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

3. 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

4. I. RNA Processing in Eukaryotes
STABILITY

5. STABILITY

6. Introns and Exons

7. 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

8. II. Structure: Unusual Bases Found in tRNA

9. 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.

10. tRNA Structure
Aminoacyl tRNA synthetase

11. 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.

12. Ribosome Structure
S = Svedberg, a measure of sedimentation in centrifuge

13. Ribosome Binding Sites: A, P, E

14. III. Translation has 3 Steps, Each Requiring Different Supporting Proteins
Initiation
Requires Initiation Factors
Elongation
Requires Elongation Factors
Termination
Requires Termination Factor

15. Overview of Prokaryotic Translation

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

17. Elongation: 1. Binding of next tRNA using EFs at A site. 2
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

18. Termination: 1. Binding of Release Factor to Stop Codon UGA, UAA, UAG
Termination: 1. Binding of Release Factor to Stop Codon UGA, UAA, UAG. 2. Disassembly

19. EM of Polyribosomes: >1 Ribosome working on the same mRNA
Rabbit Hemoglobin mRNA
Midgefly Salivary Gland
with Nascent Polypeptide
Note: occurs in cytoplasm.

20. 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.

21. 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.

22. Metabolic Pathways for Phe and Tyr
tyrosinase

23. 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

24. 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.