Microbial Genetics Unlocking the Secrets of Heredity

1. Microbial GeneticsUnlocking the Secrets of Heredity Chapter 8

2. Chromosomes • Chromosome: discrete cellular structure composed of a neatly packaged DNA molecule • Eukaryotic chromosomes • DNA wound around histones • located in the nucleus • diploid (in pairs) or haploid (single) • linear appearance • Prokaryotic chromosomes • DNA condensed into a packet by means of histone-like proteins • single, circular chromosome

4. A sampling of genes related to obesity in the human genome • http://www.obesity.chair.ulaval.ca/genemap.html

5. A closer look at chromosome 1 • Genes are SPECIFIC & DISCRETE segments of DNA

6. A map of E. coli’s ~5000 genes • Notice it is single & circular • Does E. coli have 1 or 2 alleles of each gene? How do you know? • Humans were first thought to function with 100,000 genes and now the number has dropped to ~35,000 genes although this is still a hot topic in research

7. DNA is lengthy and occupies a small part of the cell by coiling up into a smaller package. Fig. 9.3 An Escherichia coli cell disrupted to release its DNA molecule.

8. Genome • Genome: sum total of genetic material of an organism • most of the genome exists in the form of chromosomes • some appears as plasmids or in certain organelles of eukaryotes • genome of cells composed entirely of DNA • genome of viruses can contain either DNA or RNA

9. Gene • Gene • a certain segment of DNA that contains the necessary code to make a protein or RNA molecule • Three categories of genes • structural genes: code for proteins • genes that code for RNA machinery used in protein production • regulatory genes: control gene expression

10. Genetic Terms • Genotype • an organism’s genetic makeup; its entire complement of DNA • Phenotype • is the expression of the genes: the proteins of the cell and the properties they confer on the organism. • Size, shape, color, environment • Which one is easier to see?

11. The DNA Code Hydrogen bond H H H N H–N O • Nucleotide: basic unit of DNA structure • phosphate • deoxyribose sugar • nitrogenous base • Nucleotides covalently bond to each other in a sugar-phosphate linkage • Pairing of bases dictated by the formation of hydrogen bonds between bases N C N N–H H G N N O N– H Sugar 3′ H OH P D 5′ 4′ D 1′ 5′ P D C 2′ G P D 3′ P P P O O T A D D O P O P O C G D D O O P P C G D D O P P T A D D O P O P O C G D D O O P P T A P D D P 5′ D D 3′ 5′ H OH CH3 O H N–H N N N A T H– H N N N O H Sugar (a)

12. Nature of the double helix • antiparallel arrangement: one side of the helix runs in the opposite direction of the other • the order of the bond between carbon on deoxyribose and the phosphate is used to keep track of the direction of the two sides • one side runs from 5’ to 3,’ and the other side runs 3’ to 5’ • this is a significant factor in DNA synthesis and protein production

13. DNA Replication

14. DNA Replication • What you need to replicate DNA: 1. Original DNA template (parental chromosome) 2. Nucleotides (Guanine, Cytosine, Adenine, Thymine) a pool of nucleotides will be free floating in the cytoplasm waiting to be used 3. Enzymes I.e., DNA polymerase (hooks together nucleotides), ligase (ligates) 4. Energy (ATP)

15. Replication • Don’t worry about knowing the leading strand, lagging strand and 5’ and 3’ terminology for the exam. These are more in depth than we will go.

16. Certain enzymes unwind the DNA. Then, DNA polymerase can read the parent strand and attach a complementary nucleotide to the new strand of DNA. The nucleotides are free in the cytoplasm. DNA Replication in Prokaryotes

17. DNA replication is semi-conservative since each new chromosome will have one “old” and one “new” strand

18. Transcription

19. Transcription (RNA Synthesis) • What you need to synthesize RNA: 1. Original DNA template (parental chromosome) with a promoter site (DNA sequence indicating start site) and a terminator site 2. Nucleotides(G, C, A, U) Ribose (sugar) + phosphate + N base Uracil is substituted for thymine 3. Enzymes I.e., RNA polymerase (hooks together nucleotides) 4. Energy (ATP)

20. Transcription • RNA polymerase: large, complex enzyme that directs the conversion of DNA into RNA • Template strand: only one strand of DNA that contains meaningful instructions for synthesis of a functioning polypeptide

21. Transcription 4 types of RNA can be transcribed: 1. Messenger ribose nucleic acid (mRNA) mRNA (RNA molecule that serves as a message of the protein to be produced) 2. Transfer ribose nucleic acid (tRNA) tRNA (64 different tRNA molecules participate in translation) 3. Ribosomal ribose nucleic acid (rRNA) rRNA (forms the ribosome) 4. Regulatory RNA

22. The RNAs • RNA is similar to DNA in terms of its general properties, but its structure is different in several ways • single-stranded molecule that exists in helical form; can assume secondary and tertiary levels of complexity, leading to specialized forms of RNA (tRNA and rRNA) • contains uracil (U) instead of thymine; does not change the DNA code because uracil still follows the pairing rules • contains ribose rather than deoxyribose

23. The RNAs • mRNA- message from DNA, single stranded • rRNA- part of ribosome • tRNA- transfers amino acids to ribosome • Regulatory RNAs: • micro RNAs, anti-sense RNAs, riboswitches, small interfering RNAs • Primer RNAs: operative in both prokaryotic and eukaryotic cells • Ribozymes: remove unneeded sequences from other RNAs

24. Transcription: Initiation • RNA polymerase recognizes promoter region • RNA polymerase begins its transcription at a special codon called the initiation codon • As the DNA helix unwinds it moves down the DNA synthesizing RNA molecule

25. Transcription: Elongation Direction of transcription Early mRNA transcript Nucleotide pool • During elongation the mRNA is built, which proceeds in the 5’ to 3’direction (with regard to the growing RNA molecule) • the mRNA is assembled by the adding nucleotides that are complementary to the DNA template. • As elongation continues, the part of DNA already transcribed is rewound into its original helical form.

26. Transcription: Termination Elongation Late mRNA transcript At termination the polymerases recognize another code that signals the separation and release of the mRNA strand,or transcript.

27. Translation

28. Translation • Decoding the “language” of nucleotides and converting/translating that information into the “language” of proteins. • The nucleic acid “language” is in the form of codons, groups of three mRNA nucleotides.

29. Translation occurs at the RIBOSOME! The green mRNA strand is “threaded” through the ribosome. The ribosome “reads” the mRNA strand codons with the help of the genetic code and tRNA (next slides) Where does translation occur?

30. tRNA • Decoder molecule which serves as a link to translate the RNA language into protein language • One site of the tRNA has an anticodon which complements the codon of mRNA • The other site of the tRNA has an amino acid attachment site corresponding to a specific amino acid as noted in the genetic code

31. Codons • Triplet code that specifies a given amino acid • Multiple codes for one amino acid • Degenerate(repetitive) code is good to allow for a certain amount of mutation to occur without having an effect on the amino acid sequence • 1 Start codon • 3 Stop codons • 64 total possible codons • 20 amino acids

32. Translation and the “Genetic Code” • We use the “genetic code” (at right) to translate mRNA nucleotide sequence (codons) into amino acid sequence which make up proteins. • The “genetic code” is degenerate which allows for a certain amount of mutation. I.e. UUU and UUC both code for Phe

33. Translation and the “Genetic Code” • There is one start codon, AUG, that codes for the amino acid methionine. • There are 3 stop codons, UAA, UAG and UGA that signal the ribosome to stop translation and let go of the polypeptide chain (protein).

34. Translation • Ribosomes bind mRNA near the start codon (ex. AUG) • tRNA anticodon with attached amino acid binds to the start codon

35. Translation • Ribosomes move to the next codon, allowing a new tRNA to bind and add another amino acid

36. Translation • Series of amino acids form peptide bonds

37. Translation • Stop codon terminates translation

38. a single mRNA is long enough to be fed through more than one ribosome • permits the synthesis of hundreds of protein molecules from the same mRNA transcript • occurs only in prokaryotes, where there transcription and translation both occur in the cytoplasm • Would you see this in Eukaryotes? Polyribosomal Complex

39. Introns and Exons Eukaryotic mRNAs code for just one protein, unlike bacterial mRNAs, which often contain information from several genes in series

40. Transcription and translation in eucaryotes • Similar to procaryotes except • AUG encodes for a different form of methionine • Transcription and translation are not simultaneous (since eucaryotes have a nucleus----transcription occurs in the nucleus, translation occurs ?) • Eucaryotes must splice out introns to achieve a mature mRNA strand ready to go to the ribosome.

41. How are genes regulated? • Cells regulate genes in 3 major ways: 1. Feedback inhibition • The end-product inhibits the pathway (similar to a thermostat….when it reaches the desired temperature it turns off) 2. Enzyme induction • If a substrate is present, the enzyme for the substrate is induced. 3. Enzyme repression a. If a nutrient is present, the enzyme to make it is repressed. b. If a nutrient is absent, the enzyme to make it is turned on.

42. Operons • only found in bacteria • coordinated set of genes • all regulated as a single unit • either inducible or repressible

43. lac Operon

44. lac Operon

45. Phase Variation • Bacteria turn on or off a complement of genes that leads to obvious phenotypic changes • Phenotype is heritable! • Most often traits affecting the bacterial cell surface • Examples: • Neisseria gonorrhoeae: production of attachment fimbriae • Streptococcus pneumoniae: production of a capsule

46. What if a gene changes? • Mutation=a change in the sequence of DNA • Effects of mutations • none-->no change in a.a. sequence or…. • Good-->new aa. Seq-->antibiotic resistance • Increases variability in the gene pool • Bad-->new aa. Seq-->mutate active site of enzyme • For humans, cancer is the product of a combo of bad mutations.

47. Types of Mutations • Point Mutation • put the cat out--->puc the cat out • put the cat out--->put • Frameshift (reading frame of mRNA shifts) • put the cat out--->put hec ato ut • Deletion • Addition • Duplication

48. When a base is substituted in DNA the mutation may have 2 effects: Changes the amino acid Does not change the amino acid Why doesn’t a mutation always change the amino acid sequence? Because the genetic code is degenerate and has amino acids that may be coded for by different codons. (I.e., AAA and AAG both code for phenylalanine) The Effects of Base Substitution (a point mutation)

49. The addition, deletion or insertion of one or more nucleotides drastically changes the amino acid sequence. The Effects of Frameshift Mutations

50. Mutation Rates • Normal Mutation Rate=1/1 million per gene • Mutations are constantly occurring since our enzymes are not 100% perfect …These are called spontaneous mutations and increase in occurrence as we age….when do we get cancer? • Mutagen=certain chemicals or radiation that bring about mutations. • Mutagen Mutation Rate= 1/1000-1/100,000 per gene (10-1000X the normal rate)