Transcript DNA structure - Rutgers Biomedical and Health Sciences

The Dogma
• Nucleic acid (DNA/RNA) is
important…Why?
• The central dogma of Molecular Biology
– DNA (genes, chromosomes) begets itself
(replication), as well as RNA (transcription))
– RNA begets protein (translation)
– Which proteins a cell expresses (and how
much), dictates what a cell does
DNA structure
• DNA: an ideal molecule for storage of
information.
– Made of simple, stable(?) “bits” of information
(the nucleotide) (metaphor: letters)
– Easily assembled/disassembled (metabolism)
(metaphor: words, sentences, books)
– The information is easily “read” (replication,
transcription)
The nucleotide: Pentose sugar
4’
1’
Pentose Sugar (2’ OH=ribose, 2’H=deoxyribose)
The nucleotide: Nitrogenous bases
Pyrimidines (small)
Purines (BIG)
From Kimball’s biology pages:
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.html
The nucleotide
Phosphate
Base
Sugar (2’ OH=ribose, 2’H=deoxyribose)
Nucleotide =sugar+phosphate+base
The chain has polarity
DNA chains are connected by a phosphate between sugar carbons
The chain has polarity: the phosphate bridges between 5’ and 3’
carbons (almost never 5’ and 5’, or 3’ and 3’)
DNA metabolism
• Making phosphodiester bonds…
– Synthesis (Nucleotide addition, nucleotide by
nucleotide)
– Ligation (joining two polynucleotide chains
together)
• Breaking phosphodiester bonds….
– Cleavage, or hydrolysis
Synthesis
Chemistry dictates addition is always to 3’ end of chain. In
other words: synthesis is always 5’ to 3’
DNA synthesis
• Synthesis: requires...
• Substrates
– 3’ OH of existing chain (primer strand)
– template strand (see replication lecture)
– deoxynucleotide triphosphate (dNTP)
• Cofactors
– Mg2+ (metal cofactor)
• Enzyme (DNA polymerase)
• Products are…
• Chain that is longer by one nucleotide
• Pyrophosphate (PPi)
Cleavage: Exonuclease
Cleavage (hydrolysis)
• Chain is broken between phosphate and sugar (5’ carbon
usually retains phosphate)
• Requires….
• Substrate:
– DNA chain, usually double stranded
– Water
• Enzyme (nuclease)
• Co-factors; usually Mg2+
• Product: broken chain
• If chain broken from end, enzyme is exonuclease
– Exonucleases can chew from 3’ end (3’ to 5’ exo) or 5’ end (5’ to 3’ exo)
• If chain broken in middle, enzyme is endonuclease
Cleavage: Endonuclease
Restriction enzymes are endonucleases (see Lee lecture)
Ligation
Ligation
• Requires…
• Substrates
– two DNA chains
– ATP
• Cofactors
– Mg2+ (metal cofactor)
• Enzyme: ligase
• Products are…
• Two chains joined together into one chain
• AMP
• Pyrophosphate (PPi)
DNA chains form helices
• Single DNA chains will form a helix
(spiraling line; like threads on screw)
because of….
– Hydrophobic interactions between bases
• Bases are carbon rich rings that hide from water, and
therefore stack on top of each other
– Ionic interactions
• Phosphates are highly negatively charged, thus repel
each other
The double helix
• A single stranded DNA chain will form a
helix but…
• Each base has a number of hydrogen donors
and acceptors
• Donors like to form hydrogen bonds with
acceptors
• Like this…..
Watson-Crick base pairs
•
•
•
•
A with T
G pairs with C
Why?
Complementary
pattern of hydrogen
donors and acceptors
• GC stronger than AT
•From http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BasePairing.html
The double helix
The double helix
• Two chains with extended sequence that
can pair together, or is “complementary”,
may form a double helix
• Constraints of backbone structure permits
double helices only when complementary
sequence is of the opposite polarity.
• i.e….5’GGTCA3’ will pair with
5’TGACC3’, but NOT 5’CCAGT3’
The double helix
Association/disassociation of the
double helix
• Hydrogen bonds between paired bases are weak
– Sensitive to temperature, salt concentration
– Heating will separate, denature, or melt a double helix into two separate
stands (single stranded, or ssDNA)
– Denaturation occurs at a specific temperature (melting temperature, or Tm)
– Tm defined by length (longer comlementary sequence=higher Tm) and
sequence (higher GC%=higher Tm)
• Separation of strands required for replication, transcription
– instead of heat, these processes use ATP for energy to break base pairs
Secondary structure
Secondary structure
hairpins (intra-molecular pairing of single strand)
heteroduplex (double stranded DNA with the occasional
mismatch, forms “bubbles” in double helix.
Can be caused by renaturation of partially complementary sequence, or
replication errors
Helical shape
• Helical parameters…
– Screw sense: left handed, or right handed
– Twist: degrees rotation, along the horizontal
axis, between successive base pairs
– Rise: elevation, along the vertical axis,
between successive base pairs
– Tilt: degrees of inclination of base pair from
the horizontal access (in most double helices
base pairs are not significantly tilted )
Helical forms
• A form
– Formed in DNA under dehydrating conditions
– Major form of RNA double helix
• B form
– Standard DNA double helix
• Z DNA
– Forms in vitro primarily at GC rich regions
Forms of the double helix
Helical forms
• A form
– Shorter, fatter than B form DNA
– High degree of base pair tilt
• B form
– Standard DNA double helix
– About 34o twist, 3.4 angstrom rise, very little tilt
• Z DNA
– Only left handed helix
– Kinked backbone (does not smoothly conform to helical shape)
– Much greater rise, reduced twist relative to B DNA
Forms of the double helix