Lecture 18: DNA, structure, topology & recognition Flashcards
Central Dogma
What is the central Dogma of Molecular Biology?
DNA -> RNA -> Protein
The Basics of DNA
What is DNA?
What type of molecule is DNA?
What does DNA contain?
- DNA is an informational molecule
- DNA is a polymer
- Sequence of bases that specify genetic information
The Basics of DNA
How is DNA synthesized?
DNA is synthesized from dNTPs
- linked by phosphodiester bonds
- Bases available for hydrogen bonding and stacking
The Basics of DNA
What linkages are in DNA?
- Phosphodiester bonds
- 5’ to 3’ polarity
The Basics of DNA
What information is within the genes?
Nucleotides specify the amino acid sequences of proteins
The Basics of DNA
What information is outside of genes?
Regulatory sequences for DNA replication and mRNA synthesis
The Basics of DNA
What determines if a hydrogen bond is weak or strong?
Angled hydrogen bonds are weaker due to constrains in protein structures
DNA Structure
What are the two strands of DNA held together by?
What do hydrogen bonds mediate?
- base- pairing interactions
- base-pairing interactions in DNA
DNA Structure
What angle are the DNA strands?
Antiparalell
DNA Structure
What allows information in DNA to be copied?
Complementarity
DNA Structure
What is on the outside of the DNA structure?
What is on the inside?
Outside
- Sugar-phosphate backbone
Inside
- The bases are stacked
DNA Structure
What are the bases in DNA?
Adenine (A)
Cytosine (C)
Guanine (G)
Thymine (T)
DNA Structure
What is Chargaff’s Rule?
- Base pairs
- # of Hydrogen bonds
Base pairs are purine-pyrimidine
- A pairs with T
- G pairs with C
All Tigers Go Crazy
Hydrogen Bonds
A=T (2)
C≡G (3)
DNA Structure
What structure does DNA form?
A double helix (right-handed helix)
DNA Structure
Describe the double helix
What is it stabilized by?
What types of grooves does it have?
Where do most DNA binding proteins bind?
By hydrogen bonding & stacking interactions
A major and minor groove
Most proteins bind in the major groove
DNA Structure
What are the 3 types of DNA structures?
Describe them
A form (right-handed)
- DNA-RNA or RNA-RNA helix
- Found in solution
- I I bp/turn
B form (right-handed)
- Most stable
- Found in solution
- 10.5 bp/turn
Z form (left-handed)
- Maybe in cells, but certain viral proteins specifically recognize the Z form DNA
DNA Denaturation
What is denaturation?
How can it be done?
Separation of the two strands of DNA
Can be done by exposing DNA to a higher temperature or a higher pH
DNA Denaturation
What is the tm (melting temperature)?
What doe the two curves represent?
Temperature at which 50% of DNA is denatured
Curves
- Red curve = more G-C pairs
- Blue curve = less G-C pairs
- red has a higher tm
DNA Denaturation
What does a higher tm mean?
Why might this happen?
- It takes a higher temperature to denature 50% of DNA..
- the only way this can happen is if we have more G-C pairs (more hydrogen bonding)
RNA
What is RNA?
How stable is it in comparison to DNA?
Single strand helix
Much less stable than DNA
RNA
What are the bases in RNA?
- Adenine (A)
- Cytosine (C)
- Guanine (G)
- Uracil (U)
RNA
What are the base pairs in RNA?
- A pairs with U
- G pairs with C
- G also pairs with U
RNA
What is RNase P?
Catalytic RNA that cleaves the precurser to tRNA
RNA
What are the types of RNA?
- mRNA
- tRNA
- rRNA
RNA
What does mRNA do?
encodes for proteins
RNA
What does tRNA do?
Helps decode mRNA transferring AA to polypeptide chain
RNA
What type of RNA is rRNA?
Ribosomal RNA
RNA
What are the types of secondary structure elements in RNA?
- Single strands
- Bulge
- Internal loop
- Hairpin
DNA Binding Proteins
How are specific DNA sequences recognized?
- Hydrogen bonding
- Major groove interactions
DNA Binding Proteins
What can be discriminated from analyzing the major groove?
AT, TA, GC, CG can be discriminated
DNA Binding Proteins
What can be discriminated from analyzing the minor groove?
AT/TA vs. GC/CG
DNA Binding Proteins
Where does DNA typically bind?
In the Major groove
- alpha-helix fits nicely into the wide major groove
DNA Binding Proteins
What are the two types of binding proteins?
What are the functions of these proteins?
1) Sequence-Specific Proteins
2) Non-Sequence-Specific proteins
- May activate or repress transcription, unzip DNA, etc.
DNA Binding Proteins
Describe Sequence-Specific proteins
- What are the two methods used with these proteins
Is key to carrying out the steps in information transfer
1) Hydrogen bonding 2) Major groove
DNA Binding Proteins
Describe hydrogen bonding in Sequence Specific proteins
What is often found in those sequences?
- Patter of hydrogen bond donors/acceptors allows for discrimination
-Asparagine & Glutamine are often found in sequence-specific proteins because they can from specific H-bonds
DNA Binding Proteins
Describe major binding in Sequence Specific proteins
Like to bind to the major groove because can discriminate between all different base pairs
DNA Binding Proteins
How do Non-Sequence-Specific proteins bind?
What amino acids do they tend to have?
- Usually binds to DNA by their ability to recognize negatively charged phosphate backbone (alpha-helix fits in nicely)
- Tend to have amino acids such as lysine and arginine (positively charged)
DNA Binding Proteins
What is the function of Non-Sequence-Specific proteins?
May activate or repress transcription, unzip DNA etc.
DNA Binding Motifs
Where is the recognition helix positioned in a helix-turn helix?
recognition helix positioned in major groove; entirely alpha helical
DNA Binding Motifs
What is the function of the zinc ion in a Zinc Finger?
In what type of cell are zinc fingers found?
- The zinc ion has no function in recognition of the DNA, but is used in the thermodynamic
stability of the protein fold - Often found in eukaryotic transcription factors
DNA Binding Motifs
Where is the recognition helix positioned in a helix-turn helix?
During what time period if this binding motif seen?
- Has recognition helix inserted into major groove of DNA
- Often seen during development (“homeobox genes”)
DNA Binding Motifs
What type of helix is in a Leucine zipper?
What does Leu do in this zipper?
Where do Lys & Arg residues bind, why is this binding important?
- Amphipathic helix
- Leu residues stabilize dimerization
Lys & Arg residues bind in the major groove
- this binding is crucial for recognition of DNA
DNA Binding Motifs
What does a Basic Helix-Loop-Helix do?
- Dimerizes
- Basic residues bind to DNA
SUPER COILING
What is supercoiling?
Where does it occur?
Why does it occur?
- Supercoiling is an intrinsic property of DNA tertiary structure
- It occurs in all cellular DNAs and is highly regulated by each cell
- DNA is very long, so must be compacted to fit in cells
SUPER COILING
What does super coiling help do?
- Helps receive strain
- Supercoiling stablizes the strain on underwound DNA
Super Coiling
What is the structure of most DNA?
What does this mean?
Most DNA is underwound (strained)
- This means that there are less turns than the relaxed DNA would want
Super Coiling
What does underwinding facilitate?
- Underwinding facilitates strand separation
Topological Linking Number
What is the purpose of topological linking number (Lk)?
Quantifies supercoiling
- specifies number of helical turns in a closed circular DNA
Topological Linking Number
What is the unit for Lk?
bps per turn or base pairs per turn
Topological Linking Number
Why is Lk helpful in the context of unwinding?
Does the Lk increase or decrease if a base pair is underwound?
It can be used to define DNA underwinding
- If Lk decreases, DNA is underwound
Topological Linking Number
When is an Lk undefinded?
When the strands are not topologically joined
Topological Linking Number
What is a Negative supercoil?
- Hand?
- Winding?
- Left handed
- Underwound
Topological Linking Number
What is a positive supercoil?
- Hand?
- Winding?
- Right handed
- Overwound
Topoisomerase
What are Topoisomers?
Two forms of circular DNA that only differ in a topological property (i.e. Lk)
Topoisomerase
What do Topoisomerases do?
Change the Lk of DNA
Topoisomerase
What are the 2 Types of Topoisomerases?
- TYPE I
- TYPE II
Topoisomerase
Describe TYPE I Topoisomerase
- Increments
- Cleavage
- Relaxation of strands
- ATP hydrolysis
- Changes the Lk in increments of 1
- Cleaves one strand of duplex DNA
- Can relax both positive and negative supercoils
- Does NOT utilize ATP hydrolysis
Topoisomerase
Describe TYPE II Topoisomerase
- Increments
- Cleavage
- Relaxation of strands
- ATP hydrolysis
- Changes Lk in increments of 2
- Cleaves both strands of duplex DNA
- Can relax both positive and negative supercoils
- Can introduce negative supercoils (in prokaryotes only)
- Hydrolyzes ATP
Nucleosomes
How is DNA packaged?
Via nucleosomes
Nucleosomes
What are Nucleosomes composed of?
Histone proteins
Nucleosomes
What are histone proteins?
Small proteins wrapped around DNA
Nucleosomes
What do histone proteins do and help facilitate?
Where are they found?
- Help package DNA and facilitate supercoiling
- Found in chromatin of all eukaryotic cells
