CSB349H1 Lecture Notes - Lecture 4: Dna-Binding Domain, Leucine Zipper, Zinc Finger
14 May 2018
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Lecture 4(b): Transcription II
Sequence-Specific Transcription Factors:
• Proteins that recognize specific sequences within the gene (i.e. TATA box) and function to
activate or repress transcription by RNAPII
o Composed of three functional domains:
DNA Binding Domains:
• Zinc-containing motifs
• Homeodomain motif
• Basic leucine zipper and basic helix-loop-helix motifs
Transcriptional Activation Domain:
• Acidic type => regions with several acidic amino acids
• Glutamine-rich type => long stretches of glutamine
• Proline-rich type => long stretches of proline
o Some domains may form b-sheets – overall function is not clear
o Protein-protein interaction has been suggested to be mediated by these domains
§ Can either be functioning to activate or repress gene transcription (activation) via
interaction with RNAPII, or recruiting class I and class II enzymes
Protein Interaction Domain:
• Could be a dimerization domain – two transcription factors interacting in a pair
Methods for Determining Protein-DNA Interaction – In Vitro
a) Electrophoretic Mobility Shift Assay (EMSA): (gel shift assay)
o Run DNA fragment without an proteins on a gel:
§ DNA fragments are going to be found
together on the gel as one band
o Run DNA fragment with proteins on a gel
§ DNA fragments are going to shift on the gel
depending on the protein size:
• Big protein = less motility in the gel
b) Sequence Selection (SELEX):
o Technique that allows us to find the consensus sequence
by taking advantage of the EMSA
o Add gene regulatory protein of unknown DNA binding
sequence to a large random pool of DNA will be bound
and complete a gel shift assay
§ Fragments of DNA that are not bound to the protein
will be found at the bottom of the gel
§ Fragments of DNA that are bound to the protein will
have a gel shift
• Purify the gel and sequence the DNA fragment
Repeating this
experiment several
times will allow us
to identify the
most-specific
binding sequence
Document Summary
Lecture 4(b): transcription ii: proteins that recognize specific sequences within the gene (i. e. tata box) and function to activate or repress transcription by rnapii, composed of three functional domains: Dna binding domains: zinc-containing motifs, homeodomain motif, basic leucine zipper and basic helix-loop-helix motifs. Can either be functioning to activate or repress gene transcription (activation) via interaction with rnapii, or recruiting class i and class ii enzymes. Protein interaction domain: could be a dimerization domain two transcription factors interacting in a pair. Methods for determining protein-dna interaction in vitro: electrophoretic mobility shift assay (emsa): (gel shift assay, run dna fragment without an proteins on a gel: Dna fragments are going to be found together on the gel as one band: run dna fragment with proteins on a gel. Fragments of dna that are not bound to the protein will be found at the bottom of the gel.
