Week 3

Question 1

1. Major determinant in the transcription of any gene is the
2. The eukaryotic promoter is Located at
3. The eukaryotic promoter is Associated with
4. orientation of core promoter
5. The core promoter (2 points)
6. ‘proximal promoter (2 points)

Answer 1

1. Major determinant in the transcription of any gene is the promoter
2. . The eukaryotic promoter is Located at the 5’ end of the transcribed sequence
3. Associated with transcriptional initiation and nucleosome-free regions
4. Sequences in the core promoter usually work in only one orientation
5. The ‘core’ promoter surrounds the transcription start site. Comprises ~80bp of DNA sequence that contains canonical features that recruit basal transcriptional machinery
6. The ‘proximal promoter includes DNA sequence ~300 bp upstream of core promoter (but this can be variable). Provides additional information (where, when, and how much)

Question 2

• Internal mutations (e.g. linker scanning mutagenesis) help
• Base pair substitution analyses help

Answer 2

• Internal mutations (e.g. linker scanning mutagenesis) help define the precise elements
  within the promoter that are required for efficient transcription
• Base pair substitution analyses help define the precise sequence
  within the promoter that is mostly commonly used for efficient transcription

Question 3

promoter regultory sequences

• Short regulatory DNA elements are
• there is no such thing as a
• • Promoter elements are usually described as x since
• what help define the precise
  sequence of each functional element
• The more sequences that can be sampled,

Answer 3

• Short regulatory DNA elements are highly conserved across eukaryotic species, but
  there is no such thing as a universal core promoter – genes contain many
  combinations of promoter elements within their core promoters
• Promoter elements are usually described as “consensus” sequences, since it is
  possible to have slight variation at specific nucleotide positions, but still have similar
  function
• sequence alignments of many promoter regions can also help define the precise
  sequence of each functional element
• The more sequences that can be sampled, the better the most commonly
  occurring sequences can be assigned
• Much like with any type of voting, the ‘consensus’ sequence can be obtained
  that fits the majority of the sequences being studied

Question 4

Promoter sequences - 3 common examples

Answer 4

TATA box, Initiator (Inr) element, downstream promoter element

Question 5

TATA box

Answer 5

• first promoter element identified
• consensus sequence is 5’-TATA(A/T)A(A/T)(A/G)-3’
• usually located 25-30 bp upstream of TSS
• recognized by the TATA-binding protein subunit of TFIID

Question 6

Initiator (Inr) element

Answer 6

• consensus sequence 5’-PyPyAN(T/A)PyPy-3’ (Py = pyrimidine; N = any nucleotide)
• usually surrounds TSS with 10 bp
• can be identified in promoters that either contain a TATA box or are TATA-less
• typically recognized by the TAF1 and TAF2 subunits of TFIID
• frequently identified (>50%) in promoters of all animals

Question 7

• Downstream promoter element (DPE)

Answer 7

• consensus sequence G(A/T)CG
• usually identified in TATA-less promoters (common to find DPE + Inr)
• is positioned downstream of the TSS (within 50 bp)
• recognized by several subunits of TFIID, but not TBP
• also frequently identified in animal promoters (40-50%)

Question 8

Sequence-specific transcription factors

1. Transcription factors recognize
2. Composed of the following functional domains
3. Classes of DNA binding domains
4. • Transcription activation domain

Answer 8

• Transcription factors recognize particular sequences in DNA and activate or repress
  transcription by RNAPII
• Composed of the following functional domains (independently folded region of protein):
  -> DNA binding domain
  -> transcription activation domain
  -> other protein interaction domain (e.g. dimerization domain)
• Classes of DNA binding domains
  -> zinc-containing motifs (zinc fingers, zinc module in hormone receptors, complex zinc module in Gal4 and related proteins)
  -> homeodomain motif
  -> basic leucine zipper and basic helix-loop-helix motifs
  -> many others
• Transcription activation domain
  -> acidic type: contains a region with several acidic amino acids
  -> glutamine-rich type: long stretches of glutamines
  -> proline-rich type: long stretches of prolines
  ->protein-protein interaction has also been suggested to be mediated by these
  domains

Question 9

1. Transcription factors need to do two things:
2. • Generally they have two domains to
     achieve this role:

Answer 9

Transcription factors need to do two things:
1) Find DNA
2) Help recruit or repress RNA polymerase
II to sites of transcription
* Generally they have two domains to
achieve this role:
1) DNA binding domain that recognizes
specific nucleotide sequences
2) activation or repression domain that
somehow influences transcription

Question 10

C2H2 Zinc finger

Answer 10

• Anti-parallel b-strand connected to an
  a-helix by a short loop
• Two cysteines in the b-strand and two
  histidines in the a-helix coordinate the
  zinc ion in between them
• C2H2 Zinc finger is the most common
  domain in the human genome. Occurs more than 8000 times, in nearly 1000 proteins
• Proteins with multiple zinc fingers form a
  curved shape that matches the major
  groove of the DNA double helix
• The a-helix amino acids interact with the
  bases of DNA in the major groove
• The b-strand binds to DNA backbone
  and positions the recognition helix for
  optimal interaction

Question 11

Homeodomain
- What Family
- how many a-helicies
- what do the second and third helix do
- what does the n arm do
- - what is homodomain dependant on
- where does it occur

Answer 11

• Members of the helix-turn-helix family of
  DNA binding proteins
• Each homeodomain (~60 aa) contains 3
  a-helices; the second and third form the
  helix-turn-helix motif (third a-helix is the
  recognition helix that binds to major
  groove)
• N-term arm inserts into minor groove
• Dependent on other proteins to enhance
  binding and increase specificity
• Occurs in 300 proteins in the human
  genome

Question 12

Basic Helix-loop-helix

• what is it
• bHLH containing proteins also have a region that is responsible for
• The two basic domains of the dimer
  interact
• Occurs in

Answer 12

• The HLH is the specific dimerization
  region of the domain
• bHLH containing proteins also have a
  basic (b) region of the motif that is
  responsible for the majority of DNA
  binding and is part of the first helix
• The two basic domains of the dimer
  interact in the major groove of the DNA
  duplex
• Occurs in more than 150 proteins in the
  human genome

Question 13

Leucine or basic zipper

• bZIP containing proteins have a region responsible for
• what is the leucine zipper
• what is formed by
• how do the two basic domains interact
• occurs how many times in human genome

Answer 13

• bZIP containing proteins have a basic (b)
  region of the motif that is responsible
  for the majority of DNA binding
• The leucine zipper (ZIP) is a specific
  dimerization region of the domain
• Formed by two polypeptides, each one is
  an a-helix with leucine (and other
  hydrophobic amino acid residues)
  spaced 7 residues apart so that they all
  face one side of the helix and the two
  monomers interact
• The two basic domains of the dimer
  interact in the major groove of the DNA
  duplex
• Occurs 41 times in the human genome

Question 14

Dimerization and specificity of transcription factors

• transcription factors have to recognize
• many sequence specific transcription factors bind as. examples of this. * This is a way of

Answer 14

• transcription factors have to
  recognize sequences in large genomes – they need specificity
• many sequence specific transcription factors bind as dimers, each monomer typically has the same type of DNA binding domain, but each DNA binding domain could recognize slightly different sequences
• already seen this for the leucine zipper (bZIP) and bHLH proteins
• This is a way of combining more types of DNA binding motifs, without having to evolve new proteins

Question 15

Nuclear hormone receptors

• Bind to
• typicallu contain
• ligand binding causes
• example

Is a transcription factor

Answer 15

*Bind to many famous molecules, such as vitamin A, Vitamin D, thyroid hormone, estrogen and testosterone
*Typically contain a DNAbinding domain and a ligand binding domain
*Ligand binding causes a conformational change that either stabilizes or destabilizes dimerization
- Estrogen receptor