control of gene expression

Question 1

describe how DNA binding proteins (TFs) interact with DNA

Answer 1

DNA binding proteins = positively charged and reach into major groove
recognise binding sites through interactions with individual base pairs (e.g. H bonds)

Question 2

what is the core element of the Rox1 binding site?

Answer 2

TTGTT

Question 3

how to identify more genes regulated by a transcription factor?

Answer 3

find consensus sequence by aligning every known response element and finding the frequency of bases at each position + putting the most common nucleotide at each position

Question 4

how would you identify regulatory sequences of a gene?

Answer 4

align DNA sequences of a gene from closely related species + identify conserved sequence motifs
regulatory sequences = conserved

Question 5

helix turn helix DNA binding motif

Answer 5

2 alpha helices
bind to consecutive major grooves
DNA binding site = palindromic
recognition sequence inserts into major groove to make specific contacts

Question 6

zinc finger motif

Answer 6

4 cysteine or histidine AAs holding zinc atom in place
alpha helix interacts with major groove
1 protein contains multiple zinc finger motifs in a row

Question 7

leucine zipper motif

Answer 7

2 alpha helices held together as dimers by hydrophobic amino acids
interact with 2 parts of major groove
homodimers bind symmetrical sequences
heterodimers bind non-identical sequences

Question 8

basic helix loop helix motif

Answer 8

2 alpha helices connected by a loop
form homodimers or heterodimers
positively charged residues interact with charged groups on DNA

Question 9

activation domain

Answer 9

enables DNA binding protein to interact with transcription initiation complexes containing RNA polymerase

Question 10

EMSA = electrophoretic mobility shift assay

Answer 10

1. radioactively label DNA with regulatory sequence using 32P = DNA probe
2. mix with cell extract
3. run samples by gel electrophoresis
4. DNA probe with DNA binding protein bound migrates slower
5. purify protein to identify biochemically

Question 11

DNAse footprinting

Answer 11

1. radioactively label DNA with 32P = probe
2. mix with cell extract
3. add DNAse - partially digests DNA
4. heat sample to destroy DNAse + release binding proteins
5. run samples by gel electrophoresis
6. DNA binding protein bound to DNA probe protects from cleavage - can identify where a protein binds on a DNA sequence

Question 12

which technique would be used to identify DNA binding proteins?

Answer 12

EMSA

Question 13

which technique would be used to identify the sequence on which a DNA binding protein binds to?

Answer 13

DNAse footprinting

Question 14

what blocks regulatory sequences from inappropriately affecting neighbouring genes?

Answer 14

• barriers = prevents spread of heterochromatin into genes that need to be expressed
• insulators = prevents cis regulatory sequences from activating inappropriate genes

Question 15

tryptophan repressor protein

Answer 15

tryptophan binds to tryptophan repressor protein

represses genes required for tryptophan synthesis