Regulatory sequences in protein-coding genes

Question 1

describe linker scanning mutagenesis and reporter genes

Answer 1

you buy a plasmid that encodes a reporter gene, usually a gene that encodes for an enzyme

open reading frame on the plasmid has NO PROMOTER

systematically replace small pieces (10 bp) of the cloned regulatory DNA by a LINKER

produce multiple plasmids with linker substitutions

transform each individual plasmid in a cell culture and measure the activity of the reporter

loss of reporter activity = regulatory sequence

Question 2

what have regulatory experiments told us?

Answer 2

generated detailed maps of REGULATORY DNA SEQUENCES

mamalian promoters can have many regulatory elements: promoter-proximal or distal
can be positioned upstream or downstream from initiation site
CpG islands do not have strong regulatory elements and are expressed at a lower level
simple eukaryotes have simple regulatory elements

Question 3

what are enhancers

Answer 3

distal regulator elements, could be upstream or downstream of initiation site
could be included in introns
can function over a long distance
position independent
orientation independent

Question 4

what are promoters?

Answer 4

function within a short distance
immediately upstream or downstream from initiation site
position dependent

Question 5

how do enhancers communicate with promoters?

Answer 5

DNA bending

Question 6

what is a domain?

Answer 6

part of a protein that will acquire a distinct 3D structure and distinct activity within the whole proteins

transcriptional activators or repressors often have independent DNA-binding domains and effector domains

identified by deletion analysis

Question 7

what are the domains?

Answer 7

TRANSACTIVATION, DIMERIZATION, DNA BINDING, HORMONE BINDING

Question 8

what are the motifs?

Answer 8

100 DNA binding domains = ZN-FINGER

100 dimerization = LEU ZIPPER

Question 9

describe combinatorial binding to DNA of different dimer combinations of proteins

Answer 9

DNA binding proteins of the same class work as dimers
each monomer can recognize a specific DNA element, but cannot work on its own
if two proteins can combine, they will have three binding sites, three will have 6, etc

Question 10

what does adding an inhibitor do??

Answer 10

increases the possible outcomes for activation –> fine tuning

Question 11

what are enhanceosomes?

Answer 11

transcriptional activators or repressors often interact with each other and cooperate to establish a strong activator or repressor site to tightly control the expression of the target genes