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Lecture 8
Transcription Initiation
Prokaryotic
Eukaryotic
Reading:
Chapter 4 (115-116)
Chapter 11
Molecular Biology syllabus web site
Bacterial transcription initiation
• RNA polymerase initiates transcription of most genes at a
unique DNA position lying upstream of the coding sequence
• The base pair where transcription initiates is termed the
transcription-initiation site or start site
• By convention, the transcription-initiation site in the DNA
sequence is designated +1, and base pairs extending in the
direction of transcription (downstream) are assigned positive
numbers which those extending in the opposite direction
(upstream) are assigned negative numbers
• Various proteins (RNA polymerase, activators, repressors)
interact with DNA at or near the promoter to regulate
transcription initiation
Differences in E. coli promoter sequences
affect the frequency of transcription initiation
Lac Operon
• Beta Galactosidase (lacZ)
• Permease (lacY)
• Transacetylase (lacA)
Lac promoter
mRNA
protein
lacZ
lacY
lacA
LacZ (b-galactosidase)
Eukaryotes
Promoter deletions to identify transcriptional control sequences
Promoter deletions to identify transcriptional control sequences
Run-off transcription: to map transcription initiation sites
METHOD
1. Isolate nuclei from specific tissues
2. Incubate: nuclear extract, restriction fragments containing start site, 32P-UTP
3. Synthesized RNA is separated by electrophoresis
Common Eukaryotic Promoter Consensus: TATA box
Alternate promoters:
initiators- degenerate consensus
GC-rich region (giving rise to multiple start sites)
Run-on transcription
assay rate of
transcription initiation
METHOD
1. Isolate nuclei from
specific tissues
2. Label growing RNA
chains with 32P-UTP
3. Hybridize to filters
containing test cDNAs
S1 protection to assay transcription initiation rates
32P-labelled DNA probe
mRNA
S1 nuclease
32P-labelled DNA probe
mRNA
denature
gel electrophoresis
Enhancer elements
cis-acting sequences that serve to
increase rate of transcription initiation
regardless of their location near the
gene (first discovered in SV40 viral
genome).
S1 Protection Assay: to test rate
of transcription initiation
METHOD
1. Transfect construct
with/without enhancer into test
cells
2. Isolate RNA and hybridize with
32P-labeled DNA probe
3. Treat with S1 nuclease,
denature, subject to gel
electrophoresis
Typical eukaryotic gene
Gel-shift assays identify general region of
protein-DNA interaction
METHOD
1. Isolate nuclei and
prepare protein extract or
purify DNA binding
proteins
2. Incubate with restriction
fragment or short DNA
3. Separate by agarose
gel electrophoresis
Controls: non-specific
DNA; no extract
Column purified fractions of DNA-binding
proteins incubated with test promoter fragment
DNase I footprinting assays identify specific
regions of protein-DNA interactions
METHOD
1. Isolate restriction
fragment
2. Add DNA-binding
protein
3. Partially digest
with DNaseI
4. Separate by
polyacrylamide gel
electrophoresis
Controls: no protein
The footprint of RNA polymerase and lac
repressor on the lac control region
The lac control region contains three
critical cis-acting sites
Figure 10-9
How to demonstrate the specificity
of a putative transcription factor?
•In vitro
•In vivo
•In vitro
METHOD
1. Set up in vitro
transcription reaction with
DNA template, 32P-UTP,
plus or minus transcription
factor
2. Separate transcripts by
gel electrophoresis
Controls: non-specific
DNA; no protein
•In vivo
METHOD
1. Prepare two constructs:
a) encoding transcription
factor X
b) reporter gene
downstream of
transcription factor binding
site plus minimal promoter
2. Transfect cells and assay
for reporter gene activity
Controls: transfect each
plasmid alone
Transcription factors have
separate domains
•DNA binding
•Transcription
activation
Yeast Two-hybrid System- to fish out
interacting transcription factors