MCDB 108A Exam 2 Ch 8 - Nucleotides & Nucleic Acids

Nucleotides

Building blocks of nucleic acids

pentose sugar (oxy or deoxy)

Phosphate (1-3) can be on any hydroxyl

Nitrogenous bases (pyrimidine or purine)

Nucleoside

ribose with base

nucleotide without a phosphate

beta-N glycosidic bond

anomeric carbon of pentose in beta configuration

base attaches to sugar at:

• N-1 in pyrimidine

• N-9 in purine

Pentose

Ribose sugar

• deoxy ribose is mossing -OH at C-2

Because of 2’OH, the C2’ endo conformation in RNA is disfavored, causes a steric clash (however its common in DNA)

C-3’-endo is RNA

primes = counting on nucleotide

Adenosine Monophosphates

phosphates can reside on carbons other than C-5’

ex: phosphates on C-3’ and C-2’ in some adenine monophosphates

Phosphodiester Bonds

5’-phosphate from one unit is covalently linked to the 3’ hydroxyl of the next unit (aka phosphodiester linkage)

sugar-phosphate backbone is repeating unit

nitrogenous bases are flipped out to the side

Backbone is hydrophilic

• hydroxyl group of ribose for H-bonds with water

• Phosphate is completely ionized and (-) near pH 7

all bases are good at making H-bonds

nitrogenous bases are more hydrophobic, but amines allow them to H-bond

backbone is subject to non enzymatic hydrolysis (RNA more than DNA)

under alkaline conditions RNA is rapidly hydrolyzed (DNA is not)

• mRNA is meant to be short-lived so we can regulate # of proteins made

• 2’ hydroxyl acts as a nucleophile

Tautomers

bases are aromatic molecules and as such are subject to electron delocalization

bases may exist in 2+ tautomeric forms depending on pH

ex: uracil tautomerizes from ketone in bases to enol in RNA

Base Interactions help form 3-D Structure

bases are hydrophobic and can stack on one another via Van der Waals and dipole-dipole interactions (keeps DNA together)

H-bonding between carbonyls and nitrogens link two strands together via base pairs

A/T base pair

• 2 interactions (H-bonds)

• H-bond 1 = 2.8 A

• H-bond 2 - 3.0 A

• C-1’ to C-1’ = 11.1 A distance

G/C base pair

• 3 interactions (H-bonds)

• H-bond 1 = 2.9 A

• H-bond 2 = 3.0 A

• H-bond 3 = 2.9 A

• C-1’ to C-1’ = 10.8 A distance

Chargaff’s Rules

In all cellular DNAs, regardless of species:

• A = T

• G = C

• A + G = C + T

Franklin and Wilkins

used X-ray diffraction to show structure of DNA is helical

Watson and Crick DNA discoveries

right handed double helix

sugar-phosphate backbone on outside

C-2’ endo of deoxyribose

Base stacked inside perpendicular to long axis

major groove (biggest angle)

minor groove (smallest angle)

A = T

G triple bond C

DNA Double Helix

antiparallel strands (run in opposite directions)

Bases are 3.4 A apart (top to bottom)

10 bp per complete turn (in actuality its 10.5)

strands are complementary AT/GC

strands held together by:

• H-bonding between bases

• base stacking (pi stacking) between aromatic rings of bases from top to bottom

Account for transmission of genetic info by separating strands and synthesizing a new strand for each (half old half new)

Different 3-D Forms of DNA

rotation is possible around many bonds

rotation around the C-1’-N glycosyl bond yields two stable conformations for purines

Purines can be both sun (towards sugar) and anti (away from sugar), they prefer anti

Pyrimidines can only be anti due to steric clashes between oxygens in syn form

B- DNA form

most stable, favored in nucleus

right handed

~10.5 base pairs per turn

glycosyl bonds are anti

A-DNA form

favored in solutions devoid of water

right handed

~11 base pairs per turn

glycosyl bonds are anti

circular view from top, from side helix is closer together

Z-DNA form

involved in gene expression and has been found in bacteria and eukaryote

left handed

zig-zag formation. looks twisted from top

anti pyrimidines alternates with sin purines (especially C/G), making it zig-zag

Palindrome

commonly found in DNA (ex: ROTATOR)

inverted repeats: top and bottom strands are palindromes of each other

• 5’ - TTAGCACGTGCTAA - 3’

• 3’ - AATCGTGCACGATT - 5’

Mirror repeat: symmetric sequences within one strand

• 5’ - TTAGCAC | CACGATT - 3’

Hairpins

formed when one palindromic strand with base pairs in between base pairs with each other

Cruciforms

created by 2 palindromic strands with base pairs in-between form two hairpins opposite each other

mRNA

Gene expression: using the genetic information to generate a biological product

Transcription: involves creating an mRNA in the nucleus using the DNA as a template

Most mRNAs in eukaryotes are monocistronic

Monocistronic

1 promoter has only 1 gene associated with it

each gene is tightly regulated

ex: eukaryotic mRNA

Polycistronic

1 promoter controls a set of multiple genes that are typically related

ex: lac-operon in E. coli

RNA can have complex structures

mRNA is always single stranded but still forms a right-handed helical structure held together by base stacking

purine-purine base stacking is the strongest

complementary sequences within the single strand can fold and create helices

other 2ndary elements include: bulges (base not paired), hairpins, internal loops, and wobbles (mismatched G.U)

tRNA can H-bond with N, and U.G bond slightly off in a wobble instead of U-A bonding. DNA cannot do this

Double Helix can be Denatured

DNA needs to be stable to preserve code

Heat and extreme pHs can unravel the double helix

Disruption of H-bonding between base pairs → denaturing

renaturing (annealing) of DNA is rapid

Characterization of Tm (melting point)

temperature at which half the DNA is single strand

Higher G triple bond C content, higher Tm

Tm can act as an estimator of base composition

A=T rich regions will denature first

shorter strand is easier to pull apart

Sanger Method

put a tag on primer, stop at every letter, run on a gel

Producing 4 sets of fragments (base specific)

Radioactively labeled primer 5’ end (left)

use low concentration of ddNTP’s

• DNA polymerase will halt elongation

Fragment size corresponds to relative position of each nucleotide

running fragments on a gel will reveal the order in which they appear in the sequence

shortest sequence runs the fastest, will be next to primer

• 4 test tubes w/ same DNA

• add a small ddNTP amount to one, ddCTP to another, etc while doing PCR

• run gel, bands will account for letters/sequence

Automating DNA Sequencing Reactions

ddNTPs are tagged with fluorescent molecule (instead of labeling primers)

each base is a different color

all added to same tube

DNA polymerase stops when it hits ddNTP

Create fluorescent fragments of different length

labeled fragments are separated on a capillary gel

laser beam detects each fragment and creates a peak with a specific color

computer determines sequence (based on size we know order)