Chapter 19 (part 2)

Nucleic Acids

DNA• 1o Structure - Linear array of

nucleotides• 2o Structure – double helix• 3o Structure - Super-coiling,

stem-loop formation• 4o Structure – Packaging into

chromatin

Determination of the DNA 1o Structure (DNA Sequencing)• Can determine the sequence of

DNA base pairs in any DNA molecule

• Chain-termination method developed by Sanger

• Involves in vitro replication of target DNA

• Technology led to the sequencing of the human genome

DNA Replication• DNA is a double-helical molecule • Each strand of the helix must be

copied in complementary fashion by DNA polymerase

• Each strand is a template for copying • DNA polymerase requires template

and primer • Primer: an oligonucleotide that pairs

with the end of the template molecule to form dsDNA

• DNA polymerases add nucleotides in 5'-3' direction

Chain Termination Method

• Based on DNA polymerase reaction • 4 separate rxns• Each reaction mixture contains dATP,

dGTP, dCTP and dTTP• Each reaction also contains a small

amount of one dideoxynucleotide (ddATP, ddGTP, ddCTP and ddTTP).

• Each of the 4 dideoxynucleotides are labeled with a different fluorescent dye.

• Dideoxynucleotides missing 3’-OH group. Once incorporated into the DNA chain, chain elongation stops)

Chain Termination Method

• Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally

• Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule

• Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide

N

NN

N

NH2

O

H

HH

HH

NH

N

N

O

NH2N

O

H

HH

HHO

PO

O

HO

O-

N

NN

N

NH2

O

HO

HH

HH

PO

O

O-

NH

N

N

O

NH2N

O

H

HH

HHO

PO

O

HO

O-

NH

N

N

O

NH2N

O

H

HH

HHOH

OH

OH

PHO

O

O-

NH

N

N

O

NH2N

O

H

HH

HHOH

OH

PO

O

PO

O

O-

N

NN

N

NH2

O

H

OH

HH

HH

PHO

O

O-

NH

N

N

O

NH2N

O

H

HH

HHO

PO

O

HO

O-

NH

N

N

O

NH2N

O

H

HH

HHOH

NO CHAIN ELONGATION

OH

PO

O

PO

O

O-

Chain Termination Method

• Run each reaction mixture on electrophoresis gel

• Short fragments go to bottom, long fragments on top

• Read the "sequence" from bottom of gel to top

• Convert this "sequence" to the complementary sequence

• Now read from the other end and you have the sequence you wanted - read 5' to 3'

DNA Secondary structure

• DNA is double stranded with antiparallel strands

• Right hand double helix• Three different helical forms

(A, B and Z DNA.

Comparison of A, B, Z DNA

• A: right-handed, short and broad, 2.3 A, 11 bp per turn

• B: right-handed, longer, thinner, 3.32 A, 10 bp per turn

• Z: left-handed, longest, thinnest, 3.8 A, 12 bp per turn

A-DNA B-DNA Z-DNA

Z-DNA

• Found in G:C-rich regions of DNA

• G goes to syn conformation

• C stays anti but whole C nucleoside (base and sugar) flips 180 degrees

DNA sequence Determines Melting Point

• Double Strand DNA can be denatured by heat (get strand separation)

• Can determine degree of denturation by measuring absorbance at 260 nm.

• Conjugated double bonds in bases absorb light at 260 nm.

• Base stacking causes less absorbance.

• Increased single strandedness causes increase in absorbance

DNA sequence Determines Melting Point

• Melting temperature related to G:C and A:T content.

• 3 H-bonds of G:C pair require higher temperatures to denture than 2 H-bonds of A:T pair.

DNA 3o Structure

•Super coiling •Cruciform structures

Supercoils• In duplex DNA, ten bp per turn of helix

(relaxed form)• DNA helix can be over-wound.• Over winding of DNA helix can be

compensated by supercoiling.• Supercoiling prevalent in circular DNA

molecules and within local regions of long linear DNA strands

• Enzymes called topoisomerases or gyrases can introduce or remove supercoils

• In vivo most DNA is negatively supercoiled.• Therefore, it is easy to unwind short

regions of the molecule to allow access for enzymes

Each super coil compensates for one + or – turn of the double helix

•Cruciforms occur in palindromic regions of DNA

•Can form intrachain base pairing

•Negative supercoiling may promote cruciforms

DNA and Nanotechnology

DNA and Nanotechnology

DNA 4o Structure

• In chromosomes, DNA is tightly associated with proteins

Chromosome Structure

• Human DNA’s total length is ~2 meters!

• This must be packaged into a nucleus that is about 5 micrometers in diameter

• This represents a compression of more than 100,000!

• It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments

Nucleosome Structure• Chromatin, the nucleoprotein

complex, consists of histones and nonhistone chromosomal proteins

• % major histone proteins: H1, H2A, H2B, H3 and H4

• Histone octamers are major part of the “protein spools”

• Nonhistone proteins are regulators of gene expression

•4 major histone (H2A, H2B, H3, H4) proteins for octomer

•200 base pair long DNA strand winds around the octomer

•146 base pair DNA “spacer separates individual nucleosomes

•H1 protein involved in higher-order chromatin structure.

•W/O H1, Chromatin looks like beads on string

Solenoid Structure of Chromatin

RNA• Single stranded molecule• Chemically less stable than DNA• presence of 2’-OH makes RNA more

susceptible to hydrolytic attack (especially form bases)

• Prone to degradation by Ribonucleases (Rnases)

• Has secondary structure. Can form intrachain base pairing (i.e.cruciform structures).

• Multiple functions

Type of RNA

• Ribosomal RNA (rRNA) – integral part of ribosomes (very abundant)

• Transfer RNA (tRNA) – carries activated amino acids to ribosomes.

• Messenger RNA (mRNA) – endcodes sequences of amino acids in proteins.

• Catalytic RNA (Ribozymes) – catalzye cleavage of specific RNA species.

RNA can have extensive 2o structure