CSB349H1 Lecture Notes - Lecture 4: Deoxyribonuclease I, Plasmid, Chromatin
14 May 2018
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Lecture 4(a): Transcription II
Regulating Gene Expression – Role of Promoters:
• Major determinant in the transcription of any gene is the promoter
o Integrates all regulatory inputs to allow transcription to occur
• Eukaryotic promoter is located at the 5’ end of the transcribed sequence of a gene
o Associated with transcriptional initiation and nucleosome-free regions
Two major parts of a eukaryotic promoter:
1. Core Promoter:
o Surrounds the transcriptional start site (TSS)
o Consists of ~80bp of DNA sequence that allows for the recognition of basal
transcriptional machinery (i.e. mediator, RNAP…)
2. Proximal Promoter:
o ~300bp upstream of the core promoter that may contain additional regulatory
information of gene expression (i.e. when, where, how much)
Experimental Characterization of Functional Promoters:
• Functional promoter is defined as the minimum region required
for docking of the transcriptional machinery and initiation of
basal transcription
a) Take a fragment of DNA which corresponds to where we believe the
promotor is located
o Make a series of truncated pieces of the DNA fragment via
restriction enzymes
b) Small fragments can then be added into a plasmid that has a report
gene that allows us to see the expression of that gene
o Measuring reporter gene expression will allow us to identify
which region of the original fragment have the functional
elements of the promoter
ð Plasmid #3 and #4 can be concluded that they still have the core
promotor but not the proximal promotor which induces expression
_______
• Another method would be to generate mutants sequentially
along the promotor region
o Measure variable report gene expression allowing
us to identify which region of the promotor region
will have the functional elements of the
promotor
