp sterile velvet onto plate, picking up cells from each colony. Sterile velvet surface Bacteria Stamp replica plates with velvet. Complete medium containing tryptophan Medium lacking tryptophan Incubation Identify auxotroph as colony growing on complete medium but not on lacking medium. All colonies grow. Tryptophan auxotroph cannot grow. Inoculate auxotroph colony into complete medium.

Medium lacking histidine Colony of revertant (his+) Salmonella Figure 7.31 The Ames test Experimental tube Control tube Liver extract Suspected mutagen Liver extract Culture of his– Salmonella Medium lacking histidine Incubation Colony of revertant (his+) Salmonella No growth

Genetic Recombination and Transfer LEARNING OBJECTIVE Define genetic recombination. Exchange of nucleotide sequences often mediated by homologous sequences Recombinants Cells with DNA molecules that contain new nucleotide sequences Vertical gene transfer Organisms replicate their genomes and provide copies to descendants 62

Figure 7.32 Genetic recombination Homologous sequences 3´ 5´ DNA A 3´ 5´ DNA B Enzyme nicks one strand of DNA at homologous sequence. A B Recombination enzyme inserts the cut strand into second molecule, which is nicked in the process. Ligase anneals nicked ends in new combinations. Molecules resolve into recombinants. Recombinant A Recombinant B

Genetic Recombination and Transfer LEARNING OBJECTIVE Contrast vertical gene transfer. Explain the role of an F factor , F+ cell , and Hfr cells in bacterial conjugation. Describe the structures and action of simple and complex transposons . Compare and contrast crossing over, transformation, transduction, and conjugation. Horizontal Gene Transfer Among Prokaryotes Horizontal gene transfer Donor cell contributes part of genome to recipient cell Three types Transformation Transduction Bacterial conjugation 64

Genetic Recombination and Transfer Horizontal Gene Transfer Among Prokaryotes Transformation One of conclusive pieces of proof that DNA is genetic material Cells that take up DNA are competent Results from alterations in cell wall and cytoplasmic membrane that allow DNA to enter cell 65

Figure 7.33 Transformation in Streptococcus pneumoniae-overview

Genetic Recombination and Transfer Horizontal Gene Transfer Among Prokaryotes Transduction Generalized transduction Transducing phage carries random DNA segment from donor to recipient Specialized transduction Only certain donor DNA sequences are transferred 67

Figure 7.34 Transduction-overview Bacteriophage Host bacterial cell (donor cell) Bacterial chromosome Phage injects its DNA. Phage enzymes degrade host DNA. Phage DNA Phage with donor DNA (transducing phage) Cell synthesizes new phages that incorporate phage DNA and, mistakenly, some host DNA. Transducing phage Recipient host cell Transducing phage injects donor DNA. Transduced cell Donor DNA is incorporated into recipient’s chromosome by recombination. Inserted DNA

Figure 7.35 Bacterial conjugation-overview F plasmid Origin of transfer Conjugation pilus Chromosome Donor cell attaches to a recipient cell with its pilus. F+ cell F– cell Pilus may draw cells together. One strand of F plasmid DNA transfers to the recipient. Pilus The recipient synthesizes a complementary strand to become an F+ cell with a pilus; the donor synthesizes a complementary strand, restoring its complete plasmid. F+ cell F+ cell

Figure 7.36 Conjugation involving an Hfr cell-overview Donor chromosome Pilus F+ cell F plasmid integrates into chromosome by recombination. Hfr cell Pilus Cells join via a conjugation pilus. F+ cell (Hfr) F– recipient F plasmid Donor DNA Part of F plasmid Portion of F plasmid partially moves into recipient cell trailing a strand of donor’s DNA. Incomplete F plasmid; cell remains F– Conjugation ends with pieces of F plasmid and donor DNA in recipient cell; cells synthesize complementary DNA strands. Donor DNA and recipient DNA recombine, making a recombinant F– cell. Recombinant cell (still F–)

Genetic Recombination and Transfer Transposons and Transposition Transposons Segments of DNA that move from one location to another in the same or different molecule Result is a kind of frameshift insertion (transpositions) Transposons all contain palindromic sequences at each end 71

Figure 7.37 Transposition-overview

Genetic Recombination and Transfer Transposons and Transposition Simplest transposons Insertion sequences Have no more than two inverted repeats and a gene for transposase Complex transposons Contain one or more genes not connected with transposition © 2012 Pearson Education Inc. 73

Figure 7.38 Transposons-overview