Protein-DNA interactions

what are the two faces of DNA

major and minor groove

what are the places which proteins can make contacts with DNA

• phosphate backbone

• DNA base pairs

what are the two types of protein-DNA interactions

specific and non-specific

what is a specific DNA-protein interaction

protein interacts with DNA bases

what does a specific protein-DNA interaction give you information on

the DNA sequence

what is a non-specific protein-DNA interaction

interactions between protein and the phosphate backbone

does a non-specific protein-DNA interaction reveal information on the sequence

no

what is a bridged/mediated protein-DNA interaction

interaction between protein and DNA that occurs through connection with a water molecule or a metal ion

what is an indirect protein-DNA interaction

somewhere in between a specific and nonspecific protein-DNA interactionw

what does an indirect protein-DNA interaction gain information from

shape, flexibility, and conformation of the DNA, so it doesn’t directly read the bases but the bases will determine the structure of the DNA

does the major or minor groove provide more information for protein-DNA interactions

the major groove

why does the major groove provide more information for protein-DNA interactions

has more specific molecular contacts so there is more potential for information to be delivered

what is a specific molecular contact

a place on the DNA where a protein can interact

does the major or minor groove have more specific molecular contacts

the major groove

what are the types of molecular contacts

hydrogen bonding and methyl contacts

how many possible types of molecular contacts are there between base pairs in the major groove

four

how many possible molecular contacts are there for base pairs in the minor groove

three

what determines the strength of a protein-DNA interaction

the sum of the specific and non-specific interactions

what are the different types of non-specific protein-DNA interactions

• hydrogen bonding with oxygen in the deoxyribose ring

• hydrogen bonding with the phosphate backbone

  • recognzing stacking

what is stacking and how does it influence non-specific DNA interactions

stacking is the stacking of multiple ring structures from the side chains of amino acids, proteins can recognize the stacking of these rings structures, but they are non-specific because the protein only recognizes that it is a ring structure but not what amino acid it comes from

where do most non-specific protein-DNA interactions take place

the minor groove

are mediated protein-DNA interactions specific or non-specific or both

non-specific and specific

what is the main type of specific protein-DNA interaction

hydrogen bonding with acceptor or donor groups in the residues of bases

where do most specific DNA-protein interactions take place

the major groove

what is a sigma factor

a sigma factor is a protein that binds to DNA and facilitates its interactions with polymerase

does a sigma factor make specific, non-specific, bridged, or a combination of the three

all

what are the three factors which influence the specificity of a protein-DNA interaction

• length of DNA

• type of interaction - specific vs non-specific

  • structure of the protein - dimer?

how does the length of DNA influence the specificity of a protein-DNA interaction

a longer DNA strand with more bases will have more places to make connections with proteins, but these bases must have specific information of actually make a connection.

how does the structure of a protein influence its protein-DNA interactions

a dimer will make double the amount of contacts

what is EcoRV

a restriction enzyme found in bacteria w

what does EcoRV do

recognizes foreign DNA in bacteria that have a EcoRV site and will bind and cut the DNA up to remove it

what type of protein is EcoRV

IIP

what is EcoRV’s recognition site

5’ GATATC 3”

is EcoRB’s recognition site a palindrome

yes

what structure does EcoRV have

homodimer

how frequently does EcoRV’s recognition site occur in EcoRV

every 4096 bases

how many contacts does one subunit of EcoRV make

10 every six basesh

how many connections does all of EcoRV make with DNA

20 - homodimer

what is the most common motif for protein-DNA binding

helix-turn-helix motif

why is the helix-turn-helix motif in proteins best for protein-DNA structures

the helix structure fits perfectly into the major groove of B-DNA

what is the diameter of an alpha helix

1.2nm

how wide is the major groove of DNA

1.2nm

how deep is the major groove of DNA

0.6-0.8nm

how does the helix-turn-helix motif fit in DNA

the alpha helix fits on either side of the major groove

what does avidin bind to

biotin

what is the Kd of the biotin-avidin binding interaction

10^-15

what does the Kd of the biotin-avidin interaction mean

it binds very tightly and almost never comes apart

what is RPA

a replication protein

what does RPA bind to

ssDNA

what does SSB bind to

ssDNA

what does the lac repressor bind to

dsDNA specifically and non-specially

what is the Kd of RPA

1×10^-9 - 1×10^-11

what is the Kd of SSB

2×10^-7

what is the Kd of a nonspecific lac repressor

5×10^-4

what is the Kd of a specific lac repressor

2×10^-11

how will the lac repressor bind to DNA when there is no lactose present

the lac repressor will bind specifically to DNA at a certain sequence ahead of the promoter to prevent transcription of the lac gene

how will the lac repressor bind to DNA when lactose is present

the lac repressor will bind nonspecifically to DNA, and will slide around the DNA strand - it will recognize general DNA, not a specific sequence

what does it mean if a higher concentration of a protein and a ligand is needed to make a binding reaction

they have a lower affinity to one another

what is visualized in EMSA? what is not visalized?

DNA only - not protein

how do we see DNA in EMSA

P32 labelling or labelling with a fluorescent marker

what is in the first gel run of an EMSA test? what is the purpose of this?

• only DNA with no protein added

  • see how far DNA will run on its own

what are in the sequence gels after the first sequence in EMSA?

DNA and increasing amounts of protein that will bind to the DNA

what does the bottom line in an EMSA experiment represent

unbound DNA

what does the top line in an EMSA gel represent

bound protein and DNA

what kind of gel does an EMSA test use

non-denaturing polyacrylamide gel

what does “non-denaturing” in non-denaturing polyacrylamide mean

the gel is non-denaturing and will leave the protein structure in tact so we can see how it interacts with DNA

where does the fluorescent marker/P32 marker bind to DNA

anywhere as long as it doesn’t block where the protein binds

what does the 50% binding region look like on an EMSA gel sequence

the two bands are the same size

what kind of graph can we plot from an EMSA sequence

[AB] (bound protein and DNA) on y axis vs [B] (protein concentration) on x axis

what does the plateau in the graph derived from an EMSA graph mean

the point where the DNA is saturated and is bound completely to protein

where is the 50% bound point on a graph we get from an EMSA experiment

half way down from the plateau

what does the 50% bound point represent

the Kd

what is DNase1?

a non-specific nuclease

what does DNase1 do

cuts up DNA

what does adding protein do to the relationship between DNase1 and DNA

protein will protect the DNA from being cut up by DNase1

what is the first sequence we run in a quantative DNase1 footprinting

just DNA and DNase1, no protein, see how DNA will be cut up by DNase1 without protection from protein

what are the sequential gels after the first gel in quantitative DNase1 footprinting

you add DNA and DNase1, then you add increasing amounts of protein. the protein will protect DNA at certain places and you will see less cutting and fewer bands

what is quantitative DNase1 footprinting

a limited digestionw

what is a limited digestion

DNA will be cut at only a few limited places due to protection by protein instead of along the entirety of the strand

where is the fluorescent label in quantitative DNase1 footprinting

at either end of the DNA strand, doesn’t matter which end

what kind of graph does a quantitative DNase1 footprinting gel produce

an isotherm

what does the right handed arrow represent in a sequence gel

the promoter

what will the regions of DNA protected by protein from DNase1 look like on a sequence gel - quantitative DNase1 footprinting

will be blank or very light - DNA is not being cut in these regions anymore

what are the two controls we use for a quantitative DNase1 footprinting gel

• positive control - only DNA and DNase1, no protein

• negative control - no DNase1

how do we determine that the correct amount of protein was used in a quantitative DNase1 footprinting gel

we look for regions of strands that are the same for every protein concentration but DNase1 does not cut here

what does the lower plateau on the isotherm of a quantitative DNase1 footprinting graph represent

protein is not binding to DNA, indicates a low affinity interaction between protein and DNA

what does the top plateau represent in a quantitative DNase1 footprinting isotherm

all protein is bound and the DNA is saturated

is quantitative DNase1 footprinting or EMSA done in equilibrium

quantitative DNase1 footprinting w

what does it mean for quantitative DNase1 footprinting to be in equilibrium

it is sensitive to the equilibrium reaction of a protein-DNA interaction. Sometimes protein is bound to DNA and sometimes it is not, if it is not bound DNase1 is able to cut the DNA and we will see this represented in the gel

what does it mean that EMSA is not run in an equilibrium system

we cannot see the equilibrium reaction represented

what are the advantages of quantitative DNase1 footprinting

• yields equilibrium reactions

• shows information on individual binding sites - can make an “individual site binding isotherm”

what are the disadvantages of quantitative DNase1 footprinting

• time consuming

• reports larger protein binding regions than reality

• doesn’t provide information about the importance of specific positions

how can quantitative DNase1 footprinting be sterically hindered

DNase1 is a large molecule, and if it is a large protein binding to DNA it will not be able to get around the protein and will therefore report a larger binding site than is really there