BCMB 3100 - Nucleic Acids - Chapter 33 DNA is the genetic component of life

•Discovery of DNA Central Dogma for Biological Information Flow

•Nucleotides, nucleosides & bases
DNA  RNA  PROTEIN
•Polynucleotides
•DNA as genetic material
•Structure of double-stranded DNA Friedrich Miescher (1869): discovered DNA (nuclein 
•Chromatin nucleic acid 
C, H, O, N, P
•RNA
•Nucleases
1 2

Purines and Pyrimidines

DNA and RNA are made up of nucleotides
______________: base + sugar + phosphate(n)
See Fig. 33.5
deoxyribonucleotide (sugar = 2-deoxyribose)
ribonucleotide (sugar = ribose)
____________: base + sugar

__________ of nucleotides: heterocyclic rings

containing nitrogen
Two class of bases: ____________ and ________
3 4

See Fig 33.5 Major pyrimidines and purines Tautomers of adenine and cytosine
Amino versus Imino

5 6

1
 Tautomers of guanine, thymine and uracil
Lactam versus Lactim Ribose and Deoxyribose
Predominant forms
See 33.3 Figure

 
RNA DNA

7 8

Nucleosides

See Fig. 33.6 Nucleoside structures

9 10

See Fig 33.6

See Fig 33.7 Chemical structure of a __________
Two conformations of
nucleosides &
nucleotides are
possible due to
rotation around the
glycosidic bond:
syn and anti
The _________
conformation
predominates
11 12

2
 Structures of the deoxyribonucleoside-5’-
monophosphates
See Fig 33.7

13 14

(continued)

In vivo the negatively charged

phosphates on nucleotides are
complexed with cations or positively
charged proteins

***

15 16

BCMB 3100 - Nucleic Acids - Chapter 19 A. Nucleotides

joined by 3’-5’
•Discovery of DNA phosphodiester
linkages
•Nucleotides, nucleosides & bases
•Polynucleotides
•DNA as genetic material
•Structure of double-stranded DNA
•Chromatin
•RNA Structure of the
tetranucleotide
•Nucleases pdApdGpdTpdC
17 18

3
 Backbone of
Nucleic Acids
Story of DNA as Genetic Material
Discovery of the structure of double
Fig 33.4 stranded DNA, 1953
James Watson, Francis Crick, Rosalind Franklin,
Maurice Wilkins

20

S = capsular (1)
polysaccharide Evidence for
= death transforming
principle!
R= no
capsule =
live Evidence that
(2) DNA is the
Isolation &
characterization
genetic
of the
transforming material in
principle proved
cells!!
the chemical
make-up of the (3)
genetic material
Both set of phage
were infective
(4)
21

Nature (1953) 171:737 Nature (1953) 171:964

April May

4
 see Fig 33.12
BCMB 3100 - Nucleic Acids - Chapter 33

•Discovery of DNA
Adjacent •Nucleotides, nucleosides & bases
nucleotides
can •Polynucleotides
hydrogen •DNA as genetic material
bond to
•Structure of double-stranded DNA
each other
•Chromatin
•RNA
•Nucleases
25 26

DNA is double-stranded with equal ratios of

G:C and of A:T. However, the ratio of
(G+C):(A+T) varies in an species specific
manner

27 28

• Structure of B-DNA
• Sugar phosphate backbone
outside
• Stacking creates two
Complement unequal grooves (major
ary base and minor)
pairing and • Hydrophobic attraction
stacking in between the bases
DNA • Van der Waals contact
between bases
• H-bonds between bases
• Electrostatic repulsion
between phosphates
inhibited by cations (Mg++)
29 30

5
 Double helix
Fig 33.13 emphasizing the charge
on the phosphate
groups

Fig 33.11

B-DNA - Ball-and-stick model Forms of DNA

Z
Watson and Crick B
A
discovered
structure of
B-DNA.
Most common
form of DNA
under
physiological
conditions.
Dehydrated DNA in some GC
DNA in vivo rich regions
RNA:DNA hybrid
B-DNA - Space-filling model ds RNA
left-handed DNA 34

B-DNA A-DNA Z-DNA

DNA in vivo Dehydrated DNA

RNA:DNA hybrid
ds RNA

Fig 33.17 Fig 33.17

6
 Fig 33.20

38

Major groove: wider - 12Å; deeper – 8.5Å

DNA molecules vary greatly in length
depending upon the organism and organelle
Species Length Genome size

E. coli 4.2 x 106 bp same

fruit fly 62 x 106 bp 130 x 106bp
mitochondria 0.015 x 106 bp same
(from mammals; can be up to 2.5 x 108 in plants)
(circular in mammals; can be linear or circular in plants)

Minor groove: 6Å wide; 7.5Å deep Human 240 x 106 bp 3200 x 106 bp
Fig 33.19 (46 chromosomes)

How can you detect DNA in solution?

Absorption spectra of double-stranded and single- Melting curve for DNA
stranded DNA Temperature at
• Double-stranded which amount
(ds) DNA absorbance of dsDNA =
ssDNA is Tm
max 260 nm
(____________)
• _____________ absorbs
more than ds DNA
Tm for poly GC
• dsDNA can be denatured is greater than
by heat and chaotropic Tm for poly AT
agents
• Extent of denaturation
can be measured by
OD260 42

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 BCMB 3100 - Nucleic Acids - Chapter 33 Stem-loop structures in RNA

•Discovery of DNA • ssRNA can also have ds

•Nucleotides, nucleosides & bases regions

•Polynucleotides • __________ or _______

can form from short
•DNA as genetic material regions of complementary
•Structure of double-stranded DNA base pairs

•RNA • Stem: base-paired

nucleotides
•Chromatin
• Loop: noncomplementary
•Nucleases nucleotides
44

Stem Loop Structures

Four Classes of RNA in living organisms
______________ (rRNA) - ~80% of total RNA, part of
ribosomes (translation machinery)

•____________ (tRNA) - ~15% of total RNA, 73-95

nucleotides long, carry activated amino acids to
ribosomes during translation

______________ (mRNA) - linear “copies” of DNA that

encode genetic information. Encode primary structure
of protein. ~1-3% of total RNA, relatively unstable

___________ - may have catalytic activity and/or

associate with proteins to enhance activity, some
Fig 33.30 involved with RNA processing in the nucleus 46

Alternative Classification of RNA

BCMB 3100 - Nucleic Acids - Chapter 33
• RNAs involved in protein synthesis
rRNA, tRNA, mRNA, others •Discovery of DNA
• RNAs involved in post-transcriptional modification •Nucleotides, nucleosides & bases
or DNA replication
modification or DNA replication •Polynucleotides
snRNA, snoRNA, SmY, RNase P, others •DNA as genetic material
• Regulatory RNAs
•Structure of double-stranded DNA
aRNA (antisense RNA), miRNA (microRNA),
siRNA (small interfering RNA), others •RNA
• Parasitic RNAs •Chromatin
• Other RNAs
•Nucleases
47

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 Structure of supercoiled DNA. Circular B-DNA has 10.4
bases/turn of helix. If DNA is underwound (or overwound), it Human topoisomerase I bound to DNA
is supercoiled to restore 10.4 bases/turn. Supercoiling is done
by topoisomerases.
• Topoisomerases can
Supercoiled (underwound)
Relaxed; [supercoils are caused by underwinding or overwinding] add or remove
supercoils in DNA
(10.4 base pairs per turn
of the double helix)

• Cleave one or both

DNA strands, unwind
or overwind by rotating
cleaved ends, then
rejoin ends
See Fig.
33.22
49 50

• In the nucleus DNA is found as ______________

• Chromatin: an association of DNA with proteins
(mostly histones)  compact & manageable packing.
Chromatin looks like long threads of 30 nm diameter.
• Histones - the major proteins of chromatin
• Eukaryotes contain five small, basic histone proteins
containing many lysines and arginines:
H1, H2A, H2B, H3, and H4
• Positively charged histones bind to negatively-
charged sugar-phosphates of DNA
51 52

A structural unit in chromatin is the _____________ Electron micrograph of chromatin

Nucleosome: a ~200 bp DNA strand wound around a
histone core.
Chromatin treated with a low salt solution extends
into a “beads on a string” structure. Beads are the
nucleosomes; the string is DNA.

Fig. 33.24
Electron micrograph of chromatin 53

9
 Histone octamer Nucleosome core particle Fig. 33.26

Nucleosome
Nucleosome gives
10-fold packing

54 bp

146 bp

Fig. 33.25
57

Histone-depleted chromosome scaffold. Attachment

of DNA to RNA-protein scaffold gives further
Solenoid: a 200-fold packing
higher level of Protein scaffold Loops attached to scaffold
chromatin
structure in Solenoid
which give
adjacaent further
nucleosome 4-fold
associate via packing
histone H1

59

10
 Representation of the compaction of DNA Final chromosome is 1/8000 of length of B-DNA.
into a eukaryotic chromosome
This allows DNA to be packaged into cells. For
example, the largest human chromosome is 2.4 x
108 bp.

This chromosome would be 8.2 cm long if it

were not packaged as chromatin
(as opposed to 2-10 µm)!!

http://www.answers.com/topic/chromosome
Fig. 33.28 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1685232/pdf/ajhg00205-0039.pdf

BCMB 3100 - Nucleic Acids - Chapter 33

Nucleases and Hydrolysis
of Nucleic Acids
•Discovery of DNA
• Nucleases - hydrolyze phosphodiester bonds
•Nucleotides, nucleosides & bases RNases (RNA substrates)
•Polynucleotides DNases (DNA substrates)
•DNA as genetic material • May cleave either the 3’- or the 5’- ester bond
•Structure of double-stranded DNA of a 3’-5’ phosphodiester linkage
•RNA • Exonucleases start at the end of a chain
•Chromatin • Endonucleases hydrolyze sites within a chain
•Nucleases

• Nuclease cleavage sites

Cleavage of 3’ ester of Guanylate
• Cleavage at bond A generates
a 5’-phosphate and a 3’ OH 5’ ….pGpCpAp…3’ + H2O  5’….pG + pCpAp… 3’
terminus
• Cleavage at bond B
generates a 3’-phosphate and
a 5’-hydroxyl terminus Cleavage of 5’ ester of Guanylate

5’ ….pGpCpAp…3’ + H2O  5’….p +GpCpAp… 3’

A = cleavage of 3’- ester bond
B = cleavage of 5’- ester bond

11
 RNA is less stable than DNA
because RNA is sensitive to
hydrolysis in basic solutions

Alkaline Hydrolysis of RNA

DNA is stable in
basic solution

RNA is unstable in
base

Ribonuclease-Catalyzed Hydrolysis of RNA

RNase A cleaves 5’ ester to right of pyrimidines
5’…pApGpCpAp…3’ → 5’…pApGpCp + Ap…3’

(From previous page)

Enzyme-catalyzed cleavage by RNAse A results,

specifically, in a 3’-phosphate product

71

12
_____________________: site-specific
Most restriction enzymes recognize
endodeoxyribonucleases causing cleavage of both
palindromes: inverted sequences with
strands of DNA at points within or near the
two-fold symmetry over two strands
specific site recognized by the enzymes; important
tools in genetic engineering

5’AAGAATTCGG3’
______________________: catalyze both
methylation of host DNA and cleavage of non- 3’ATCTTAAGCC5’

methylated DNA at recognition site

______________________: cleave non-methylated

DNA at recognition site

• Methylation
and restriction
at the EcoR1
site

EcoRI

GAATTC
CTTAAG

EcoRI D. EcoR1 Binds Tightly to DNA

GAATTC
CTTAAG
• EcoR1 has 2
identical subunits
(purple and
yellow)
• Bound to a
fragment of DNA
(strands blue and
green)

78

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 Uses of Restriction Endonucleases top

• Developing restriction maps (indicates specific • Restriction digest of

cleavage sites in a DNA fragment) bacteriophage 
• Map of bacteriophage  showing cleavage sites • Four restriction
of some restriction enzymes enzymes used
• Sizing gel separates
fragments (smallest
move fastest)

bottom
80

• DNA Fingerprinting

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