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 basepairing: 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.
EPA
EPA
2. Peptide Bond
formation between 2
amino acids.
EPA
EPA
EPA
3. Translocation of
ribosome.
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 Gland
with 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.