Announcements
1. Homework - problem set 5 - due this Friday
2. 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
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
I. RNA Processing in Eukaryotes
STABILITY
STABILITY
Introns and Exons
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
II. Structure: Unusual Bases Found in tRNA
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.
tRNA Structure
Aminoacyl tRNA synthetase
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.
Ribosome Structure
S = Svedberg, a measure of sedimentation in centrifuge
Ribosome Binding Sites: A, P, E
III. Translation has 3 Steps, Each Requiring Different Supporting Proteins
• Initiation– Requires Initiation Factors
• Elongation– Requires Elongation Factors
• Termination– Requires Termination Factor
Overview of Prokaryotic Translation
Initiation:
1. Binding of initiation factors to small subunit.
2. Binding of first tRNA and mRNA to small subunit.
3. Binding of large subunit.
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
Termination:
1. Binding of Release Factor to Stop Codon
UGA, UAA, UAG.
2. Disassembly
EM of Polyribosomes: >1 Ribosome working on the same mRNA
Rabbit Hemoglobin mRNA Midgefly Salivary Glandwith Nascent Polypeptide
Note: occurs in cytoplasm.
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.
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.
Metabolic Pathways for Phe and Tyr
tyrosinase
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
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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