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Molecular Genetics Part II


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  • 1. Molecular Genetics Part II From Gene to Protein
  • 2. History
    • Archibald Garrod – 1909
    • First to suggest that genes dictate phenotype through production of proteins
    • Believed that genetic diseases resulted from the inability to make particular enzymes
    • “ Inborn errors of metabolism”
  • 3. One Gene – One Enzyme
    • Beadle & Ephrussi – 1930’s
    • Studied mutations affecting eye color in Drosophila
    • Concluded that each mutation blocks pigment synthesis at a specific step by preventing production of the enzyme that catalyzes that step
    • Specific pathways were not known, so results were inconclusive
  • 4. Beadle & Tatum
    • Treated Neurospora (a mold) with X-rays
    • Looked for mutations in nutritional requirements
      • Wild type Neurospora grows on minimal medium (agar enriched with a few nutrients)
      • All mutants will grow on complete medium (agar plus all 20 amino acids & other nutrients)
    • Identified the specific amino acid required for growth by each mutant
      • That identified the defective synthetic pathway
      • Looked at each intermediate step in the blocked synthetic pathway
    • Concluded that mutation in a single gene blocked production of a single enzyme
  • 5.  
  • 6. One Gene – One Polypeptide
    • Not all proteins are enzymes
    • Can extend one gene = one enzyme doctrine to one gene = one polypeptide
    • Many proteins are comprised of two or more polypeptides
  • 7. Central Dogma
    • How does the sequence of a strand of DNA correspond to the amino acid sequence of a protein?
    • The central dogma of molecular biology, states that:
  • 8. Transcription & Translation
    • DNA is first copied ( transcribed ) to an RNA intermediate
    • The RNA intermediate is then translated to protein
    • Why have an intermediate between DNA and the proteins it encodes?
  • 9. Why RNA?
    • The DNA remains protected in the nucleus, away from caustic enzymes in the cytoplasm.
    • Gene information can be amplified
      • Many copies of an RNA can be made from one copy of DNA.
    • Greater regulation of gene expression
      • Specific controls can act at each step in the pathway between DNA and proteins.
      • The more elements there are in the pathway, the more opportunities there are for control
  • 10. What is RNA?
    • RNA has the same primary structure as DNA
      • consists of a sugar-phosphate backbone, with nucleotides attached to the 1' C of the sugar.
    • Differences between DNA and RNA :
      • Contains the sugar ribose instead of deoxyribose
      • The nucleotide, uracil, is substituted for thymine
      • RNA exists as a single-stranded molecule.
        • Because of the extra hydroxyl group on the sugar, RNA is too bulky to form a a stable double helix.
        • Regions of double helix can form where there is some base pair complementation resulting in hairpin loops .
  • 11. Types of RNA
    • mRNA - messenger RNA
      • A copy of a gene.
      • Has a sequence complementary to one strand of the DNA & identical to the other strand.
      • Carries the information stored in DNA in the nucleus to the ribosomes in the cytoplasm where protein is made.
    • tRNA - transfer RNA
      • A small RNA with a very specific structure that can bind an amino acid at one end, and mRNA at the other end.
      • Acts as an ‘adaptor’ to carry & attach amino acids to the appropriate place on the mRNA.
  • 12. Types of RNA (Cont.)
    • rRNA - ribosomal RNA
      • One of the structural components of the ribosome.
      • Has a sequence complimentary to regions of the mRNA
      • Allows ribosome to bind to an mRNA
    • snRNA - small nuclear RNA
      • Is involved in the machinery that processes RNA's as they travel between the nucleus and the cytoplasm.
  • 13. The Genetic Code
    • How does mRNA specify an amino acid sequence?
    • It would be impossible for each amino acid to be specified by one nucleotide
      • there are only 4 nucleotides and 20 amino acids.
      • two nucleotide combinations could only specify 16 amino acids.
    • Each amino acid is specified by a combination of three nucleotides, called a codon
  • 14.  
  • 15.  
  • 16. The Code is Redundant, Not Ambiguous
    • Each amino acid may be specified by up to six codons
      • In many cases, codons that are synonyms differ only in the third base of the triplet
    • Different organisms have different frequencies of codon usage.
      • A giraffe might use CGC for arginine much more often than CGA, and the reverse might be true for a sperm whale.
    • Some codons specify “stop” (or “start)
    • There is no ambiguity
      • the same codon ALWAYS codes for the same amino acid
  • 17. Codons & Anticodons
    • How do tRNAs recognize to which codon to bring an amino acid?
    • The tRNA has an anticodon on its mRNA-binding end
    • The anticodon is complementary to the codon on the mRNA.
    • Each tRNA only binds the appropriate amino acid for its anticodon
  • 18. t-RNA Structure
  • 19. Transcription
    • How does the sequence information from DNA get transferred to mRNA?
    • How is this information carried to the ribosomes in the cytoplasm?
    • This process is called transcription
    • Highly similar to DNA replication.
    • Different enzymes are used in transcription.
    • The most important is RNA polymerase
  • 20. RNA Polymerase
    • RNA polymerase is a holoenzyme
      • an agglomeration of many different factors
    • Together, direct the synthesis of mRNA
    • Pries the DNA strands apart
    • Strings complimentary RNA nucleotides on the DNA template
    • Like DNA polymerase, can only add to the 3’ end
    • So only one mRNA is made, elongating 5’  3’
  • 21. Stages of Transcription
    • Initiation
    • Elongation
    • Termination
  • 22.  
  • 23. Initiation
    • RNA polymerase must recognize the beginning of a gene to know where to start synthesizing mRNA.
    • One part of the enzyme has a high affinity for a particular DNA sequence that appears at the beginning of genes.
    • The sequence where RNA polymerase attaches to the DNA and begins transcription = the promoter
      • a unidirectional sequence on one strand of the DNA
    • Tells RNA polymerase both where to start and in which direction (that is, on which strand) to continue synthesis.
  • 24. The Promoter
    • In prokaryotes, RNA polymerase recognizes and binds the promoter
    • The bacterial promoter almost always contains some version of the following elements:
  • 25. Eukaryotic Promoters
    • In eukaryotes special proteins, transcription factors , mediate binding RNA polymerase and the promoter
    • RNA polymerase binds to the promoter only after transcription factors bind
    • Transcription factors + RNA polymerase, bound to the promoter = transcription initiation complex
    • Eukaryotic promoters usually include a TATA box
      • A nucleotide sequence containing TATA about 25 nucleotides prior to the start point
  • 26.  
  • 27. Elongation
    • 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
    • The RNA polymerase recruits RNA nucleotides in the same way that DNA polymerase recruits dNTPs.
    • Since synthesis only proceeds in the 5' to 3' direction, there is no need for Okazaki fragments.
  • 28.  
  • 29. Sense & Antisense
    • Synthesis only occurs in the 5’ to 3’ direction
    • In transcription, only one DNA strand is copied
    • We call the strand that is copied the antisense or template strand
    • The other strand, which is identical to the mRNA made (substituting U for T), is the sense or coding strand.
  • 30. Termination of Transcription
    • How does RNA polymerase know when to stop transcribing a gene?
    • Sequence that signals the end of transcription = terminator
    • RNA polymerase transcribes the terminator
      • The transcribed terminator actually ends the process
    • In prokaryotes there is no nucleus, so ribosomes can begin making protein from an mRNA immediately
    • The terminator sequence of the mRNA allows it to form a hairpin loop, which blocks the ribosome.
      • The ribosome falls off the mRNA,
      • That signals termination by the RNA polymerase.
      • RNA polymerase falls off the DNA and transcription ceases.
  • 31. Eukaryotic Termination
    • RNA polymerase continues for hundreds of nucleotides beyond the termination signal
    • AAUAAA
    • At a point 10 to 35 nucleotides past the AAUAAA, the forming m-RNA is cut free
    • The cleavage site is the point of addition of a poly-A tail
  • 32. Post Transcription Modification
    • In eukaryotes, enzymes modify pre-mRNA before it is sent to the cytoplasm
    • Both ends of the transcript are altered
    • The 5’ end is capped with modified guanine
      • Protects mRNA from degradation
      • Helps attach the ribosome
    • At the 3’ end an enzyme makes a poly-A tail formed from 50 to 250 adenine nucleotides
      • Inhibits degradation and helps ribosome attach
      • May also help export mRNA out of the nucleus
    • Interior sections are cut out, and the remaining parts are spliced together
  • 33. RNA Processing
  • 34. Introns & Exons
    • Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides = introns
    • Noncoding sequences are interspersed between coding sections
    • Coding sections = exons
    • That is, the sequence of eukaryotic DNA that codes for a polypeptide is not continuous
    • RNA polymerase transcribes both introns and exons
  • 35. RNA Splicing
    • Introns are cut out and exons are spliced together before mRNA exits the nucleus
    • Short nucleotide sequences at the end of introns are signals for RNA splicing
    • Small nuclear ribonucleoproteins (snRNPs) recognize splice sites
      • Composed of snRNA & protein
    • Several snRNPs and additional proteins form a complex = spliceosome
    • At splice sites at the end of an intron, cuts out the intron and fuses the exons
  • 36. The Spliceosome
  • 37. Why Introns?
    • Introns may play regulatory role in the cell
    • Split genes allow a single gene to code more than one kind of polypeptide
    • Outcome depends on which sections are treated as exons during RNA processing
      • Alternative RNA splicing
    • May facilitate evolution of new proteins
    • Increase possibility of potentially beneficial crossing-over of genes
  • 38.  
  • 39. Translation
    • How do messenger RNAs direct protein synthesis?
    • The message encoded in the mRNA is an amino acid sequence
    • mRNA travels to ribosome in the cytoplasm, where the message is read
    • The specified amino acids are assembled on the mRNA template on the ribosome
    • Enzymes help form the sequenced amino acids into a polypeptide
  • 40.  
  • 41. The Ribosome
    • The cellular factory where proteins are synthesized
    • Consists of structural RNA and ~ 80 different proteins.
    • In its inactive state, it exists as two subunits
      • a large subunit and a small subunit.
    • When the small subunit encounters an mRNA, it begins translation of the mRNA to protein.
    • There are three sites in the large subunit
      • The A site accepts a new tRNA bearing an amino acid
      • the P site bears the tRNA attached to the growing chain.
      • The E site contains the exiting tRNA
  • 42.  
  • 43. Charging the tRNA
    • tRNA (transfer RNA) acts as a translator between mRNA and protein
    • Each tRNA has a specific anticodon and an amino acid acceptor site.
    • Each tRNA also has a specific charger protein;
      • This protein can only bind to that particular tRNA and attach the correct amino acid to the acceptor site.
      • These charger proteins are called aminoacyl tRNA synthetases
    • The energy to make this bond comes from ATP .
  • 44.  
  • 45. Aminoacyl-tRNA Synthases
    • Each tRNA must match with the correct amino acid
      • Each tRNA must attach only the amino acid specified by the mRNA codon to which the tRNA anticodon binds
    • The amino acid is joined to the tRNA by an aminoacyl-tRNA synthase
      • There are 20 of these enzymes; one for each amino acid
    • Catalyzes the covalent bond between the amino acid and tRNA
    • The active site of each aminoacyl-tRNA synthase fits only a specific amino acid and tRNA
    • Once the amino acid is bound, the tRNA is aminoacyl tRNA
  • 46.  
  • 47. Wobble
    • If there was one tRNA for each mRNA codon, there would be 61 different tRNAs
    • Actually, there are fewer
    • Some tRNAs have anticodons that recognize 2 or more different codons
    • Base pairing rules between the third base of a codon and its tRNA anticodon are not a rigid as DNA to mRNA pairing
      • Example: U in tRNA can pair with either A or T in the third position of an mRNA codon
    • This flexibility is called wobble
  • 48.  
  • 49. Initiation of Translation
    • The start signal for translation is the codon ATG
      • Codes for methionine.
      • Not every protein starts with methionine,
      • Often this first amino acid will be removed in post-translational processing.
    • A tRNA charged with methionine binds to the translation start signal.
    • The large subunit binds to the mRNA and the small subunit
    • Elongation begins.
  • 50.  
  • 51. Elongation of the New Protein
    • After the first charged tRNA appears in the A site, the ribosome shifts so that the tRNA is in the P site.
    • New charged tRNAs, corresponding the codons of the mRNA, enter the A site, and a peptide bond is formed between the two amino acids.
    • The first tRNA is now released
    • The ribosome shifts again so that a tRNA carrying two amino acids is now in the P site
    • A new charged tRNA can bind to the A site.
    • This process of elongation continues until the ribosome reaches a stop codon.
  • 52.  
  • 53. Termination of the Protein
    • When the ribosome reaches a stop codon, no aminoacyl tRNA binds to the empty A site.
    • This is the ribosome’s signal to break into its large and small subunits,
    • Releasing the new protein and the mRNA.
  • 54.  
  • 55. Polyribosomes
    • A single mRNA can be used to make many copies of a polypeptide at the same time
    • Multiple ribosomes can read the same mRNA strand, like beads on a string
    • These strings are called polyribosomes
  • 56. Polyribosomes
  • 57. Post-Translational Processing
    • This isn't always the end of the story for the new protein.
    • Often it will undergo post-translational modifications .
    • Modifications include:
    • Cleavage by a proteolytic (protein-cutting) enzyme at a specific place.
    • Having some amino acids altered.
      • For example, a tyrosine residue might be phosphorylated.
    • Become glycosylated.
      • Many proteins have carbohydrates covalently attached to asparagine residues.
  • 58.  
  • 59. Mutations
    • What kinds of errors can occur in DNA?
    • What causes them?
    • What are their effects?
    • Types of mutations:
      • Chromosomal mutations
      • Point mutations
      • Frameshift mutations
  • 60.  
  • 61. Chromosomal Mutations
    • Mutations that occur at a macroscopic level.
    • Large sections of chromosomes can be altered or shifted, leading to changes in the way genes are expressed.
    • Types of chromosomal mutations:
      • Translocations
      • Inversions
      • Deletions
      • Nondisjunction
  • 62. Translocations & Inversions
    • Translocation
      • The interchange of large segments of DNA between two chromosomes.
      • Can change gene expression if a gene is at the translocation breakpoint or if it is reattached so that it is incorrectly regulated
    • Inversion
      • Occurs when a region of DNA flips its orientation with respect to the rest of the chromosome.
      • Rotates, end for end
      • This can lead to the same problems as translocations.
  • 63. Deletions & Nondisjunction
    • Deletion
      • Sometimes large regions of a chromosome are deleted.
      • This can lead to a loss of important genes.
    • Nondisjunction
      • Sometimes chromosomes do not divide correctly in cell division
      • When large regions of a chromosome are altered (such as translocation), the chromosome may not segregate properly during cell division
      • One daughter cell will end up with extra genetic material, one will end up with less than its share
      • This is called nondisjunction.
      • When there are extra or too few copies of a gene, the cell will have problems
  • 64. Point Mutations
    • Point mutations are single base pair changes.
    • Three possible outcomes:
    • Nonsense mutation
      • Creates a stop codon where none previously existed.
      • This shortens the resulting protein, possibly removing essential regions.
    • Missense mutation
      • Changes the code of the mRNA.
      • Which changes the resulting amino acid
      • This may alter the shape and properties of the protein.
    • Silent mutation
      • Has no effect on protein sequence.
      • Because the genetic code is redundant, some changes have no effect
  • 65.  
  • 66. Frameshift Mutations
    • Insertions or deletions have a disastrous effect
    • mRNA is “read” as a series of three letter words
    • Insertions or deletions that are not multiples of three, shift the reading frame
  • 67. Frameshift Example
    • Given the coding sequence:
    • corresponding to the protein:
    • arginine - serine - threonine - leucine - serine
    • 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
    • which would result in a non-functional protein: arginine - serine - histidine - valine - lysine
    • Every amino acid after the insertion will be wrong.
    • The frame shift might even generate a stop codon which would prematurely end the protein.
  • 68.  
  • 69. DNA Repair
    • If replication of DNA proceeded as was described previously, DNA polymerase would make a mistake on average about every 1000 base pairs.
    • This level would be unacceptable, because too many genes would be rendered non-functional.
    • Organisms have elaborate DNA proofreading and repair mechanisms, which can recognize false base-pairing and DNA damage, and repair it.
    • The actual error rate is more in the region of one in a million to one in a billion.
  • 70. The Beauty of Mutations
    • Why mutations?
    • Our environment constantly changes, the Earth and its ecosystems change.
    • Populations must change to survive
    • Evolutionary change requires variation, the raw material on which natural selection works
    • One mechanism for variation and change is at the DNA level.
    • Mutations can be beneficial and enable the organism to adapt to a changing environment.
    • However, most mutations are deleterious, and cause varied genetic diseases