Download Recombinant DNA I

Announcements
1. Tuesday afternoon lab section: lab start time
next week is 3pm. 2-3 pm might be a good time to do
problem set 6!
2. No advance reading for next week’s lab; focus on your
lab report.
3. Problem set 5 due at start of class today.
4. Reading - Ch. 16: skip btm. 442- top 444.
Review of Last Lecture
1.The lac operon - in detail; know roles of all components
- lactose
- repressor protein from I gene
- Operator sequence
- Promoter sequence
- Regulation of expression of 3 structural genes:
lacY, lacZ, lacA
- CAP + cAMP
- Glucose
**Clarification: maximal transcription= repressor bound by
lactose and CAP bound to CAP-binding site
2. The trp operon - very briefly
Learning Check
Will B-galactosidase be made: a-always, b-never,
c-sometimes (in presence of lactose)
1. I+O+Z+
2. I+OCZ+
3. ISO+Z+
Outline of Lecture 27
I. Eukaryotic Gene Expression: it’s more complicated being
multicellular
II. The Promoter
III. Enhancers
IV. Methylation
V. Alternative splicing
I. Problems of Multicellularity
• All of our genes are present in every cell, but only
certain proteins are needed.
Pancreatic cell
+ insulin
- neurotransmitter
Neuron
- insulin
+neurotransmitter
• Expression of a gene at the wrong time, in the wrong
type of cell, or in abnormal amounts can lead to
deleterious phenotypes or death - even when the
gene itself is normal.
Levels of Regulation
of Eukaryotic Gene Expression
• “What is true of E. coli is only partly true of elephants.”
• Prokaryotic control primarily at the transcription level.
• Eukaryotic control at several levels
Levels of
gene
regulation
***focus today
The interphase nucleus
Chromosome structure is continuously rearranged, so that
transcriptionally active genes are cycled to edge of
territories.
Organization/
packaging of DNA
Nucleus= 5-10 m
(0.01mm)
Diploid genome=
6.4x109 bp
0.34nm/bp
DNA=2 meters
(2000 mm)
Chromatin remodeling
II. Promoters: Eukaryotic vs. Prokaryotic
RNA pol II
RNA pol
Promoters: sequences adjacent to genes, where
RNA pol binds to initiate transcription
Euk. - Chromatin and TFs
Prok. - Naked DNA and no TFs needed
“Promoter-Bashing” Mutations
Determine the Critical Regions of DNA
for Gene Expression
Transcription
factors
TBP-TATA binding protein
TAFs- TATA assoc. factors
III. Eukaryotic Enhancers and Promoters
Promoters- needed for basal level transcription
Enhancers- needed for full level transcription; location and orientation variable
Two types of transcription factors bind enhancers and affect levels of txn: true
activators and anti-repressors
Combinatorial Model of Gene Expression
Liver
Brain
Regulatory
TFs increase
transcription
activity
No reg.TFs
in this cell
for albumin
enhancer
Binding of True Activator TFs to Enhancers
Greatly Stimulates Transcription
Looping of DNA allows Activator TF bound to Enhancer to interact
with Promoter, facilitating binding of Basal TF complex.
Types of Regulatory Transcription Factors
• True activators are modular proteins: one domain
binds DNA in enhancer; one domain interacts with
protein at promoter
• Classified by DNA-binding motifs in the protein:
–
–
–
–
Helix-Turn-Helix
Zinc Finger
Leucine Zipper
Helix-Loop-Helix
Helix-Turn-Helix (HTH) TFs
• Two -helical stretches of
AA’s linked by non-helical
sequence of AA’s
• e.g. lac repressor protein
(binds operator DNA)
• also eukaryotic
homeodomain TFs,
important in embryonic
development
Zinc Finger TFs
• Zn2+ is coordinated between His and Cys residues in protein, forming
“finger” of protein
• 2-13 fingers may be present
• Found in pseudooncogenes and in Drosophila Kruppel TF in
embryonic development
Leucine Zipper TFs
• Dimeric, held together
by interactions between
leucine residues
• includes AP-1 TF, a
heterodimer of Jun and
Fos proteins, important
in control of cell
division; mutation can
cause cancer.
Helix-Loop-Helix (HLH) TFs
• Dimeric: two subunits of
two helices linked by loops
• includes mammalian TFs
for muscle differentiation MyoD, myogenin and Myf5
Antirepressor Transcription Factors
Access
Slow txn
TFs can recruit
HATs or HDs
IV. Control of gene expression
by DNA Methylation
• Addition of CH3 to selected C’s in DNA can inactivate
genes, e.g. high levels are seen in inactivated X
chromosome of female mammals.
• Mammals have about 5% methylation.
• Not essential in eukarotyes, since Drosophila has 0%
methylation.
• First observed in lac operon: methylation of operator
DNA sequence affects binding by repressor
V. Post transcriptional gene regulation
If humans have approximately the same number of
genes as a fruit fly, and we require more complex
cellular functions (presumably with a larger number
of proteins) - how do we accomplish this?
Alternative splicing
1. Chromosomal ratio
activates txn of Sxl in
females only
2. SXL controls splicing of
tra-2 mRNA
3. Females: exon 2 (which
has a stop codon) is
removed via SXL
Males: exon 2 is not
removed.
4. Males: no active TRA
Females: TRA is made.
5. TRA directs splicing of dsx
mRNA in specific
manner; in males default
splicing occurs.