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Announcements. Homework - problem set 5 - due this Friday Reading Ch. 14: Skim btm 391 -top 397. Skip rest of 397- 403. Review of Last Lecture. I. tRNA and the genetic code II. Transcription - prokaryotes III. Transcription - eukaryotes. Outline of Lecture 25.

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slide1

Announcements

Homework - problem set 5 - due this Friday

Reading Ch. 14: Skim btm 391 -top 397.

Skip rest of 397- 403.

slide2

Review of Last Lecture

I. tRNA and the genetic code

II. Transcription - prokaryotes

III. Transcription - eukaryotes

slide3

Outline of Lecture 25

I. RNA processing in eukaryotes

II. Translation of mRNA into protein - tRNA and ribosomes

III. Three steps of translation

IV. First evidence that proteins are important to heredity

V. One gene- one enzyme hypothesis

slide7

Eukaryotic vs. Prokaryotic Transcription

  • In eukaryotes, transcription and translation occur in separate compartments.
  • In bacteria, mRNA is polycistronic; in eukaryotes, mRNA is usually monocistronic.
    • Polycistronic: one mRNA codes for more than one polypeptide
    • moncistronic: one mRNA codes for only one polypeptide
  • 3 RNA polymerases in euk., 1 in prok.
  • Binding of Basal Transcription Factors required for euk. RNA Pol II binding.
  • “Processing” of mRNA in eukaryotes, no processing in prokaryotes
slide9

Function of Unusual Bases

  • Created post-transcriptionally.
  • Purpose is sometimes to allow for promiscuous base-pairing: Inosine in the 1st “wobble” position of anticodon can bind to 3rd U, C or A in codon.
  • This means that fewer different tRNAs are required.
  • Others play a structural role.
slide10

tRNA Structure

Aminoacyl tRNA synthetase

slide11

Aminoacyl tRNA Synthetases

  • Enzymes which bond specific amino acids to their cognate tRNAs.
  • There are 20 different synthetases, one for each amino acid.
  • Covalent linkage through an ester bond (amino acid activation) requires ATP.
  • tRNA linked to amino acid is charged.
slide12

Ribosome Structure

S = Svedberg, a measure of sedimentation in centrifuge

slide14

III. Translation has 3 Steps, Each Requiring Different Supporting Proteins

  • Initiation
    • Requires Initiation Factors
  • Elongation
    • Requires Elongation Factors
  • Termination
    • Requires Termination Factor
slide16

Initiation:1. Binding of initiation factors to small subunit.2. Binding of first tRNA and mRNA to small subunit.3. Binding of large subunit.

slide17

Elongation:1. Binding of next tRNA using EFs at A site.2. Peptide Bond formation between 2 amino acids.3. Translocation of ribosome.

E P A

E P A

E P A

E P A

E P A

slide19

EM of Polyribosomes: >1 Ribosome working on the same mRNA

Rabbit Hemoglobin mRNA

Midgefly Salivary Gland

with Nascent Polypeptide

Note: occurs in cytoplasm.

slide20

IV. Inborn Errors of Metabolism Provided First Evidence that Genes Encode Proteins

Alkaptonuria is an inherited disorder

first described by Garrod (1902) and Willliam Bateson.

  • Infants have black urine, darkened ears and nose due to homogentisic acid deposits.
  • Garrod increased the amino acids phenylalanine and tyrosine in the diet and saw increased deposits in affected individuals only.
  • He concluded that “unit factors control ferments” (genes control enzymes); results ignored for 30 years.
slide21

Phenylketonuria (PKU)

  • Autosomal recessive human metabolic disorder, first described in 1934.
  • 1/11,000 live births, results in mental retardation due to high [Phe] in body fluids.
  • Homozygotes cannot convert Phe to Tyr, since enzyme phenylalanine hydroxylase is lost.
  • Treatment: detection in newborns, low Phe diet; prevents mental retardation
  • Thousands of disorders have been found that result from genetic factors rather than pathogens.
slide23

Other Metabolic Disorders in the Pathway

  • Albinism
    • Autosomal recessive
    • Results from loss of tyrosinase enzyme in skin, which converts Tyr to DOPA and DOPA to Melanin pigments
    • Loss of tyrosinase in brain causes Parkinson’s Disease (loss of DOPA+ neurons).
  • Tyrosinemia
    • Results from loss of tyrosine transaminase
slide24

V. Beadle and Tatum: One Gene - One Enzyme (Polypeptide)

  • From mutations in fungus Neurospora
  • True in many cases, but there are many exceptions:
    • Some proteins have multiple subunits, each a polypeptide coded by a different gene.
    • Some genes code for more than one polypeptide, through differential splicing out of introns; e.g. secreted vs. membrane-bound forms of antibody molecules.
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