Lecture 21 & 22 Flashcards
which RNA pol requires the most diverse control mechanisms to allow for the variety of gene expression observed in protein coding genes
RNA pol III
How do transcription factors recruit RNA pol
DNA-Protein interactions
What are the four experiments that can be used to investigate DNA binding domains
ChIP
DNAse I Footprinting
EMSA
X-ray crystallography
What do sequence alignments do
If a number of genes are activated by a transcription factor, sequence alignments can show the diversity possible in the bound region
What does a weight matrix do
A weight matrix shows the consensus sequence, and the variation observed in functional alternate binding sites
What are basic amino acids
Basic amino acids are positively charge and will attach to negatively charge phosphate backbones
how do proteins bind DNA
Basic amino acids are positively charged and will attach to the negatively charged phosphate backbone
Some amino acid side-chains will fit very well into a major/minor groove and make specific recognition pairings with the side of a base-pair
some flexibility in the protein and in DNA will allow bending/folding to make more stable contacts
what king of bonds can be between amino acids and base pairs
hydrogen bonds and weaker van der Waals interactions
Can you determine how a DNA-binding protein and it’s targe sequence interact
we can determine at the atomic level how a DNA-binding protein and it’s target sequence interact
T/F there is a limited set of core protein sequences found to bind DNA
true
How many DNA-binding proteins do we have
~3000
4 super classes
each with 3 to 13 classes
each with 7 to 24 families
80% of DBD are of 3 major types
What is a helix-turn-helix (structure, binding, dimer, example)
An alpha helix, flexible linker (turn) and another alpha helix (about 20 amino acids in total)
the first helix fits into the major groove with sequence specificity
the remaining protein outside this domain will add to the specificity and stability of the interaction
May function as monomer or dimer
example: tryptophan repressor
describe the protein-dna interaction of tryptophan repressor
Homodimer binds trp-control region through dual helix turn helix domains
Binds and turns off the tryptophan operon in E. coli when bound to trp
What are eukaryotic homeodomains
Helix-turn helix
What are homeobox genes
homeobox genes are master control transcription factors for anterior–> posterior pattern regulation in animals
determine the identity of a bodily unit
Hox proteins contain homeodomains which are HtH
major vs minor groove
Major groove “information content” allows for
full sequence discrimination (all 4 base pairs)
Minor groove “information content” contains less detail, so A-T vs C-G
What are Zinc fingers (structure, subtypes, binding, example)
type of dna binding domain
20-30 amino acids
Defined positions of particular amino acids will hold a zinc, 3bp per finger
Alpha helix- beta strand structure can lie across the major groove to bind DNA
common to find several zinc fingers in succession in one protein (extends affinity/stability)
Multiple subtypes the most common is C2H2
Specific binding along major groove, specificity can’t be determined yet
TFIIIA of Xenopus has 9 zinc fingers (1,2,3 and 7,8,9 wrap around major groove)
What is TFIII
transcription factor that recruits RNA pol III
What is the leucine zipper (structure, dimer, N-terminus, C-terminus, what is the leucine for)
DNA binding domain
Doesn’t actually zipper, more like magnets
Dimer of elongated alpha-helices
Homodimer or heterodimer
N-terminus is enriched for basic amino acids and will contact the DNA
C-terminus contains repeating leucines every 7th amino acid
slightly overwound
Pairs of hydrophobic leucines pair up to stabilize the coiled-coil of two alpha-helices wrapped together
recognition site is ~4bp per contact
What is a helix-loop-helix? (similarity to?, structure, dimer)
DNA binding functionally similar to leucine zippers as a pair of proteins have specificity through two alpha helix
the second helix is amphipathic with dimerizing through the hydrophobic faces of the helix
Basic aa’s assist binding DNA
may act as homodimer or heterodimer
Why are dimers important
allow for combinatorial gene expression
Binding locations at the DNA can have different combinations of monomeric binding sequences
from 3 proteins that can form homodimers or heterodimers we can get six novel binding sites
How does a sequence of multiple A’s affect a DNA molecule
bends the molecule
Can protein bind by shape specificity
yes
what are two examples of proteisn that bind by shape specificity
It is well established that a run of 4-6 A’s will generate a bend in the DNA molecule.
The insulator protein su(Hw) binds the specific
sequence in the central box, but the stability of this
interaction depends on the presence of the polyA:T
runs to either side
Example 2: BRCA1
Tumor suppressor gene
important for mismatch and DSB repair
structural specificty is was tested using EMSA
Affinity of shape binding
Cruciform>G:C>Bulge>G:T (linear)
What are the four DNA binding proteins
HtH, Zn finger, Leu-zip, bHLH
one protein may have more than one DBD
T/F the closer the promoter sequence to the consensus, the probability of binding the target protein and recruiting RNA pol II is greater
true
