Download Regulation of gene expression: Prokaryotic

Announcements
1. Grading of Mendel papers: A= completeness of answer
(40); F = flow and organization (15); R = references (10);
S = spelling and grammar (10).
2. 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:
Review of Last Lecture
1. Eukaryotic DNA replication is complex
2. The “end” problem and telomerase: aging and cancer
3. The Genetic Code - theoretical evidence for triplet code;
genetic evidence using mutagens, ie. insertions and deletions
can cause frameshift mutations
Outline of Lecture 23
I.
The Genetic Code - biochemical evidence
II.
Transcription
I. Biochemical Evidence
• 1961: Nirenberg, Matthaei used synthetic mRNAs
and an in vitro translation system to decipher the
code.
• Polynucleotide Phosphorylase enzyme links NTPs to
make RNA without a template
• Homopolymers:
– poly(U) codes for Phe-Phe-Phe-Phe-…
– poly(A) codes for Lys-Lys-Lys-Lys-…
– poly(C) codes for Pro-Pro-Pro-Pro-...
Repeating Copolymers
• Khorana, early 1960’s
• UGUGUGUGUGUGUGUGU...
– Cys-Val-Cys-Val-Cys-Val-...
– Therefore GUG or UGU codes for either Cys or Val
• UUCUUCUUCUUCUUC…
– Phe-Phe-Phe-Phe-... or
– Ser-Ser-Ser-Ser-… or
– Leu-Leu-Leu-Leu-...
In Vitro Triplet Binding Assay
• Nirenberg and Leder (1964) mixed all 20
amino acids with ribosomes, different RNA
triplets:
– Ribosomes + UAU -> Tyr binds
– Ribosomes + AUA -> Ile binds
– Ribosomes + UUU -> Phe binds, etc.
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
Degeneracy and the Wobble Hypothesis
•
•
•
•
•
Codon in mRNA
Anticodon in tRNA
Codon: 5’-1-2-3-3’
Anticodon: 3’-3-2-1-5’
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
Making Sense of the Strands
• DNA coding strand = Sense Strand
• DNA template strand = Antisense Strand
• mRNA formed = Sense Strand
Coding strand
5’
mRNA 5’
3’
3’
3’
Template strand
5’
Prokaryotic Promoter Lies Just Upstream
(5’) of Transcribed Region; RNA
Polymerase Binds Two Places
-35 Region
-10 TATA Box
Initiation of Prokaryotic Transcription
Requires Binding of Sigma Factor to Pol
Note: No primer needed
5’ to 3’
Termination of Transcription in
Prokaryotes
• Occurs when hairpin
loops form from
intramolecular GC base
pairing in mRNA.
• Sometimes a special
protein called
termination factor, rho is
required for termination.
Isolating Eukaryotic RNA Polymerases
• Roeder and Futter (1974): Are there enzymes in the
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
RNA
Synthesis
+ 1 ug/ml 0
 amanitin
II
III
10
20
30
Fraction #
40
50
Eukaryotic RNA Polymerases
• RNA Pol I
– 1000 g/ml -amanitin inhibits
– makes three rRNAs (28S, 18S, 5.8S)
• RNA Pol II
– 1 g/ml -amanitin inhibits
– makes mRNA and snRNA (small nuclear RNA)
• RNA Pol III
– 50 g/ml -amanitin inhibits
– makes tRNA and 5S rRNA
• Each recognizes different core promoter regions.
Anatomy of a Eukaryotic Gene
(Protein Encoding)
Pol II, Basal TFs bind
CAAT Box
TATA Box
Cis-regulatory Elements may be located
thousands of bases away; Regulatory TFs bind.
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:
– 5’ 7-methylguanosine (7mG) cap added
– 3’ Poly-A tail added
– Splicing out of introns
TF
Binding of
Eukaryotic
RNA Pol II
Requires
Binding of
Basal TFs to
Core
Promoter
RNA Processing in
Eukaryotes
Pre-mRNA (primary transcript)
5’ cap
Poly A tail
Splicing
Mature mRNA
Introns and Exons