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 Pentose Sugar (2’ OH=ribose, 2’H=deoxyribose) 1’ 4’

The nucleotide: Nitrogenous bases Purines (BIG) Pyrimidines (small) From Kimball’s biology pages:

The nucleotide Nucleotide =sugar+phosphate+base Phosphate Base Sugar (2’ OH=ribose, 2’H=deoxyribose)

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

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

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 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 Secondary structure

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 34 o 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