Molecular Genetics Part II


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Molecular Genetics Part II

  1. 1. Molecular Genetics Part II From Gene to Protein
  2. 2. History <ul><li>Archibald Garrod – 1909 </li></ul><ul><li>First to suggest that genes dictate phenotype through production of proteins </li></ul><ul><li>Believed that genetic diseases resulted from the inability to make particular enzymes </li></ul><ul><li>“ Inborn errors of metabolism” </li></ul>
  3. 3. One Gene – One Enzyme <ul><li>Beadle & Ephrussi – 1930’s </li></ul><ul><li>Studied mutations affecting eye color in Drosophila </li></ul><ul><li>Concluded that each mutation blocks pigment synthesis at a specific step by preventing production of the enzyme that catalyzes that step </li></ul><ul><li>Specific pathways were not known, so results were inconclusive </li></ul>
  4. 4. Beadle & Tatum <ul><li>Treated Neurospora (a mold) with X-rays </li></ul><ul><li>Looked for mutations in nutritional requirements </li></ul><ul><ul><li>Wild type Neurospora grows on minimal medium (agar enriched with a few nutrients) </li></ul></ul><ul><ul><li>All mutants will grow on complete medium (agar plus all 20 amino acids & other nutrients) </li></ul></ul><ul><li>Identified the specific amino acid required for growth by each mutant </li></ul><ul><ul><li>That identified the defective synthetic pathway </li></ul></ul><ul><ul><li>Looked at each intermediate step in the blocked synthetic pathway </li></ul></ul><ul><li>Concluded that mutation in a single gene blocked production of a single enzyme </li></ul>
  5. 6. One Gene – One Polypeptide <ul><li>Not all proteins are enzymes </li></ul><ul><li>Can extend one gene = one enzyme doctrine to one gene = one polypeptide </li></ul><ul><li>Many proteins are comprised of two or more polypeptides </li></ul>
  6. 7. Central Dogma <ul><li>How does the sequence of a strand of DNA correspond to the amino acid sequence of a protein? </li></ul><ul><li>The central dogma of molecular biology, states that: </li></ul>
  7. 8. Transcription & Translation <ul><li>DNA is first copied ( transcribed ) to an RNA intermediate </li></ul><ul><li>The RNA intermediate is then translated to protein </li></ul><ul><li>Why have an intermediate between DNA and the proteins it encodes? </li></ul>
  8. 9. Why RNA? <ul><li>The DNA remains protected in the nucleus, away from caustic enzymes in the cytoplasm. </li></ul><ul><li>Gene information can be amplified </li></ul><ul><ul><li>Many copies of an RNA can be made from one copy of DNA. </li></ul></ul><ul><li>Greater regulation of gene expression </li></ul><ul><ul><li>Specific controls can act at each step in the pathway between DNA and proteins. </li></ul></ul><ul><ul><li>The more elements there are in the pathway, the more opportunities there are for control </li></ul></ul>
  9. 10. What is RNA? <ul><li>RNA has the same primary structure as DNA </li></ul><ul><ul><li>consists of a sugar-phosphate backbone, with nucleotides attached to the 1' C of the sugar. </li></ul></ul><ul><li>Differences between DNA and RNA : </li></ul><ul><ul><li>Contains the sugar ribose instead of deoxyribose </li></ul></ul><ul><ul><li>The nucleotide, uracil, is substituted for thymine </li></ul></ul><ul><ul><li>RNA exists as a single-stranded molecule. </li></ul></ul><ul><ul><ul><li>Because of the extra hydroxyl group on the sugar, RNA is too bulky to form a a stable double helix. </li></ul></ul></ul><ul><ul><ul><li>Regions of double helix can form where there is some base pair complementation resulting in hairpin loops . </li></ul></ul></ul>
  10. 11. Types of RNA <ul><li>mRNA - messenger RNA </li></ul><ul><ul><li>A copy of a gene. </li></ul></ul><ul><ul><li>Has a sequence complementary to one strand of the DNA & identical to the other strand. </li></ul></ul><ul><ul><li>Carries the information stored in DNA in the nucleus to the ribosomes in the cytoplasm where protein is made. </li></ul></ul><ul><li>tRNA - transfer RNA </li></ul><ul><ul><li>A small RNA with a very specific structure that can bind an amino acid at one end, and mRNA at the other end. </li></ul></ul><ul><ul><li>Acts as an ‘adaptor’ to carry & attach amino acids to the appropriate place on the mRNA. </li></ul></ul>
  11. 12. Types of RNA (Cont.) <ul><li>rRNA - ribosomal RNA </li></ul><ul><ul><li>One of the structural components of the ribosome. </li></ul></ul><ul><ul><li>Has a sequence complimentary to regions of the mRNA </li></ul></ul><ul><ul><li>Allows ribosome to bind to an mRNA </li></ul></ul><ul><li>snRNA - small nuclear RNA </li></ul><ul><ul><li>Is involved in the machinery that processes RNA's as they travel between the nucleus and the cytoplasm. </li></ul></ul>
  12. 13. The Genetic Code <ul><li>How does mRNA specify an amino acid sequence? </li></ul><ul><li>It would be impossible for each amino acid to be specified by one nucleotide </li></ul><ul><ul><li>there are only 4 nucleotides and 20 amino acids. </li></ul></ul><ul><ul><li>two nucleotide combinations could only specify 16 amino acids. </li></ul></ul><ul><li>Each amino acid is specified by a combination of three nucleotides, called a codon </li></ul>
  13. 16. The Code is Redundant, Not Ambiguous <ul><li>Each amino acid may be specified by up to six codons </li></ul><ul><ul><li>In many cases, codons that are synonyms differ only in the third base of the triplet </li></ul></ul><ul><li>Different organisms have different frequencies of codon usage. </li></ul><ul><ul><li>A giraffe might use CGC for arginine much more often than CGA, and the reverse might be true for a sperm whale. </li></ul></ul><ul><li>Some codons specify “stop” (or “start) </li></ul><ul><li>There is no ambiguity </li></ul><ul><ul><li>the same codon ALWAYS codes for the same amino acid </li></ul></ul>
  14. 17. Codons & Anticodons <ul><li>How do tRNAs recognize to which codon to bring an amino acid? </li></ul><ul><li>The tRNA has an anticodon on its mRNA-binding end </li></ul><ul><li>The anticodon is complementary to the codon on the mRNA. </li></ul><ul><li>Each tRNA only binds the appropriate amino acid for its anticodon </li></ul>
  15. 18. t-RNA Structure
  16. 19. Transcription <ul><li>How does the sequence information from DNA get transferred to mRNA? </li></ul><ul><li>How is this information carried to the ribosomes in the cytoplasm? </li></ul><ul><li>This process is called transcription </li></ul><ul><li>Highly similar to DNA replication. </li></ul><ul><li>Different enzymes are used in transcription. </li></ul><ul><li>The most important is RNA polymerase </li></ul>
  17. 20. RNA Polymerase <ul><li>RNA polymerase is a holoenzyme </li></ul><ul><ul><li>an agglomeration of many different factors </li></ul></ul><ul><li>Together, direct the synthesis of mRNA </li></ul><ul><li>Pries the DNA strands apart </li></ul><ul><li>Strings complimentary RNA nucleotides on the DNA template </li></ul><ul><li>Like DNA polymerase, can only add to the 3’ end </li></ul><ul><li>So only one mRNA is made, elongating 5’  3’ </li></ul>
  18. 21. Stages of Transcription <ul><li>Initiation </li></ul><ul><li>Elongation </li></ul><ul><li>Termination </li></ul>
  19. 23. Initiation <ul><li>RNA polymerase must recognize the beginning of a gene to know where to start synthesizing mRNA. </li></ul><ul><li>One part of the enzyme has a high affinity for a particular DNA sequence that appears at the beginning of genes. </li></ul><ul><li>The sequence where RNA polymerase attaches to the DNA and begins transcription = the promoter </li></ul><ul><ul><li>a unidirectional sequence on one strand of the DNA </li></ul></ul><ul><li>Tells RNA polymerase both where to start and in which direction (that is, on which strand) to continue synthesis. </li></ul>
  20. 24. The Promoter <ul><li>In prokaryotes, RNA polymerase recognizes and binds the promoter </li></ul><ul><li>The bacterial promoter almost always contains some version of the following elements: </li></ul>
  21. 25. Eukaryotic Promoters <ul><li>In eukaryotes special proteins, transcription factors , mediate binding RNA polymerase and the promoter </li></ul><ul><li>RNA polymerase binds to the promoter only after transcription factors bind </li></ul><ul><li>Transcription factors + RNA polymerase, bound to the promoter = transcription initiation complex </li></ul><ul><li>Eukaryotic promoters usually include a TATA box </li></ul><ul><ul><li>A nucleotide sequence containing TATA about 25 nucleotides prior to the start point </li></ul></ul>
  22. 27. Elongation <ul><li>The RNA polymerase stretches open the double helix at the start point in the DNA and begins synthesis of a complementary RNA strand on one of the DNA strands </li></ul><ul><li>The RNA polymerase recruits RNA nucleotides in the same way that DNA polymerase recruits dNTPs. </li></ul><ul><li>Since synthesis only proceeds in the 5' to 3' direction, there is no need for Okazaki fragments. </li></ul>
  23. 29. Sense & Antisense <ul><li>Synthesis only occurs in the 5’ to 3’ direction </li></ul><ul><li>In transcription, only one DNA strand is copied </li></ul><ul><li>We call the strand that is copied the antisense or template strand </li></ul><ul><li>The other strand, which is identical to the mRNA made (substituting U for T), is the sense or coding strand. </li></ul>
  24. 30. Termination of Transcription <ul><li>How does RNA polymerase know when to stop transcribing a gene? </li></ul><ul><li>Sequence that signals the end of transcription = terminator </li></ul><ul><li>RNA polymerase transcribes the terminator </li></ul><ul><ul><li>The transcribed terminator actually ends the process </li></ul></ul><ul><li>In prokaryotes there is no nucleus, so ribosomes can begin making protein from an mRNA immediately </li></ul><ul><li>The terminator sequence of the mRNA allows it to form a hairpin loop, which blocks the ribosome. </li></ul><ul><ul><li>The ribosome falls off the mRNA, </li></ul></ul><ul><ul><li>That signals termination by the RNA polymerase. </li></ul></ul><ul><ul><li>RNA polymerase falls off the DNA and transcription ceases. </li></ul></ul>
  25. 31. Eukaryotic Termination <ul><li>RNA polymerase continues for hundreds of nucleotides beyond the termination signal </li></ul><ul><li>AAUAAA </li></ul><ul><li>At a point 10 to 35 nucleotides past the AAUAAA, the forming m-RNA is cut free </li></ul><ul><li>The cleavage site is the point of addition of a poly-A tail </li></ul>
  26. 32. Post Transcription Modification <ul><li>In eukaryotes, enzymes modify pre-mRNA before it is sent to the cytoplasm </li></ul><ul><li>Both ends of the transcript are altered </li></ul><ul><li>The 5’ end is capped with modified guanine </li></ul><ul><ul><li>Protects mRNA from degradation </li></ul></ul><ul><ul><li>Helps attach the ribosome </li></ul></ul><ul><li>At the 3’ end an enzyme makes a poly-A tail formed from 50 to 250 adenine nucleotides </li></ul><ul><ul><li>Inhibits degradation and helps ribosome attach </li></ul></ul><ul><ul><li>May also help export mRNA out of the nucleus </li></ul></ul><ul><li>Interior sections are cut out, and the remaining parts are spliced together </li></ul>
  27. 33. RNA Processing
  28. 34. Introns & Exons <ul><li>Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides = introns </li></ul><ul><li>Noncoding sequences are interspersed between coding sections </li></ul><ul><li>Coding sections = exons </li></ul><ul><li>That is, the sequence of eukaryotic DNA that codes for a polypeptide is not continuous </li></ul><ul><li>RNA polymerase transcribes both introns and exons </li></ul>
  29. 35. RNA Splicing <ul><li>Introns are cut out and exons are spliced together before mRNA exits the nucleus </li></ul><ul><li>Short nucleotide sequences at the end of introns are signals for RNA splicing </li></ul><ul><li>Small nuclear ribonucleoproteins (snRNPs) recognize splice sites </li></ul><ul><ul><li>Composed of snRNA & protein </li></ul></ul><ul><li>Several snRNPs and additional proteins form a complex = spliceosome </li></ul><ul><li>At splice sites at the end of an intron, cuts out the intron and fuses the exons </li></ul>
  30. 36. The Spliceosome
  31. 37. Why Introns? <ul><li>Introns may play regulatory role in the cell </li></ul><ul><li>Split genes allow a single gene to code more than one kind of polypeptide </li></ul><ul><li>Outcome depends on which sections are treated as exons during RNA processing </li></ul><ul><ul><li>Alternative RNA splicing </li></ul></ul><ul><li>May facilitate evolution of new proteins </li></ul><ul><li>Increase possibility of potentially beneficial crossing-over of genes </li></ul>
  32. 39. Translation <ul><li>How do messenger RNAs direct protein synthesis? </li></ul><ul><li>The message encoded in the mRNA is an amino acid sequence </li></ul><ul><li>mRNA travels to ribosome in the cytoplasm, where the message is read </li></ul><ul><li>The specified amino acids are assembled on the mRNA template on the ribosome </li></ul><ul><li>Enzymes help form the sequenced amino acids into a polypeptide </li></ul>
  33. 41. The Ribosome <ul><li>The cellular factory where proteins are synthesized </li></ul><ul><li>Consists of structural RNA and ~ 80 different proteins. </li></ul><ul><li>In its inactive state, it exists as two subunits </li></ul><ul><ul><li>a large subunit and a small subunit. </li></ul></ul><ul><li>When the small subunit encounters an mRNA, it begins translation of the mRNA to protein. </li></ul><ul><li>There are three sites in the large subunit </li></ul><ul><ul><li>The A site accepts a new tRNA bearing an amino acid </li></ul></ul><ul><ul><li>the P site bears the tRNA attached to the growing chain. </li></ul></ul><ul><ul><li>The E site contains the exiting tRNA </li></ul></ul>
  34. 43. Charging the tRNA <ul><li>tRNA (transfer RNA) acts as a translator between mRNA and protein </li></ul><ul><li>Each tRNA has a specific anticodon and an amino acid acceptor site. </li></ul><ul><li>Each tRNA also has a specific charger protein; </li></ul><ul><ul><li>This protein can only bind to that particular tRNA and attach the correct amino acid to the acceptor site. </li></ul></ul><ul><ul><li>These charger proteins are called aminoacyl tRNA synthetases </li></ul></ul><ul><li>The energy to make this bond comes from ATP . </li></ul>
  35. 45. Aminoacyl-tRNA Synthases <ul><li>Each tRNA must match with the correct amino acid </li></ul><ul><ul><li>Each tRNA must attach only the amino acid specified by the mRNA codon to which the tRNA anticodon binds </li></ul></ul><ul><li>The amino acid is joined to the tRNA by an aminoacyl-tRNA synthase </li></ul><ul><ul><li>There are 20 of these enzymes; one for each amino acid </li></ul></ul><ul><li>Catalyzes the covalent bond between the amino acid and tRNA </li></ul><ul><li>The active site of each aminoacyl-tRNA synthase fits only a specific amino acid and tRNA </li></ul><ul><li>Once the amino acid is bound, the tRNA is aminoacyl tRNA </li></ul>
  36. 47. Wobble <ul><li>If there was one tRNA for each mRNA codon, there would be 61 different tRNAs </li></ul><ul><li>Actually, there are fewer </li></ul><ul><li>Some tRNAs have anticodons that recognize 2 or more different codons </li></ul><ul><li>Base pairing rules between the third base of a codon and its tRNA anticodon are not a rigid as DNA to mRNA pairing </li></ul><ul><ul><li>Example: U in tRNA can pair with either A or T in the third position of an mRNA codon </li></ul></ul><ul><li>This flexibility is called wobble </li></ul>
  37. 49. Initiation of Translation <ul><li>The start signal for translation is the codon ATG </li></ul><ul><ul><li>Codes for methionine. </li></ul></ul><ul><ul><li>Not every protein starts with methionine, </li></ul></ul><ul><ul><li>Often this first amino acid will be removed in post-translational processing. </li></ul></ul><ul><li>A tRNA charged with methionine binds to the translation start signal. </li></ul><ul><li>The large subunit binds to the mRNA and the small subunit </li></ul><ul><li>Elongation begins. </li></ul>
  38. 51. Elongation of the New Protein <ul><li>After the first charged tRNA appears in the A site, the ribosome shifts so that the tRNA is in the P site. </li></ul><ul><li>New charged tRNAs, corresponding the codons of the mRNA, enter the A site, and a peptide bond is formed between the two amino acids. </li></ul><ul><li>The first tRNA is now released </li></ul><ul><li>The ribosome shifts again so that a tRNA carrying two amino acids is now in the P site </li></ul><ul><li>A new charged tRNA can bind to the A site. </li></ul><ul><li>This process of elongation continues until the ribosome reaches a stop codon. </li></ul>
  39. 53. Termination of the Protein <ul><li>When the ribosome reaches a stop codon, no aminoacyl tRNA binds to the empty A site. </li></ul><ul><li>This is the ribosome’s signal to break into its large and small subunits, </li></ul><ul><li>Releasing the new protein and the mRNA. </li></ul>
  40. 55. Polyribosomes <ul><li>A single mRNA can be used to make many copies of a polypeptide at the same time </li></ul><ul><li>Multiple ribosomes can read the same mRNA strand, like beads on a string </li></ul><ul><li>These strings are called polyribosomes </li></ul>
  41. 56. Polyribosomes
  42. 57. Post-Translational Processing <ul><li>This isn't always the end of the story for the new protein. </li></ul><ul><li>Often it will undergo post-translational modifications . </li></ul><ul><li>Modifications include: </li></ul><ul><li>Cleavage by a proteolytic (protein-cutting) enzyme at a specific place. </li></ul><ul><li>Having some amino acids altered. </li></ul><ul><ul><li>For example, a tyrosine residue might be phosphorylated. </li></ul></ul><ul><li>Become glycosylated. </li></ul><ul><ul><li>Many proteins have carbohydrates covalently attached to asparagine residues. </li></ul></ul>
  43. 59. Mutations <ul><li>What kinds of errors can occur in DNA? </li></ul><ul><li>What causes them? </li></ul><ul><li>What are their effects? </li></ul><ul><li>Types of mutations: </li></ul><ul><ul><li>Chromosomal mutations </li></ul></ul><ul><ul><li>Point mutations </li></ul></ul><ul><ul><li>Frameshift mutations </li></ul></ul>
  44. 61. Chromosomal Mutations <ul><li>Mutations that occur at a macroscopic level. </li></ul><ul><li>Large sections of chromosomes can be altered or shifted, leading to changes in the way genes are expressed. </li></ul><ul><li>Types of chromosomal mutations: </li></ul><ul><ul><li>Translocations </li></ul></ul><ul><ul><li>Inversions </li></ul></ul><ul><ul><li>Deletions </li></ul></ul><ul><ul><li>Nondisjunction </li></ul></ul>
  45. 62. Translocations & Inversions <ul><li>Translocation </li></ul><ul><ul><li>The interchange of large segments of DNA between two chromosomes. </li></ul></ul><ul><ul><li>Can change gene expression if a gene is at the translocation breakpoint or if it is reattached so that it is incorrectly regulated </li></ul></ul><ul><li>Inversion </li></ul><ul><ul><li>Occurs when a region of DNA flips its orientation with respect to the rest of the chromosome. </li></ul></ul><ul><ul><li>Rotates, end for end </li></ul></ul><ul><ul><li>This can lead to the same problems as translocations. </li></ul></ul>
  46. 63. Deletions & Nondisjunction <ul><li>Deletion </li></ul><ul><ul><li>Sometimes large regions of a chromosome are deleted. </li></ul></ul><ul><ul><li>This can lead to a loss of important genes. </li></ul></ul><ul><li>Nondisjunction </li></ul><ul><ul><li>Sometimes chromosomes do not divide correctly in cell division </li></ul></ul><ul><ul><li>When large regions of a chromosome are altered (such as translocation), the chromosome may not segregate properly during cell division </li></ul></ul><ul><ul><li>One daughter cell will end up with extra genetic material, one will end up with less than its share </li></ul></ul><ul><ul><li>This is called nondisjunction. </li></ul></ul><ul><ul><li>When there are extra or too few copies of a gene, the cell will have problems </li></ul></ul>
  47. 64. Point Mutations <ul><li>Point mutations are single base pair changes. </li></ul><ul><li>Three possible outcomes: </li></ul><ul><li>Nonsense mutation </li></ul><ul><ul><li>Creates a stop codon where none previously existed. </li></ul></ul><ul><ul><li>This shortens the resulting protein, possibly removing essential regions. </li></ul></ul><ul><li>Missense mutation </li></ul><ul><ul><li>Changes the code of the mRNA. </li></ul></ul><ul><ul><li>Which changes the resulting amino acid </li></ul></ul><ul><ul><li>This may alter the shape and properties of the protein. </li></ul></ul><ul><li>Silent mutation </li></ul><ul><ul><li>Has no effect on protein sequence. </li></ul></ul><ul><ul><li>Because the genetic code is redundant, some changes have no effect </li></ul></ul>
  48. 66. Frameshift Mutations <ul><li>Insertions or deletions have a disastrous effect </li></ul><ul><li>mRNA is “read” as a series of three letter words </li></ul><ul><li>Insertions or deletions that are not multiples of three, shift the reading frame </li></ul>
  49. 67. Frameshift Example <ul><li>Given the coding sequence: </li></ul><ul><li>AGA UCG ACG UUA AGC </li></ul><ul><li>corresponding to the protein: </li></ul><ul><li>arginine - serine - threonine - leucine - serine </li></ul><ul><li>The insertion of a C-G base pair between bases 6 and 7 would result in the following new code: AGA UCG CAC GUU AAG C </li></ul><ul><li>which would result in a non-functional protein: arginine - serine - histidine - valine - lysine </li></ul><ul><li>Every amino acid after the insertion will be wrong. </li></ul><ul><li>The frame shift might even generate a stop codon which would prematurely end the protein. </li></ul>
  50. 69. DNA Repair <ul><li>If replication of DNA proceeded as was described previously, DNA polymerase would make a mistake on average about every 1000 base pairs. </li></ul><ul><li>This level would be unacceptable, because too many genes would be rendered non-functional. </li></ul><ul><li>Organisms have elaborate DNA proofreading and repair mechanisms, which can recognize false base-pairing and DNA damage, and repair it. </li></ul><ul><li>The actual error rate is more in the region of one in a million to one in a billion. </li></ul>
  51. 70. The Beauty of Mutations <ul><li>Why mutations? </li></ul><ul><li>Our environment constantly changes, the Earth and its ecosystems change. </li></ul><ul><li>Populations must change to survive </li></ul><ul><li>Evolutionary change requires variation, the raw material on which natural selection works </li></ul><ul><li>One mechanism for variation and change is at the DNA level. </li></ul><ul><li>Mutations can be beneficial and enable the organism to adapt to a changing environment. </li></ul><ul><li>However, most mutations are deleterious, and cause varied genetic diseases </li></ul>