The Genetics of Viruses and Prokaryotes

1. The Genetics of Viruses and Prokaryotes

2. Probing the Nature of Genes • Prokaryotes and viruses have advantages for the study of genetics: • They have small genomes. • They quickly produce large numbers of individuals. • They are usually haploid, making genetic analyses easier.

3. Probing the Nature of Genes • The ease of use of bacteria and viruses in genetic research has propelled the science of genetics and molecular biology during the last 50 years. • Prokaryotes continue to play a central role as tools for biotechnology and for research on eukaryotes. • Prokaryotes play important ecological roles, including the cycling of elements. • Many prokaryotes and viruses are pathogens.

4. Viruses • TMV - tobacco mosaic virus • 1st virus to be discovered in the 1890s • Direct observation of viruses requires electron microscopes • The simplest infective agents are viroids, which are made up only of genetic material.

5. Viruses • Viruses are acellular • Composed of a nucleic acid and a few proteins • DNA or RNA • Coat proteins • Viral enzymes (e.g. reverse transcriptase) • Do not carry out metabolism • obligate intracellular parasites • Reproduce only in living cells • use host cell’s transcription/translation machinery • often integrate into host cell’s chromosome(s) • Progeny released from host cell • often destroy the host cell in the process

6. Virions Come in Various Shapes Adenovirus TMV phage lambda Influenza A

7. Virus Nomenclature • Viruses are categorized by four criteria: • DNA or RNA genome • Single-stranded or double-stranded nucleic acid • Shape of the virion • Presence or absence of lipid capsule around capsid

8. Viruses of Prokaryotes • Bacteriophage - viruses of bacteria • Bacteriophage recognize hosts via specific interaction of viral capsid proteins and proteins on host cell. • Virions are equipped with tail assemblies that inject the phage’s DNA into the host cell.

9. Bacteriophage • Reproduction of a phage involves • Replication of phage DNA • Expression of phage genes needed for capsid • Two types of reproductive cycles • lytic cycle • Immediate reproduction and lysis of host cell • lysogenic cycle • Integration into host chromosome with reproduction and lysis occurring later • Some phage are only lytic other are both (temperate) • Most well studied is phage Lambda ()

10. The Lytic and Lysogenic Cycles of Bacteriophage

11. Plant & Animal Viruses • Plant viruses are usually only capsid + nucleic acid • Majority of plant viruses have an RNA genome • Many animal viruses have a lipid membrane derived from the host cell’s - envelope • Some animal viruses have DNA, and some have RNA • Most viruses are species specific, but many can gain (jump) host species • Influenza • HIV • Arboviruses infect both insects and vertebrates

12. Figure 13.4 The Reproductive Cycle of the Influenza Virus (Part 1)

13. Figure 13.4 The Reproductive Cycle of the Influenza Virus (Part 2) cDNA

14. Figure 13.5 The Reproductive Cycle of HIV (Part 1)

15. Figure 13.5 The Reproductive Cycle of HIV (Part 2)

16. Viruses can lead to unusual transmission of traits • Normal genetic transmission • Vertical transmission • transfer of genes (traits) from parent to offspring • Viral mediated gene transfer • Horizontal transmission • spread of genes to unrelated individuals • Horizontal transmission inferred from presence of transposable elements • DNA sequences which can move themselves into/out of & between genomes • Perhaps incorporated into viruses or perhaps originated from viruses

17. Prokaryotes • Bacteria and archaea • Single, circular chromosome • E. coli – 4.65Mbp • Plasmids • Extrachromosomal, ds DNA circles • 1-10Kbp • Replicated independently of chromosomal DNA • Contain genes that encode resistance to antibiotics, metabolic pathways, or conjugation • Clonal expansion • Binary fission (prokaryotic cell division) • Formation of visible colonies on solid media

18. Figure 13.6 Growing Bacteria in the Laboratory

19. Prokaryotes: Reproduction and Recombination • Transformation • Uptake of DNA in their environment (extracellular DNA) and incorporation into genome • Frederick Griffith • Used by Avery to show DNA was genetic material • Conjugation • Prokaryotic “sexual” reproduction • Physical contact between bacteria and transfer of plasmids or portions of genome

20. Figure 13.10 Transformation

21. Figure 13.7 Lederberg and Tatum’s Experiment - Conjugation

22. Figure 13.11 Gene Transfer by Plasmids

23. Figure 13.9 Recombination Following Conjugation

24. Prokaryotes: Reproduction and Recombination • Transduction • viruses carry genes from one cell to another (horizontal transfer) • Excision of a prophage to enter a lytic cycle sometime allows host DNA to be incorporated into the bacteriophage genome • Cells infected by such phage get a segment of another bacterium’s DNA • This bacterial DNA recombines with the chromosomal DNA of the host and alters its genetic composition.

25. Figure 13.10 Transformation and Transduction

26. Prokaryotes: Reproduction and Recombination • Transposable elements • “jumping genes” • transposons • segments of DNA that can move within the genome • often contain gene encoding the enzyme transposase

27. Gods François Jacob & André Lwoff – 1953 CSH Symposium Jacques Monod – Paris 1961

28. Regulation of Gene Expression in Prokaryotes • Metabolic carbohydrate C-sources • Glucose – feeds directly into glycolysis • Lactose, Arabinose, Galactose – Feed indirectly into glycolysis • E. coli only uses secondary sugars once glucose is depleted • Jacques Monod demonstrated that proteins were induced upon switching C source • Lactose metabolism - hydrolysis of lactose disaccharide into galactose and glucose monosaccharides • The enzyme used is -galactosidase • Two other enzymes are also involved in lactose metabolism • A permease to transport lactose into the cell • An acetylase to modify lactose (unknown biochemical relevance) • Lactose induces the expression of -galactosidase, permease and acetylase

29. -Galactosidase Induction by Lactose Lag

30. Regulation of Gene Expression in Prokaryotes • Prokaryotes conserve resources by making proteins only when needed. • Lactose metabolic enzymes not made when lactose not present • Two main ways to regulate metabolic pathways • Allosteric regulation • Shape / activity of enzyme • Protein already present when induction occurs • Regulation of protein synthesis • Transcription and /or translation • Protein made/destroyed when induction occurs

31. Figure 13.14 Two Ways to Regulate a Metabolic Pathway

32. Prokaryotic Gene Structure Shine-Delgarnobox Shine-Delgarnobox Stop CodonTAA, TAG, TGA +1 DNA Cis-RegulatoryElements Coding Sequence= ORF Coding Sequence= ORF Protein B USE/Promoter/Operator Protein A Terminatorsequence 5’ UTR = Leadersequence Spacer = 5’UTR of 2nd cistron ATG ATG Regulatory Sequences Structural or Coding Sequences Cistron 1 Cistron 2 Regulatory and Coding Sequence Unit = Operon Stop CodonTAA, TAG, TGA

33. Shine-Delgarnobox Stop CodonTAA, TAG, TGA Shine-Delgarnobox Stop CodonTAA, TAG, TGA +1 DNA Cis-RegulatoryElements Coding Sequence= ORF Coding Sequence= ORF Protein B USE/Promoter/Operator Protein A Terminatorsequence 5’ UTR = Leadersequence Spacer = 5’UTR of 2nd cistron ATG ATG TRANSCRIPTION Cistron 2 Cistron 1 5’ UTR = Leadersequence Spacer PolycistronicmRNA ORF Protein A ORF Protein B +1 AUG AUG Stop CodonUAA, UAG, UGA Stop CodonUAA, UAG, UGA Shine-Delgarnobox Shine-Delgarnobox Prokaryotic Gene Structure I

34. The Lac Operon Regulatory sequences Transcription & translation of an operon

35. Transcriptional Regulatory Sequences of Prokaryotes • Promoter – • DNA sequences to which RNA polymerase physically binds • Two 6 bp elements – -10 box & -35 box TGTACA TATAAT

36. Transcriptional Regulatory Sequences of Prokaryotes • Operator • DNA sequence to which a repressor binds • When repressor is bound, DNA can not be transcribed • When repressor is not bound transcription proceeds Repressor

37. Repressor Proteins: Allosteric Proteins • The Lac repressor protein has two binding domains • DNA binding domain • The inducer (ligand) binding domain • The lac repressor is a homotetramer protein • Each monomer provides ½ of a lactose binding site and ½ of a DNA binding site • The tetramer binds to DNA in two places or binds to two lactose molecules • The repressor can not bind to DNA and lactose simultaneously • Binding to lactose alters the shape of the repressor tetramer causing it to release DNA.

38. Lac Repressor Tetramer

39. Lac Repressor-DNA Complex Lac repressor protein (violet) forms a tetramer which binds to two operator sites (red) located 93 bp apart in the DNA causing a loop to form in the DNA. As a result expression of the lac operon is turned off. This model also shows the CAP (CRP) protein (dark blue) binding to the CAP site in the promoter (dark blue DNA). The -10 and -35 sequences of the promoter are indicated in green.

40. Figure 13.17 The lac Operon: An Inducible System (Part 1) • Lactose absent – Transcription repressed • No lacZ, lacY, lacA produced RNA poly

41. Figure 13.17 The lac Operon: An Inducible System (Part 2)

42. Summary of lac operon Transcriptional Control • When no lactose (inducer) is present, lac operon is off. • The LacI gene produces the repressor protein • The repressor prevents transcription of the operon • The operator is the DNA sequence to which the repressor binds (binding site) • The promoter is the DNA sequence to which the RNA polymerase binds (-10 & -35 boxes) • Adding inducer (lactose) allows the operon to be transcribed.

43. Regulation of Gene Expression in Prokaryotes • If synthesis of an enzyme can be turned off, it is said to be repressible. • The trp operon in E. coli is repressible. • In the absence of tryptophan, RNA polymerase transcribes the trp operon, leading to production of enzymes that synthesize tryptophan. • When tryptophan is present, it binds to a repressor, which becomes active. • The repressor binds to the operator of the trp operon, blocking production of enzymes for tryptophan synthesis.

44. Figure 13.18 The trp Operon: A Repressible System (Part 1) trp repressor gene aporepressor

45. Figure 13.18 The trp Operon: A Repressible System (Part 2) tryptophan

46. Inducible or Repressible? • inducible systems • Require presence of substrate to activate expression of operon • Usually for operons encoding proteins in catabolic pathways • repressible systems • The presence of the substrate inactivates expression of the operon • Usually for operons encoding proteins in anabolic pathways

47. Control of Transcription in Viruses • Viruses also have gene regulation mechanisms. • Bacteriophage l is a temperate phage, meaning that it can undergo either a lytic or a lysogenic cycle. • When host bacteria are growing in rich medium, the prophage remains lysogenic; when the host is less healthy, the prophage becomes lytic. • A “genetic switch” determines the prophage behavior.

48. Lysis vs Lysogeny • Control of phage gene expression determines life cycle route • Study of lytic induction led to the early understanding of transcriptional regulation

49. Map of Phage  • Phage genome contains a variety of promoters that attract host RNA polymerase to differing degrees • Viral control proteins specify which promoters are used

50. Phage Molecular Biology: Gene Regulation • Three stages of phage “development” • Immediate early • viral genes adjacent to the promoters are transcribed. • Delayed early • Proteins of early genes compete to activate/inhibit transcription of late genes • Late stages • Lysis – lytic control proteins win in DE stage – activate lytic proteins in late stage • Lysogeny – lysogenic control proteins win in DE stage – activate prophage formation