L21 Gene regulation by transcription initiation

Question 1

DNA binding proteins

Answer 1

• trans-acting regulatory genes
• +ve acting
• -ve acting
• cis-acting DNA binding sites e.g. enhancers, UAS, URS (upstream repression sites), silencers

Question 2

Regulation by transcription initiation mechanism

Answer 2

see onenote

DNA binding proteins may bind some distance away from transcription start site

In larger eukaryotes, regulatory sequences does not need to be close to their target gene. Can be genes between enhancer and its target gene. Enhancers can also be at the 3’ end of the gene, operate well at both 5’ and 3’ end of gene.
Regulatory genes in yeast must be upstream of the gene in order to work.

Question 3

Tissue/cell specific expression

Co-activators

Answer 3

see onenote

Question 4

Insulators

Answer 4

see onenote diagram

• restrict range of enhancers
• proteins bound to insulator elements block action of activators bound to enhancers

Question 5

Enhancers can act in trans

Answer 5

see onenote

Odorant receptors
Genes on different chromosomes

Question 6

Determining DNA binding sites for regulatory proteins - in vitro

Unknown DNA binding site or promoter

Answer 6

see onenote

Random oligonucleotide probe

Question 7

Determining DNA binding sites for regulatory proteins - in vitro

known DNA binding site or promoter

Answer 7

see onenote

Question 8

Determining DNA sequence bound by proteins - Chromatin immunoprecipitation (ChIP)

Answer 8

see onenote slides

• in vivo
• single gene or whole genome
• antibody targets can be RNA pol, regulatory protein, GTFs, mediator complexes etc.

Question 9

Transcription factors

Answer 9

• DNA binding domain
• dimerisation domain
• nuclear localisation domain
• interaction domain - modulate action or interaction domain e.g. ligand, coactivator/corepressor, histone modifying enzymes

Question 10

DNA binding motifs

Answer 10

see onenote

Bind sequence specific sequences

Structural motifs that make specific molecular interactions in DNA

Question 11

Zinc finger

Answer 11

see onenote slides

• contacts between aa and nucleotides

Question 12

Leucine zipper

Answer 12

see onenote

dimerisation

Question 13

DNA binding motifs - combinatorial control

Answer 13

see onenote

two DNA binding proteins, three possible sites

Question 14

Regulating activity of regulatory proteins

Answer 14

see onenote

Question 15

A.nidulans nitrogen metabolite expression

Answer 15

see onenote

areA gene produces a +ve acting transcriptional regulator

AreA activates genes in the absence of NH4+

AreA differentially nuclear localised

Question 16

Genetic analysis of gene regulation

Answer 16

see onenote

• Isolate mutants affected in particular phenotype
• distinguish between structural and regulatory mutants

structural mutant => loss of one gene product, recessive

regulatory mutant => loss of structural gene expression, misregulated expression, recessive/dominant, cis/trans

Question 17

Galactose utilisation in yeast

Answer 17

see onenote

• Gal non-utilising mutants, gal- mutants

Structural genes

• GAL1,2,7,10
• MEL1