Lecture 21 & 22

Question 1

which RNA pol requires the most diverse control mechanisms to allow for the variety of gene expression observed in protein coding genes

Answer 1

RNA pol III

Question 2

How do transcription factors recruit RNA pol

Answer 2

DNA-Protein interactions

Question 3

What are the four experiments that can be used to investigate DNA binding domains

Answer 3

ChIP
DNAse I Footprinting
EMSA
X-ray crystallography

Question 4

What do sequence alignments do

Answer 4

If a number of genes are activated by a transcription factor, sequence alignments can show the diversity possible in the bound region

Question 5

What does a weight matrix do

Answer 5

A weight matrix shows the consensus sequence, and the variation observed in functional alternate binding sites

Question 6

What are basic amino acids

Answer 6

Basic amino acids are positively charge and will attach to negatively charge phosphate backbones

Question 7

how do proteins bind DNA

Answer 7

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

Question 8

what king of bonds can be between amino acids and base pairs

Answer 8

hydrogen bonds and weaker van der Waals interactions

Question 9

Can you determine how a DNA-binding protein and it’s targe sequence interact

Answer 9

we can determine at the atomic level how a DNA-binding protein and it’s target sequence interact

Question 10

T/F there is a limited set of core protein sequences found to bind DNA

Answer 10

true

Question 11

How many DNA-binding proteins do we have

Answer 11

~3000
4 super classes
each with 3 to 13 classes
each with 7 to 24 families
80% of DBD are of 3 major types

Question 12

What is a helix-turn-helix (structure, binding, dimer, example)

Answer 12

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

Question 13

describe the protein-dna interaction of tryptophan repressor

Answer 13

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

Question 14

What are eukaryotic homeodomains

Answer 14

Helix-turn helix

Question 15

What are homeobox genes

Answer 15

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

Question 16

major vs minor groove

Answer 16

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

Question 17

What are Zinc fingers (structure, subtypes, binding, example)

Answer 17

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)

Question 18

What is TFIII

Answer 18

transcription factor that recruits RNA pol III

Question 19

What is the leucine zipper (structure, dimer, N-terminus, C-terminus, what is the leucine for)

Answer 19

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

Question 20

What is a helix-loop-helix? (similarity to?, structure, dimer)

Answer 20

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

Question 21

Why are dimers important

Answer 21

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

Question 22

How does a sequence of multiple A’s affect a DNA molecule

Answer 22

bends the molecule

Question 23

Can protein bind by shape specificity

Answer 23

yes

Question 24

what are two examples of proteisn that bind by shape specificity

Answer 24

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)

Question 25

What are the four DNA binding proteins

Answer 25

HtH, Zn finger, Leu-zip, bHLH

one protein may have more than one DBD

Question 26

T/F the closer the promoter sequence to the consensus, the probability of binding the target protein and recruiting RNA pol II is greater

Answer 26

true