Genetics

1. Genetics

2. What is genetics? • The science of heredity; includes the study of genes, how they carry information, how they are replicated, how they are expressed

3. Adaptation and Natural Selection • How do organisms adapt to change? • Two basic options: regulate gene expression or change the genetic code • Change in genetic code = mutation

4. Why use bacteria to study mutations? Only have one chromosome…one copy of each gene Easy to grow

5. Direct selection Testing for traits that are easily identified Colony color Motility Resistance to antibiotics

6. Indirect selection A way to look at traits that are not easily identified, at changes in metabolic pathways Replica plating A way to identify AUXOTROPHS from PROTOTROPHS

7. Vertical Gene transfer

8. Horizontal gene transfer

9. Chapter 7

10. What do you know about DNA? • Chromosomes made of DNA make up an organism’s genome • DNA codes for genes = functional unit of the genome • Genes code for proteins • Chemical composition = nucleotides

11. Replication: duplication of the genome prior to cell division Gene expression: decoding of DNA in order to synthesize gene products (proteins): Transcription: DNA →RNA Translation: RNA → protein

12. Diagrammatic representation of DNA

13. DNA Structure • Double helix formed by complementary strands • Strands composed of deoxyribonucleotide subunits = nucleotides • Antiparallel strands held together by hydrogen bonds between base pairs • 5’ P04 binds to 3’ OH • Thymine pairs with adenine • Guanine pairs with cytosine

15. DNA Replication

16. Enzymes necessary for DNA replication • Primase: synthesizes the RNA primer • Helicase: “unzips” 2 strands of DNA • DNA Polymerase: synthesize 5’→3’ • DNA gyrase: releases tension during uncoiling of circular DNA • Produced by prokaryotes and some simple eukaryotic organisms only, so potential target for antibiotics **target of quinolones and aminocoumarins** • DNA ligase: seals the gaps between Okazaki fragments (forms covalent bonds)

17. Gene Expression • Transcription • Post-transcriptional modification • Translation • Post-translational modification

18. Transcription: DNA to RNA • RNA polymerase • Does not require a primer to initiate synthesis • Recognition of the promoter via sigma factor (bacterial transcription factor) • Process begins at the promoter region and ends at the terminator sequence • Process proceeds in the direction 5’→3’ • Base pairing: thymine replaced with uracil; U-A, G-C

19. RNA synthesis

20. What are the possible products from transcription? • Messenger RNA (mRNA) • Transfer RNA (tRNA) • Ribosomal RNA (rRNA)

21. Translation: RNA to protein • What is needed for the process? • mRNA: has the code • Ribosomes: present the codons to tRNA, align the amino acids • Protein + rRNA • Amino acids • tRNA: anticodon ; initiates the protein sythesis at the P-site brings the correct amino acid to add at the A-site

22. Translation: RNA to protein • What is needed for the process? • mRNA • Ribosomes • Amino acids • tRNA

23. Initiation of Translation • Ribosome binds ribosome binding site • on mRNA molecule • In bacteria: binding occurs during mRNA synthesis – so translation and transcription occur simultaneously • Ribosome completes assembly while bound to the mRNA • Initiating tRNA binds to start codon: AUG • N-formylmethionine = f-Met) • Also codon for normal methionine

24. Elongation of the Polypeptide Chain • 2 binding sites on ribosome for tRNA: • P-site: • A-site: • Initiation tRNA binds to P-site and provides f-Met • tRNA recognizing the next codon binds to A-site and provides coded AA • Ribosomal enzyme creates a peptide bond between

25. Termination of Translation • Ribosome gets to stop codon • No tRNA recognizes the stop codon →enzymatic cleavage of bond that binds the polypeptide to the mRNA • Ribosome falls off and dissociates into 2 subunits • Subunits are ready to reassemble and initiate translation at another site

26. Post-Translational Modification • Synthesized polypeptides are straight chains of amino acids • Modifications to make them into functional proteins, ready them for transport out of the cell = PTMs • Folding: chaperone-assisted • Tag removal: export signal sequence is removed in the process of crossing the cytoplasmic membrane

27. The reading frame determines the protein

28. The Genetic code

29. Translation

30. Both processes occur at the same time in bacteria (why not in eukaryotic cells?)

31. Ribosomes are 80s – 40s and 60s subunits 5’ end of mRNA is capped Methylated guanine added to pre-mRNA Stabilizes transcript, enhances translation Polyadenylation of 3’ end of mRNA Poly A tail added to pre-mRNA Stabilizes transcript , enhances translation? Splicing: removal of non-coding sequences = introns; exons spliced together Translation is monocystronic Eukaryotic cells differ in transcription and translation

32. Is protein synthesis regulated? • Three types of protein regulation • Enyme inhibition (ex: feedback inhibition) • Repression (ex: tryptophan operon) • Induction (ex: lactose operon)

33. Does regulation occur at the level of transcription? • Some gene expression is constitutive: proteins encoded by these genes are continuously synthesized • Other genes are induced: proteins only made when needed • Other genes are repressed: proteins produced routinely, but turned off when not needed

34. Models for transcriptional regulation with repressors

35. Transcriptional regulation by activators

36. Used to understand control of gene expression in bacteria Operon consists of three genes needed to degrade lactose Repressor gene (codes for repressor protein) outside of operon coding region inhibits transcription unless something else binds to the repressor protein Lactose Operon as a model

37. Lactose Operon

38. What conditions are needed for the lactose operon to be turned “on”? • No glucose • Lactose present • Increasing levels of cAMP • cAMP binds to CAP, then complex binds next to lactose operon promoter at the activator region • RNA polymerase binds to promoter

39. If E. coli is growing in a flask with glucose and lactose…

40. Gene regulation systems in bacteria • Signal transduction: transmission of information from outside to inside cell • Quorum sensing: ability to sense the density of cells within the same population • Communication occurs via molecular signals • In quorum sensing, response to the signal is concentration dependent • Critical level → induction of gene expression

41. Chapter 8

43. Adaptation and Natural Selection • How do bacteria adapt to change? • Like any organisms, they have 2 basic options: • Regulate gene expression • Change the genetic code • Change in genetic code = mutation • Bacteria can also utilize HORIZONTAL GENE TRANSFER

44. Vertical Gene transfer

45. Horizontal gene transfer

46. What are mutations? Changes in the base sequence of the DNA Do they always change the genetic code?

47. What can cause mutations? Chemicals (nitrous acid) Physical mutagens (uv light) Biological mutagens (transposons) Spontaneous mutations (errors in replication) Random occurrences Low frequency; usually at a constant within a given population Essential for a population to adapt to change

48. Causes of mutations in bacteria Most are spontaneous Errors made by DNA Polymerase UV light exposure Oxidative injury induced by reactive oxygen species (ROS) – superoxide, hydrogen peroxide

49. Types of Mutations Base substitution: replacement of one nucleotide base with another Missense mutation: altered codon specifies a different amino acid Nonsense mutation: altered codon is a stop codon, resulting in formation of a truncated, usually non-functional protein Silent mutation: the strict definition = a change in the codon does not change the encoded amino acid; a more broad definition = a change that does not change the function of the encoded protein by this definition a silent mutation could be any of these types of base substitions, as long as the function of the protein (phenotype) was not affected)

50. Base-pair mutation: missense