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Central dogma: Information flow in cells

Central dogma: Information flow in cells. Nucleotides. Pyrimidine bases: Cytosine (C), Thymine (T), Uracil (U, in RNA) Purine bases: Adenine (A), Guanine (U). Prokaryotic gene coding. Eukaryotic processing of rRNA. DNA Replication: addition of a nucleotide. DNA duplex formation.

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Central dogma: Information flow in cells

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  1. Central dogma: Information flow in cells

  2. Nucleotides • Pyrimidine bases: Cytosine (C), Thymine (T), Uracil (U, in RNA) • Purine bases: Adenine (A), Guanine (U)

  3. Prokaryotic gene coding

  4. Eukaryotic processing of rRNA

  5. DNA Replication: addition of a nucleotide

  6. DNA duplex formation

  7. A-T hydrogen bonding

  8. G-C hydrogen bonding

  9. 3D structure of DNA

  10. Inverted repeats in DNA

  11. Formation of Stem-loops

  12. “Sticky ends”

  13. Hairpins

  14. Genetic Elements • Prokaryotes: Chromosome, plasmid, viral genome, transposable elements • Eukaryotes: Chromosomes, plasmid, mitochondrion or chloroplast genome, viral genome, transposable elements

  15. Melting of DNA • Melting means separation of two strands from the heteroduplex • Melting temperature of DNA is dependent on the relative number of AT and GC pairs • Melted DNA can hybridize at temperatures below melting temperature • This process can be used to test relatedness between species (interspecies DNA-DNA hybridization) • It is also possible to reanneal DNA with rRNA to test relatedness of one species rRNA with the rRNA genes of another species

  16. Reannealing DNA

  17. **DNA structure overview** • complementary strands (antiparallel) • 3 Angstrom separationof hydrogen bonds • sugar phosphate backbone held together with hydrogen bonding between bases • size is expressed in nucleotide bases pairs. E. coli has 4600 kbp. (E. coli chromosome is > 1mm, about 500X longer than the cell itself. How can the organism pack so much DNA into its cell? • each bp takes up to 0.34nm, and each helix turn is 10bp(or 34 Angstroms), therefore how long is l kb of DNA? and how many turns does it have? • inverted repeats, stem-loop, hairpins, sticky ends • supercoiled DNA (DNA-binding proteins) • relaxed, nicked circular DNA

  18. Supercoiled and relaxed DNA

  19. DNA Organization • In prokaryotes: naked circular DNA with negative supercoiling • Negative supercoiling is introduced by DNA gyrase (topoisomerase II) • Topoisomerase I relaxes supercoiling by way of single-strand nicks • In eukaryotes: linear DNA packaged around histones in units called nucleosomes • The coiling around histones causes negative supercoiling

  20. Restriction and modification

  21. DNA Replication: addition of a nucleotide

  22. Semiconservative replication

  23. Initiation of DNA replication Origin of replication= oriC = ~300bp Templates, primers, polymerase, primase

  24. DNA Replication

  25. Bidirectional replication

  26. Okazaki fragments

  27. Proofreading by DNA polymerase III

  28. Replication overview • 1. origin of replication+ 300 bases, recognized by specific initiation proteins = replication fork • 2. bidirectional, therefore leading and lagging strands • helicase unwinds the DNA a little (ATP-dependant) • single-strand binding protein prevents single strand from reannealing • Primase, DNA polymerase III and DNA polymerase I (also 5' to 3' exonuclease activity), ligase • Okazaki fragments • Topoisomerases, and supercoiling regulation • 3. Proofreading (3 to 5' exonuclease activity by DNA pol III)

  29. DNA Sequencing

  30. Transcription • RNA plays an important role • tRNA, mRNA, rRNA • Name three differences between chemistry of RNA and DNA • RNA has both functional and genetic roles

  31. Initiation of Transcription Pribnow box=tataat

  32. Transcription

  33. Completion of transcription

  34. Example of termination sequence

  35. More transcription • Polycistronic mRNA • How can mRNA be used in microbial ecology? • Antibiotics and RNA polymerases

  36. RNA processing • Removal of introns • Ribozymes (nobel prize-Tom Cech and Sid Altman) • RNA-splicing enzymes • Origins of life? Which came first RNA or DNA?

  37. The genetic code • Notice that the wobble base generally makes minor changes in the amino acid • AUG is the start code (formyl methionine) for bacteria • UAA, UAG, UGA are stop codons • Specific tRNA for each other codon

  38. Codon and Anticodon; Wobble

  39. tRNA associated with codon ~60 specific tRNAs in prokaryotes

  40. mRNA, tRNA and ribosomes Shine Dalgarno sequence GTP and Elongation Factors (EF)

  41. Growing protein polymer

  42. Translocation

  43. Role of rRNA in protein synthesis • Structural and functional role • 16S rRNA involved in initiation • Base pairing occurs between ribosome binding sequence on the mRNA and a complementary seq on the 16S rRNA • 23S rRNA involved in elongation • Interacts with EFs

  44. Chaperones (heat-shock proteins)

  45. Overview of today • Summarized basic DNA structure • DNA replication • DNA sequencing • Transcription • RNA processing • Translation • Role of rRNA in protein synthesis

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