Download Regulation of gene expression: Eukaryotic

Announcements
1. Specifics on reading assignments: Ch. 11: Skip, p. 304,
btm. 309- top 312; Ch. 12: skim 327-328; skip btm 335336; skip recombination on 338-341; Ch. 13: skim btm
353-354; 358-top 360; skip splicing mechanism, editing,
and EM (367-370).
2. Problems to look over: Ch. 11: 4, 5, 8, 15; Ch. 12: 3, 10,
21, 22; Ch. 13: 2, 6, 7, 13, 15, 23.
3. Homework due next Friday, 11/1.
4. Last day to withdraw is next Fri. Nov. 1. If you have
concerns, talk to me.
Review of Last Lecture
1. Small group discussion on Watson-Crick paper
2. The Genetic Code - biochemical evidence for triplet code
Outline of Lecture 24
I. tRNA and the genetic code
II. Transcription - prokaryotes
III. Transcription - eukaryotes
IV. RNA Processing
Nucleic Acid to Protein
• How does the information in
codons of mRNA get
translated into amino acids
in polypeptides?
• Through adapter molecules:
tRNA
• tRNA has anticodon that
base pairs with the codon in
mRNA and carries an amino
acid corresponding to that
codon.
Note that 3rd Base Position is Variable
The genetic code is
nearly universal.
Exceptions:
yeast
mitochondria
Tetrahymena
Mycoplasma
Degeneracy and the Wobble Hypothesis
C
G
•
•
•
•
Codon in mRNA
Anticodon in tRNA
Codon: 5’- 1-2-3 -3’
Anticodon: 3’- 3-2-1 -5’
U
G
• First two bases of codon are
more critical than 3rd base
• Base-pairing rules are relaxed
between 3rd base of codon and
1st base of anticodon (third
base “wobble”)
Special Anticodon-Codon Base-Pairing Rules
II. Transcription: RNA from DNA
What is the enzyme that can direct RNA synthesis?
RNA polymerase - first isolated in liver of rats
- requires NTPs with ribose as sugar
- NO primer is needed
DNA
n(NTP)
(NMP)n + n (Ppi)
enzyme
RNA polymerase well characterized in E. coli - 4 subunits
Making Sense of the Strands
• DNA coding strand = Sense Strand
• DNA template strand = Antisense Strand
• mRNA formed = Sense Strand
Coding or Partner strand
5’
mRNA 5’
3’
3’
3’
Template strand
5’
Components of Prokaryotic Transcription
3 main steps of transcription: initiation, elongation, termination
NO primer required
5’ to 3’
50 nt/second
Prokaryotic Promoter Lies Just Upstream
(5’) of Transcribed Region
Two Consensus sequences
-35 Region
-10 TATA Box
Effect of mutations here?
Termination of Transcription in
Prokaryotes
• A specific nucleotide sequence acts as a
termination signal, about 40 base pairs in length
• Sometimes a special protein called termination
factor, rho is required for termination
• At termination, RNA dissociates from DNA and
enzyme (RNA polymerase) falls off too
III. Isolating Eukaryotic RNA Polymerases
Transcription in euk. much more complex
• Roeder and Futter (1974): Are there enzymes in the
eukaryotic nucleus that make RNA?
• From cultured frog cells, isolated nuclei.
• Separated proteins by Ion-Exchange
Chromatography:
Add nuclei, containing
proteins
Elute with Na+ gradient
Beads with negative
charge; some
proteins bind
strongly, most don’t.
Results of Experiment
[NaCl]
I
Total
Protein
RNA
Synthesis
Activity
II
III
0
10
20
30
40
50
Fraction #
How many euk. RNA polymerases are there?
Eukaryotic RNA Polymerases
• RNA Pol I
– makes three rRNAs (28S, 18S, 5.8S)
• RNA Pol II
- makes mRNA and snRNA (small nuclear RNA)
• RNA Pol III
– makes tRNA and 5S rRNA
• How does each pol know which DNA to bind?
Each recognizes different core promoter regions.
Anatomy of a Eukaryotic Gene
Pol II, Basal TFs bind
CAAT Box TATA Box
Cis-regulatory Elements may be located
thousands of bases away; Regulatory TFs bind.
TF
Binding of
Eukaryotic RNA
Pol II Requires
Binding of
Basal TFs to
Core Promoter
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