Microbial Genetics ht10 DNA Structure & Function

Rosalind Franklin’s DNA image

Double helix structure of DNA was proposed in 1953 by James Watson and Francis Crick
DNA structure
1. primary base sequence
2. secondary - helical form
forces that hold it together
environmental factor effects
alternative helices & structures
supercoiling
- local structure
static (intrinsic) structure
dynamic structure
3. tertiary structure / protein binding
4. Protein-nucleic interactions – where, what, how?
5. Amino acid recognition of bases
6. DNA-binding motifs / DNA distortion upon protein binding
7. Wet lab techniques – identifying DNA and proteins that interact
Features of DNA nucleotides
DNA has a phosphodiester backbone Structure of a nucleotide Principle bases in nucleic acid

RNA versus DNA

1. Primary DNA structure. Base sequence
1.

DNA sequence is not random – Erwin Chargaff

Chargaff’s rules:
1. First parity rule: for duplex DNA %A  %T and %G  %C (Watson-
Crick base pairing)

2. Cluster rule: bases cluster non-randomly

Base clustering is often related to direction of transcription. The
template strand tends to be pyrimidine rich. The mRNA
synonymous strand tends to be purine rich
Y = pyrimidines 2.
R = purines

3. Second parity rule: %R  %Y also in single DNA strands

Associated with ability of DNA and RNA to form stem structures
Loops in RNA tend to be purine rich - May reduce probability of
dsRNA formation between mRNA’s (Part of a defence
mechanism against foreign DNA?)´. Later lecture we will discuss
regulation by small antisense RNA’s

4. The GC rule: (G+C%) is species specific

Codon choice
Mutation pressure
Thermophilicity?
2. DNA secondary structure – helical form
B-DNA: most common physiological form, right-handed helix, 10 bp per helical turn
A-DNA: RNA/DNA & RNA/RNA helices, DNA at low ionic strength, 11 bp per helical turn
Z-DNA: alternating R & Y or methylated C’s, left-handed helix, 6 bp per helical turn
Triple-helical DNA: polyR, polyY, polyY

B DNA – the classic double helix

Hydrogen bonding A-T or G-C holds the two strands together

A-T = 2 H bonds
G-C = 3 H bonds
Z DNA Alternating R & Y or methylated C’s, left-handed helix, 6 bp per helical turn

Does left-handed Z-DNA have any

biological purpose?

Z DNA commonly forms near

transcription start sites.

Z-DNA regions are stabilized by the

negative supercoiling generated by
transcription:

Suggests Z DNA is associated with

transient localized conformational
changes.

Certain classes of proteins bind to Z-

DNA with high affinity and
specificity. Indicates a biological
role.

Still very little known about Z-DNA’s

role or significance!
 Triple helical DNA = Triplex DNA polyR, polyY, polyY

Intermolecular triplexes = Y:RY, or R:RY (TFO = triplex forming oligonucleotides)

Intramolecular triplexes = H-DNA (occurs naturally in supercoiled DNA)

Intermolecular triplex Intramolecular triplex

H-DNA (intramolecular triplex DNA).

PolyR–PolyY tract with mirror repeat symmetry, one of the
single strands (shown in blue) folds back and forms a triplex
structure and the other strand (yellow) is left unpaired.

A DNA triplex is formed when pyrimidine or Watson-Crick base

purine bases occupy the major groove of the pairing is illustrated
DNA double Helix forming Hoogsteen pairs by dotted lines.
with purines of the Watson-Crick basepairs.
TFOs are being investigated as gene-drugs, to Hoogsteen base
modulate gene activity in vivo. pairing by broken
lines
 In addition to the helical coiling of the strands to form a double helix,
Supercoiling of DNA the double stranded DNA molecule can also twist upon itself.

Relaxed DNA has no supercoils

(10.4 bp per turn in B-DNA)

Negatively supercoiled DNA is underwound (favors unwinding of the helix)

DNA isolated from cells is always negatively supercoiled
Positively supercoiled DNA is overwound
L = linking number = number of times one DNA strand winds in a right-handed direction around the other in the molecule.
Topological property (cannot change without breaking a covalent bond – topoisomerase activity).

T = twist = The number of complete revolutions that one DNA strand makes around the duplex axis.
Geometric property (can change by physical deformation of the molecule, without breaking any covalent bonds).

W = writhing number = the number of times the duplex axis turns around the superhelical axis.

 = superhelical density = number of supercoils per turn  0.05 in DNA in vivo.

Supercoiling of DNA
Topoisomerases

Type I topoisomerases:
Relax negatively supercoiled DNA by nicking then closing one strand of duplex.
Cuts one strand of the double helix, passes the other strand through, then rejoins the cut ends.
Type II topoisomerases: breaks both strands and changes the linking number in steps of ±2.
e.g. DNA Gyrase: introduces negative supercoiling.

Negatively supercoiled DNA = Undertwisted DNA

Easier for proteins to access bases
Easier to separate strands (replication, transcription).
Local secondary structure of DNA

minor

major
Local secondary structure of DNA
DNA-Protein Interactions often occur in Minor and Major Grooves

minor

major

The number and type on

possible DNA protein
interactions depends on base
sequence and also the major
and minor grooves of DNA
Tertiary structure of DNA – protein binding

Proteins interact with DNA during: replication (recognition of ori and ter)
transcription (promoters, operators, terminators)
recombination (homologous & site specific)
defence (restriction enzymes)

Proteins interact with: bases (but these are buried inside the helix)
structure
phosphodiester backbone

Protein interactions with DNA involve:

hydrogen bonds between aa side chains and bases or phosphate: 51%
van der Waals interactions (molecular fit) 22%
hydrophobic interactions (aromatic aa to sugar) 19%
electrostatic interactions (salt bridges) 8%
interactions mediated by water (aa to base or phosphate)
Protein binding

One typical contact of

Protein and DNA
Protein binding

• Protein can bind the DNA through the base, sugar, and the phosphate groups
• Hydrogen bonds with phosphate are not specific, but are important in stabilizing
protein-DNA complexes
• Guanine exposes the greatest number of potential hydrogen-bonding atoms on
the base edge (4 positions)
• Polar and charged residues of amino acids play a central role in DNA binding
Arg > Lys > Ser > Thr; Asn and Gln
• Acidic residues are used sparingly Asp and Glu
• Relatively few interactions are produced by hydrophobic residues

Example of an Arginine interaction with Guanine

Distribution of single hydrogen bonds with DNA-bases
Types of protein-DNA recognition mechanisms used for specificity.

Two main classes of recognition:

Base readout and Shape readout, which are further subdivided as illustrated.
 Base recognition in the major and minor groove.

Sequence-specific patterns on the edges of the bases in the major groove underlie the ability of
proteins to readout base pairs through hydrogen bonds and hydrophobic contacts (hydrogen bond
acceptors in red, donors in blue, thymine methyl group in yellow, and base carbon hydrogens in white).

Note that A:T versus T:A and C:G versus G:C are indistinguishable in the minor groove.

The three panels show successive rotations of 90° around the helix axis.
Common Motifs in DNA binding proteins:
1. Helix-turn-Helix
2. Zinc Finger
3. Leucine Zipper
4. Helix-loop-Helix
Helix-turn-Helix C-terminal helix binds to major groove
N-terminal helix helps to position the complex
Examples of Helix-turn-Helix DNA-Binding Proteins
Example of a Helix-turn-Helix Binding Protein
Homeodomain Protein in Drosophila utilizing helix-turn-helix motif
Example of a Helix-turn-Helix Binding Protein -  repressor (also in P22)

Helix-2 lies in the major groove of its

DNA target

Critical amino acid residues in the recognition helix

are positioned to facilitate hydrogen bonding with
the edges of base pairs in the DNA
Example of a Helix-turn-Helix Binding Protein -  repressor (also in P22)

The recognition helix of the phage  repressor

* * *
*
* are amino acids facing one side of the helix
Example of a Helix-turn-Helix Binding Protein -  repressor (also in P22)

Details of -Repressor Binding to

Operator

The helix-turn-helix motif is

inserted into the major groove of
the DNA

The arm of the lower monomer

reaches around to “embrace”
the DNA
Example of a Helix-turn-Helix Binding Protein -  repressor (also in P22)

Geometry of the  repressor-operator complex

1. Recognition helices: 3 & 3

2. Protein-protein interaction
helices: 5 & 5’

3. Bending of DNA.
Example of a Helix-turn-Helix Binding Protein -  repressor (also in P22)

Amino terminal of  Repressor + DNA Details of hydrogen bonds:

base pair 2

base pair 4

base pair 6
Example of a Helix-turn-Helix Binding Protein -  repressor (also in P22)

Amino-acid DNA Backbone

Interactions

hydrogen bonds form

between peptide NH
groups and phosphates
Example of a Helix-turn-Helix Binding Protein -  repressor (also in P22)

Changes of DNA conformation associated with repressor binding

DNA alone Shape when repressor is bound

Zinc Finger – DNA binding protein domain Utilizing a zinc in the center of
an alpha helix and two beta sheets
DNA-binding by a Zinc Finger Protein Three zinc-fingers forming a
recognition site

Alpha helix amino acids in each zinc finger interacts with DNA bases
Zinc Finger: beta-sheet amino acids can also recognize DNA

A dimer of the zinc finger domain of the glucocorticoid receptor bound to its
specific DNA sequence.
Zinc fingers: Structure alone does not detect binding Site – three examples

The specific amino acids in the

C-terminal alpha helix of the
zinc finger motif determine
the interaction specificity of
DNA recognition

CS 6463: (P) Control of Gene Expression 32

 Helix-loop-helix and Leucine Zippers

Heterodimerization of a Leucine Zipper

Homodimers and heterodimers can recognize

different DNA patterns

Leucine Zipper Dimer

The motif mediates both DNA binding and protein dimerization
Helix-Loop-Helix (HLH): Helix-loop-helix
Helix-loop-Helix motif and its dimer

The helix-loop-helix motif consists of a short alpha helix connected by a loop to a

longer alpha helix. Part of this motif is a dimerization domain that interacts with other
helix-loop-helix proteins to form homo- or heterodimers; the dimerization partner
often determines DNA binding affinity and specificity because two alpha-helices, one
from each monomer, bind to the major groove of the target DNA
Helix-Loop-Helix (HLH): Helix-loop-helix

Truncation of a HLH tail (DNA binding domain)

inhibits binding
Wet-lab Techniques: Gene Mobility Shift Assay

One can identify the sizes of proteins associated/bound with the desired DNA fragment
Wet-lab Techniques:DNA Affinity Chromatography
After obtaining the protein, run mass spectroscopy, identify the amino acid sequence, check
against the genome, identify the gene sequence
Wet-lab Techniques: Detecting DNA Binding Sites
Assay to determine the gene sequence Chromatin Immunoprecipitation
recognized by a specific protein In vivo genes bound to a known protein