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IX: DNA Function: Protein Synthesis

IX: DNA Function: Protein Synthesis. IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information. IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information

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IX: DNA Function: Protein Synthesis

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  1. IX: DNA Function: Protein Synthesis

  2. IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information

  3. IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. Why a two-step process? a. Historical contingency… That’s how it evolved from an RNA Protein system…

  4. IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. Why a two-step process? - it evolved that way…. - because it is more productive…. tRNA

  5. IX: DNA Function: Protein Synthesis A. Overview: 1. The central dogma of genetics: unidirectional flow of information 2. The code is: 3. Why a two-step process? 4. Players: ds-DNA; GENE (recipe) RNA polymerases make: m-RNA (gene transcript) r-RNA (reader in ribosome) t-RNA (AA carrier) tRNA

  6. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription:

  7. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: In a given region (gene), only one strand is transcribed; only one strand carries a message that makes ‘sense’. The sequence on the other strand is limited to being complementary to the first strand. 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  8. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: In a given region (gene), only one strand is transcribed; only one strand carries a message that makes ‘sense’. The sequence on the other strand is limited to being complementary to the first strand. There is a region ‘upstream’ from the gene called the PROMOTER. This is where the RNA Polymerase binds. The polymerase is attracted to particular sequences. Many are consensus sequences found upstream from different genes, and across many many species. 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  9. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: In a given region (gene), only one strand is transcribed; only one strand carries a message that makes ‘sense’. The sequence on the other strand is limited to being complementary to the first strand. There is a region ‘upstream’ from the gene called the PROMOTER. This is where the RNA Polymerase binds. The polymerase is attracted to particular sequences. Many are consensus sequences found upstream from different genes, and across many many species. In all bacteria, the sequence TATAAT lies 10 bases upstream from all bacterial genes, and TTGACA lies 35 bases upstream. Two binding sites create a directionality. Promoters can be 40 bases long. Frequency of binding is affected by variation in the rest of the sequence…. 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  10. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: In a given region (gene), only one strand is transcribed; only one strand carries a message that makes ‘sense’. The sequence on the other strand is limited to being complementary to the first strand. In all eukaryotes, there is a consensus sequence of TATA in all promoters at -35, and a CAAT box at -80. There are also enhancer regions that can modulate binding, and Transcription Factors that bind to the promoter and increase/decrease the efficacy of polymerase binding. 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  11. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: In a given region (gene), only one strand is transcribed; only one strand carries a message that makes ‘sense’. The sequence on the other strand is limited to being complementary to the first strand. In all eukaryotes, there is a consensus sequence of TATA in all promoters at -35, and a CAAT box at -80. There are also enhancer regions that can modulate binding, and Transcription Factors that bind to the promoter and increase/decrease the efficacy of polymerase binding. Why is there consensus in promoter sequences across all life? What does that say about how well mutations are tolerated in these regions? 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  12. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: In a given region (gene), only one strand is transcribed; only one strand carries a message that makes ‘sense’. The sequence on the other strand is limited to being complementary to the first strand. There is region downstream called a TERMINATOR (40 bases long). 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  13. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: In a given region (gene), only one strand is transcribed; only one strand carries a message that makes ‘sense’. The sequence on the other strand is limited to being complementary to the first strand. So, the polymerase binds at the promoter, transcribes the whole gene, and decouples at the terminator. 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  14. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: In a given region (gene), only one strand is transcribed; only one strand carries a message that makes ‘sense’. The sequence on the other strand is limited to being complementary to the first strand. In bacteria, the gene is a continuous coding sequence…. 3’ ‘sense’ strand 5’ Continuous recipe for a protein 5’ 3’ ‘anti-sense’ strand

  15. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: In a given region (gene), only one strand is transcribed; only one strand carries a message that makes ‘sense’. The sequence on the other strand is limited to being complementary to the first strand. In bacteria, the gene is a continuous coding sequence…. In eukaryotes, genes contain non-coding, intervening sequences called introns; the coding sequences are called exons. 3’ ‘sense’ strand 5’ Disconti nuous recipe for a protein 5’ 3’ ‘anti-sense’ strand

  16. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: In a given region (gene), only one strand is transcribed; only one strand carries a message that makes ‘sense’. The sequence on the other strand is limited to being complementary to the first strand. In bacteria, the gene is a continuous coding sequence…. In eukaryotes, genes contain non-coding, intervening sequences called introns; the coding sequences are called exons. Although transcription is continuous and every base is transcribed, RNA processing is required to splice out the non-coding introns and create a continuous reading frame for translation. 3’ ‘sense’ strand 5’ Disconti nuous recipe for a protein 5’ 3’ ‘anti-sense’ strand

  17. TANGENT

  18. There are two alternate hypotheses for the evolution of introns: Archaea (introns in r-RNA and t-RNA genes) Eubacteria (no introns at all) Eukarya (introns)

  19. There are two alternate hypotheses for the evolution of introns: “Introns Early”: The ancestral structure was a split gene structure, favored because evolution could proceed rapidly by the shuffling of functional exons to create new genes (exon shuffling hypothesis). Archaea (introns in r-RNA and t-RNA genes) Eubacteria (no introns at all) Eukarya (introns)

  20. There are two alternate hypotheses for the evolution of introns: “Introns Early”: The ancestral structure was a split gene structure, favored because evolution could proceed rapidly by the shuffling of functional exons to create new genes (exon shuffling hypothesis). Archaea (introns in r-RNA and t-RNA genes) Eubacteria (no introns at all) Eukarya (introns) Introns were lost from prokaryotes because of the extreme selective advantage for rapid division.

  21. Mitochondria have primitive spliceosomal apparatus that may have invaded eukaryotic host genome… leading to conserved phylogenetic patterns of intron position through eukaryotic evolution. VIDEO

  22. There are two alternate hypotheses for the evolution of introns: “Introns Late”: The ancestral structure was a continuous gene structure. Introns evolved as “transposeable elements” and inserted themselves and multiplied only in eukaryotic ancestors. Archaea (introns in r-RNA and t-RNA genes) Eubacteria (no introns at all) Eukarya (introns)

  23. The split-gene structure of eukaryotes was discovered in 1977: Philip Sharp and coworkers found that viral genes in eukaryotes and initial transcripts were longer than the functional m-RNA or proteins.

  24. The split-gene structure of eukaryotes was discovered in 1977: Philip Sharp and coworkers found that viral genes in eukaryotes and initial transcripts were longer than the functional m-RNA or proteins. 1978: Walter Gilbert coins the terms ‘intron’ and ‘exon’.

  25. Heteroduplex analyses of DNA and m-RNA show “loops” of RNA that have no complement in the DNA template strand:

  26. All eukaryotic genes except those coding for histones have introns; some make up the vast majority of the ‘gene’, itself:

  27. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: In bacteria, there is only one enzyme; consisting of a ‘core enzyme’ (responsible for polymerization), and subunits that affect different functions. For example, the sigma subunit is responsible for initiation, and the rho subunit stimulates termination. 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  28. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: In bacteria, there is only one enzyme; consisting of a ‘core enzyme’ (responsible for polymerization) with two polypeptides, and subunits that affect different functions. For example, the sigma subunit is responsible for initiation, and the rho subunit stimulates termination. There are different sigma subunits that affect polymerase binding; complementing the variations in promoters. 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  29. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: In bacteria, there is only one enzyme; consisting of a ‘core enzyme’ (responsible for polymerization) with two polypeptides, and subunits that affect different functions In eukaryotes, there are three RNA Polymerases: 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  30. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand A U A U

  31. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  32. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter 3’ ‘sense’ strand 5’ …C A T… 5’ …G T A… 3’ ‘anti-sense’ strand

  33. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter b. Helicases separate strands; first bases linked 5’  3’ 3’ ‘sense’ strand 5’ C C C A G T C A T G G G T…. G G G 3’ 5’ 3’ ‘anti-sense’ strand 5’

  34. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter b. Helicases separate strands; first bases linked 5’  3’ c. Sigma subunit dissociates, polymerization continues 3’ ‘sense’ strand 5’ C C C A G T C A T G G G T…. G G G UC A GU A C C C A… 3’ 5’ 3’ ‘anti-sense’ strand 5’

  35. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter b. Helicases separate strands; first bases linked 5’  3’ c. Sigma subunit dissociates, polymerization continues d. Termination: sequences rich in C’s and G’s, followed by A’s and T’s 3’ ‘sense’ strand 5’ C C C C G C A A G C G G G G A A T T…. G G G GC G UU C G C C C C U U A A… 3’ 5’ 3’

  36. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter b. Helicases separate strands; first bases linked 5’  3’ c. Sigma subunit dissociates, polymerization continues d. Termination: sequences rich in C’s and G’s, followed by A’s and T’s - Rho Independent: - the C’s and G’s in the m-RNA for a ‘stem-loop’ structure; and binds to a protein bound to the Polymerase (nusA) 3’ ‘sense’ strand 5’ C C C C G C A A G C G G G G A A T T…. 3’ 5’ 3’ U C G C C C C G G G GC G U

  37. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter b. Helicases separate strands; first bases linked 5’  3’ c. Sigma subunit dissociates, polymerization continues d. Termination: sequences rich in C’s and G’s, followed by A’s and T’s - Rho Independent: - the C’s and G’s in the m-RNA for a ‘stem-loop’ structure; and binds to a protein bound to the Polymerase (nusA) - this causes the polymerase to pause, just as it is reading the area rich in A’s… which have fewer h-bonds. The pausing and the destabilization of the polymerase caused by the ‘stem-loop’ causes the m-RNA/polymerase to detach. 3’ ‘sense’ strand 5’ C C C C G C A A G C G G G G A A T T…. 3’ 5’ 3’ U C G C C C C G G G GC G U

  38. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter b. Helicases separate strands; first bases linked 5’  3’ c. Sigma subunit dissociates, polymerization continues d. Termination: sequences rich in C’s and G’s, followed by A’s and T’s - Rho Independent: - recent research has identified certain RNA’s that loop and bind a small protein produced BY their own code. So, if the product concentration is high, the m-RNA binds the protein, loops, and shuts down transcription (down-regulating the gene). These RNA’s that turn off their own gene are “riboswitches” 3’ ‘sense’ strand 5’ C C C C G C A A G C G G G G A A T T…. 3’ 5’ 3’ U C G C C C C G G G GC G U

  39. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter b. Helicases separate strands; first bases linked 5’  3’ c. Sigma subunit dissociates, polymerization continues d. Termination: sequences rich in C’s and G’s, followed by A’s and T’s - Rho-dependent: Rho – a circular hexamer, binds the m-RNA in C-G rich regions 3’ ‘sense’ strand 5’ C C C C G C A A G C G G G G A A T T…. G G G GC G UU C G C C C C U U A A… 3’ 5’ 3’

  40. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter b. Helicases separate strands; first bases linked 5’  3’ c. Sigma subunit dissociates, polymerization continues d. Termination: sequences rich in C’s and G’s, followed by A’s and T’s - Rho-dependent: Rho – a circular hexamer, binds the m-RNA in C-G rich regions it slides up the strand; decoupling the polymerase from the m-RNA at A-T rich sites. 3’ ‘sense’ strand 5’ C C C C G C A A G C G G G G A A T T…. G G G GC G UU C G C C C C U U A A… 3’ 5’ 3’

  41. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter b. Helicases separate strands; first bases linked 5’  3’ c. Sigma subunit dissociates, polymerization continues d. Termination: e. PolycistronicDNA: In bacteria, proteins involved in the same metabolic process are often encoded by neighboring genes that are read as a UNIT (operon), Three genes ‘read’ as a single unit

  42. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter b. Helicases separate strands; first bases linked 5’  3’ c. Sigma subunit dissociates, polymerization continues d. Termination: e. PolycistronicDNA: In bacteria, proteins involved in the same metabolic process are often encoded by neighboring genes that are read as a UNIT (operon), producing one m-RNA that has the transcript of all three genes… Three genes ‘read’ as a single unit

  43. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria a. Polymerase (with sigma) lands on ds-DNA at promoter b. Helicases separate strands; first bases linked 5’  3’ c. Sigma subunit dissociates, polymerization continues d. Termination: e. Polycistronic DNA: In bacteria, proteins involved in the same metabolic process are often encoded by neighboring genes that are read as a UNIT (operon), producing one m-RNA that has the transcript of all three genes… then, in TRANSLATION, ribosomes attach at ‘start codons’ along the strand, synthesizing all proteins simultaneously. Three genes ‘read’ as a single unit

  44. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria: Translation of m-RNA by ribosomes occurs even before m-RNA is complete!

  45. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria: 5. Process in Eukaryotes:

  46. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria: 5. Process in Eukaryotes: - The chromatin must be unwound = chromatin remodeling

  47. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria: 5. Process in Eukaryotes: - The chromatin must be unwound = chromatin remodeling - Initiation is regulated by ‘enhancer sequences’ upstream and downstream from the gene, and transcription factor binding.

  48. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria: 5. Process in Eukaryotes: - The chromatin must be unwound = chromatin remodeling - Initiation is regulated by ‘enhancer sequences’ upstream and downstream from the gene, and transcription factor binding. The enhancer sequences are “cis-acting regulatory elements” (CRE’s), because they are on the same chromosome as the gene. Since the transcription factors are proteins encoded elsewhere in the genome, even on different chromosomes, they are called “trans-acting regulatory elements”.

  49. IX: DNA Function: Protein Synthesis A. Overview: B. Transcription: 1. The DNA template: 2. RNA Polymerase: 3. RNA triphosphate precursors: 4. Process in Bacteria: 5. Process in Eukaryotes: - The chromatin must be unwound = chromatin remodeling - Initiation is regulated by ‘enhancer sequences’ upstream and downstream from the gene, and transcription factor binding. - Because the m-RNA is bound in the nucleus—separated from the ribosomes—translation does not take place immediately.

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