Download Bio 402/502 Section II, Lecture 1

Bio 402/502
Section II, Lecture 2
Nuclear Processes: DNA
replication and transcription
Dr. Michael C. Yu
General concepts of DNA replication
• Purpose of DNA replication?
• Duplicate a cell’s genetic material - with accuracy
- why?
• How does a cell accomplish this?
• Via a complex set of cellular machineries - how
do you identify these proteins?
• Rate: as high as 1KB per second - difference
eukaryotic cells have different genome size
• Biological significance of DNA replication?
Cells die if they can’t replicate their DNA properly
Prokaryotic vs. eukaryotic DNA replication
Prokaryotes:
• Single origin of DNA replication (circular chromosome)
Eukaryotes:
• Multiple origins of DNA replication
• Occurs during S phase of the
cell cycle
DNA replication is “semi-conservative”
DNA replication:
1) Separation of the two
strands
2) Complete replication using
each strand as a template
for the synthesis of a new
“daughter” strand
(Figure obtained at www.sparknotes.com)
DNA replication is also bi-directional
• Replication starts at “origins of replication” or
“replication fork”
• Can have multiple origins within a chromosome - efficient
Identify origins of replication: using yeast as a model organism
(Figure obtained from Alberts, 4th ed.)
DNA polymerase: the enzyme that makes DNA
Prokaryotes (E. coli):
• Five DNA polymerases: I, II,
III, IV, and V
Eukaryotes:
• Many more DNA polymerases
• Can be broadly categorized into
replication or repair
Substrate: deoxyribonuceloside triphosphate
Common structure of DNA polymerase
Enzyme has independent domains
Responsible
for positioning
template
correctly at
the active site
Binds DNA as it
exits the enzyme
Conserved sequence motif for catalytic active site
Understanding the function of domains within DNA polymerase
In vivo mutagenesis?
• Create mutations within the
domain and test its function in
vivo
In vitro assays?
• Biochemical reconstitution of
replication assay (bacteria)
• Not really able to do so with
eukaryotes - too complex.
Process of DNA replicatiton
1. Helicase
separates both
strands of the DNA
2. Single-stranded
proteins bind and
maintain separated
strands
5. Ligation of
Okazaki fragments
by ligase (lagging
strand only)
4. Synthesis of DNA
by DNA polymerase
3. Prime with 3’-OH
end (difference
between leading &
lagging strand)
(Figure obtained at Ohio State Biosci website)
A different view of DNA replication process
Things to also consider: chromatin access by trans-factors
How do you identify “essential” genes involved in DNA replication?
The use of conditional mutants - (e.g. temperature sensitive)
Loss of CFU at non-permissive
temperature
37°C
In vitro DNA
synthesis
Mutagenized cells
grown on petri dish
30°C
Good model organisms: yeast and bacteria
Transcription by RNA polymerase II: making of messenger RNAs
3’
5’
5’
3’
Only one of the two strands of DNA is transcribed into mRNA
Transcription in Prokaryotes
• Lacks nucleus: couples
transcription with translation
Transcription in the eukaryotes
• The predominant form of eukaryotic gene expression
• Three RNA polymerases in eukaryotes:
RNA Pol I: 5.8S, 18S, and 28S rRNA genes
RNA Pol II: all protein-encoding genes, snoRNA genes, some snRNA genes
RNA Pol III: tRNA genes, 5S rRNA genes, some snRNA and small RNA genes
• Major players involved in regulating transcription are
transcription factors
Several types - basal, activators, co-activators, etc.
Cell/tissue specific - achieve cell/tissue specificity
Basic Concept of Eukaryotic Gene Expression
Activation of gene structure (i.e. chromatin)
Transcription initiation
Majority of the
eukaryotic gene
expression controls
Transcription elongation
Process of transcripts
Export of mRNAs to the cytoplasm
Translation of mRNAs to proteins
Basic Concept of Eukaryotic Gene Expression
What is the role of chromatin
during transcription?
• Euchromatin vs. heterochromatin
First step - open up chromatin
structure
General structure of chromatin resulting in its compactness
Subunit of all chromatin: nucelosome
A nucleosome is composed of DNA &
histones
A nucleosome wraps approx. 200bp DNA
How do you know if a segement of
DNA is protected/wrapped within a
nucelosome? Difference in their
susceptibility to MN
(Lewin, Genes IX)
Histone N-terminal tails are post-translationally modified
(Lewin, Genes IX)
Two copies of each core
histones per nucleosome
N-terminal tails of histones are
positioned outside of a nucleosome
Model of chromatin regulation during transcription
Histone PTMs: a
major control in
chromatin structure
Modification status
indicative of
transcriptional state
(Li et al, Cell, 2007)
Which histone modifications correlates with gene activation or silencing?
Initiating transcription at the promoter region is a multi-step process
Normally, chromatin is silent
Histone modifications and their
implications on gene expression:
Acetylation- transcriptionally active
Methylation (K,R)- transcriptionally silent
Phosphorylation (S) - activation
Ubiquintination - signals methylation
Sumolyation - transcriptionally silent
(Lewin, Genes IX)
Events leading to transcription initiation at the promoter
1. Recruitment of activator (trans) to
the cis-element
2. Activators recruit chromatin
remodelers
3. Modification of chromatin,
reorganization of nucleosome, release
of chromatin compactness
(Lewin, Genes IX)
Recognition of core promoter elements by transcription factors
Selectivity of promoters
determines which transcription
factors are recruited to activate
gene transcription.
How would you determine the
consensus promoter sequences
of a gene?
(Thomas & Chiang, 2007)
Identify cis-acting elements using mutagenesis approach
• Approaches: linker scan, alanine substitution, deletion, etc.
a) Linker scan
approach
b) Assays to
determine the
effect of mutation
(Hou et al, 2002)
Recruitment of RNA polymerase II: start of transcription initiation
Transcription factors
specifies the location of
transcription and recruits
RNA pol II.
How would you determine which factors
are responsible for transcription in a cell?
(Lodish et al, 2000)
Protein-protein interactions between transcription factors
Principles of a two-hybrid assay:
• Use a known transcription factor as a “bait”
Pros & cons:
• Let cells do the work
• More in vivo context
• False positives
• Organism specific (lacking PTM)
Identifying protein-protein interactions between transcription factors
• Detect biochemical association between proteins using Co-IP
Use mass spec to
identify co-IP’ed
proteins
• Can also use epitope-tagged proteins such as Myc, HA,
FLAG, etc
Modes of transcription factor activation
How would you determine
binding of a TF to a promoter?
(Lewin, Genes IX)
Detecting DNA-protein interactions in vitro
• Can use EMSA (electrophoresis mobility shift assay) or gelshift assay:
or PAGE
(Sigma website)
Radio-labeled DNA probes
( i.e. specific promoter elements)
An example of EMSA
• Use of EMSA involved biochemically purified components/extracts
• How would you use this method to determine which mode of
transcriptional activation
Detecting DNA-protein interactions in vivo
1. Crosslink Protein-DNA complexes in situ
2. Isolate nuclei
and fragment DNA (sonication or digestion)
3. Immunoprecipitate with antibody 4. Identify
against target nuclear protein
and reverse crosslinks
DNA sequence
by quantitative or real-time
PCR
5. Detection of PCR
products by PAGE or
real-time machine