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Nucleic Acid
Chemistry
Andy Howard
Introductory Biochemistry
1 May 2008
Nucleic Acid Chemistry
01 May 2008
What we’ll discuss
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RNA (concluded)
Chromatin
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Packaging of DNA
Nucleosomes
Histones
Higher levels
Bacterial
packaging
Nucleic Acid Chemistry
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Nucleases
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Alkaline hydrolysis
RNAses
Restriction
Endonucleases
Applications of
restriction endos
p.2 of 43
01 May 2008
RNA physics & chemistry
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RNA molecules vary widely in size, from a few
bases in length up to 10000s of bases
There are several types of RNA found in cells
Type
%%turn- Size,
RNA over
by
mRNA 3
25 50-104
tRNA 15
21 55-90
rRNA 80
50 102-104
sRNA
2
4 30-103
Nucleic Acid Chemistry
Partly
DS?
no
yes
no
?
Role
protein template
aa activation
transl. catalysis &
scaffolding
various
p.3 of 43
01 May 2008
Messenger RNA
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mRNA: transcription vehicle
DNA 5’-dAdCdCdGdTdAdTdG-3’
RNA 3’- U G G C A U A C-5’
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typical protein is ~500 amino acids;
3 mRNA bases/aa: 1500 bases (after splicing)
Additional noncoding regions (see later) brings it
up to ~4000 bases =
4000*300Da/base=1,200,000 Da
Only about 3% of cellular RNA but instable!
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Nucleic Acid Chemistry
p.4 of 43
01 May 2008
Transfer RNA
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tRNA: tool for engineering protein
synthesis at the ribosome
Each type of amino acid has its
own tRNA, responsible for
positioning the correct aa into the
growing protein
Roughly T-shaped or Y-shaped
molecules; generally 55-90 bases
long
15% of cellular RNA
Nucleic Acid Chemistry
p.5 of 43
Phe tRNA
PDB 1EVV
76 bases
yeast
01 May 2008
Ribosomal RNA
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rRNA: catalyic and scaffolding
functions within the ribosome
Responsible for ligation of new
amino acid (carried by tRNA)
onto growing protein chain
Can be large: mostly 500-3000
bases
a few are smaller (150 bases)
Very abundant: 80% of cellular
RNA
Relatively slow turnover
Nucleic Acid Chemistry
p.6 of 43
23S rRNA
PDB 1FFZ
602 bases
Haloarcula
marismortui
01 May 2008
Small RNA
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sRNA: few bases / molecule
often found in nucleus; thus it’s
often called small nuclear RNA,
snRNA
Involved in various functions,
including processing of mRNA in
the spliceosome
Some are catalytic
Typically 20-1000 bases
Not terribly plentiful: ~2 % of total
RNA
Nucleic Acid Chemistry
p.7 of 43
Protein Prp31
complexed to U4
snRNA
PDB 2OZB
33 bases +
85kDa
heterotetramer
Human
01 May 2008
Relative quantities
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Note that we said there wasn’t much
mRNA around at any given moment
The amount synthesized is much
greater because it has a much shorter
lifetime than the others
Ribonucleases act more avidly on it
We need a mechanism for eliminating it
because the cell wants to control
concentrations of specific proteins
Nucleic Acid Chemistry
p.8 of 43
01 May 2008
mRNA processing in Eukaryotes
Genomic DNA
Unmodified mRNA produced therefrom
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# bases (unmodified mRNA) =
# base-pairs of DNA in the gene…
because that’s how transcription works
BUT the number of bases in the unmodified
mRNA > # bases in the final mRNA that actually
codes for a protein
SO there needs to be a process for getting rid of
the unwanted bases in the mRNA: that’s what
splicing is!
Nucleic Acid Chemistry
p.9 of 43
01 May 2008
Splicing:
quick summary
Genomic DNA
transcription
Unmodified mRNA produced therefrom
exon
intron
exon
intron
exon
intron
splicing
exon
exon
(Mature transcript)
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exon
translation
Typically the initial eukaryotic message
contains roughly twice as many bases as the
final processed message
Spliceosome is the nuclear machine
(snRNAs + protein) in which the introns are
removed and the exons are spliced together
Nucleic Acid Chemistry
p.10 of 43
01 May 2008
Heterogeneity via
spliceosomal flexibility
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Specific RNA sequences in the initial
mRNA signal where to start and stop
each intron, but with some flexibility
That flexibility enables a single gene to
code for multiple mature RNAs and
therefore multiple proteins
Nucleic Acid Chemistry
p.11 of 43
01 May 2008
iClicker quiz
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1. Shown is the lactim
form of which nucleic
acid base?
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Uracil
Guanine
Adenine
Thymine
None of the above
Nucleic Acid Chemistry
HN
O
N
OH
lactim
p.12 of 43
01 May 2008
iClicker quiz #2
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Suppose someone reports that he has
characterized the genomic DNA of an organism
as having 29% A and 22% T. How would you
respond?
(a) That’s a reasonable result
(b) This result is unlikely because [A] ~ [T] in
duplex DNA
( c) That’s plausible if it’s a bacterium, but not if
it’s a eukaryote
(d) none of the above
Nucleic Acid Chemistry
p.13 of 43
01 May 2008
Chromatin
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Discovered long before we
understood molecular
biology
Seen to be banded objects
in nuclei of stained
eukaryotic cells
In resting cell it exists as
long slender threads, 30
nm diameter
Nucleic Acid Chemistry
From answers.com
p.14 of 43
01 May 2008
Squishing the DNA
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If the double helix were fully extended,
the largest human chromosome
(2.4*108bp) would be 2.4*108 *0.33nm ~
0.8*108nm=80 mm;
much bigger than the cell!
So we have to coil it up a lot to make it fit.
Longest chromosome is 10µm long
So the packing ratio is 80mm/10µm =
8000
Nucleic Acid Chemistry
p.15 of 43
01 May 2008
Nucleosomes
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DNA-protein complexes that
hold together the DNA in coiled
forms at the second- and thirdlevels of organization
(first is helicity itself)
The proteins involved are
histones
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Proteins rich in basic aa’s (R,K)
These interact closely with DNA to
facilitate appropriate coiling
Nucleic Acid Chemistry
p.16 of 43
Nucleosome
core particle
PDB 1KX5
143 bp +
108 kDa
heterooctamer
Xenopus
01 May 2008
Histones
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Characterized as H1, H2A, H2B, H3, H4
H1 involved in higher level of
organization; others in nucleosome itself
All are small, K&R-rich proteins
Highly conserved
Nucleic Acid Chemistry
p.17 of 43
01 May 2008
Categories of histones
Type
MW,kDa #aa’s
#basic
#acidic
H1
21
213
65
10
H2A
14
129
30
9
H2B
13.8
125
31
10
H3
15.3
135
33
11
H4
11.3
102
27
7
Nucleic Acid Chemistry
p.18 of 43
01 May 2008
Unfolded chromatin
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Treat chromatin with low ionic strength;
that disrupts higher level interactions so
the individual nucleosomes are strung
out relative to one another like beads on
a string
Image
courtesy
U. Maine
Nucleic Acid Chemistry
p.19 of 43
01 May 2008
O
HN
Histone
deactivation
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ONH3+
O
acylated lysine
Histones interact with DNA via
+charges on lys and arg residues.
If we neutralize those charges by
acetylation, the histones don’t bind as
tightly to the DNA
Carefully-timed enzymatic control of
histone acylation is a crucial element in
DNA organization
Nucleic Acid Chemistry
p.20 of 43
01 May 2008
CoASH
Histone
acetylation
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Active histone + Acetyl CoA
 inactive (acetylated)
histone + CoASH
Without the positive
charges, the affinity for DNA
goes down
Nucleic Acid Chemistry
p.21 of 43
Histone H1
PDB 1GHC
8.3 kDa monomer
Chicken
Histone
acetyltransferase
PDB 1QSO
66 kDa
tetramer
yeast
01 May 2008
Histone
deacetylation
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Type III deacetylases use
a non-trivial reaction:
Prot-lys-NAc + NAD+ 
Prot-lys-NH3+ + nicotinamide +
2’-O-acetyl-ADP-ribose
Part of the NAD salvage
pathway
Histone/protein deacetylase +
histone H4 active peptide
PDB 1SZD; 34 kDa “heterodimer”
yeast
Nucleic Acid Chemistry
p.22 of 43
01 May 2008
Nucleosome
structure
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Core octamer is two molecules
each of H2A, H2B, H3, H4
Typically wraps around
~200bp of DNA
DNA between
nucleosomes is ~54 bp long
H1 binds to linker and to core
particle; but in beads-on-a-string
structure, it’s often absent
Nucleic Acid Chemistry
p.23 of 43
01 May 2008
How much does this coil up?
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200 bp extended would be about 50nm
The width of the core-particle disk is 5nm
So this is a tenfold reduction
Nucleosomal organization corresponds to
negative supercoiling
… so DNA ends up supercoiled when we
take away the histones
Nucleic Acid Chemistry
p.24 of 43
01 May 2008
Courtesy answers.com
Next level of
organization
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H1 interacts with
DNA along linker
region
Individual histones
spiral along to form
30 nm fiber
See fig.19.25
Nucleic Acid Chemistry
Courtesy
Johns
Hopkins
Univ
p.25 of 43
01 May 2008
Even higher…
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The 30nm fibers are attached to
an RNA-protein scaffold that
holds the 30nm fibers in large
loops
Typical chromosome has ~200
loops
Loops are attached to scaffold at
their base
Ends can rotate so it can be
supercoiled
Nucleic Acid Chemistry
p.26 of 43
01 May 2008
What about prokaryotes?
No actual histones
 Histone-like proteins involved
 Bacterial DNA attached to scaffold
in large loops (~100kb)
 This makes a nucleoid
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Nucleic Acid Chemistry
p.27 of 43
01 May 2008
How many loops in bacteria?
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Typical bacterial genome (E.coli) has
3000 open reading frames ~ 3000
genes.
Assume 500 amino acids per protein =
1500 bases per gene (ignores
transcriptional elements)
Then genome is 1500 bp/gene * 3000
genes = 4.5*106 base-pairs
That’s (4.5*106 bp)/(1*105 bp/loop) = 45
loops
Nucleic Acid Chemistry
p.28 of 43
01 May 2008
O
Nucleases
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HN
O
N
Enzymes that hydrolyze
phosphodiester bonds in
nucleic acids
Can clip on the 3’ end or the
5’ end of the phosphorus
Can operate on DNA or RNA
DNA tends to be more
resistant to degradation
O-
O
O
P
O
O
OH
O
O
O
N
P
-O
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Nucleic Acid Chemistry
N
O-
p.29 of 43
HO
NH2
OH
N
N
01 May 2008
Alkaline hydrolysis
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RNA can be readily hydrolyzed
nonenzymatically, particularly at high pH
DNA considerably less so
RNA will be completely degraded at pH
13 (0.1N NaOH) in hours;
DNA untouched
This will still happen at lower pH, but
much more slowly
Nucleic Acid Chemistry
p.30 of 43
01 May 2008
Mechanism for alkaline
hydrolysis of RNA (fig. 19.28)
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Cyclic phosphate intermediate stabilizes
cleavage product
Nucleic Acid Chemistry
p.31 of 43
01 May 2008
Results of creating cyclic
phosphate
O
H
N
O
O
O-
O
P
N
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Hydroxyl or water can
attack five-membered
P-containing ring on
either side and leave
the –OP on 2’ or on 3’.
Nucleic Acid Chemistry
O
O-
H
O
O
P
O
H
p.32 of 43
O-
O-
H
O
H
O
H
01 May 2008
Consequences
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So RNA is considerably less stable
compared to DNA, owing to the
formation of this cyclic phosphate
intermediate
DNA can’t form this because it
doesn’t have a 2’ hydroxyl
In fact, deoxyribose has no free
hydroxyls!
Nucleic Acid Chemistry
p.33 of 43
01 May 2008
Enzymatic
hydrolysis of RNA
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Ribonucleases operate through
a similar 5-membered ring
intermediate: see fig. 19.29 for
bovine RNAse A:
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His-119 donates proton to 3’-OP
His-12 accepts proton from 2’-OH
Cyclic intermediate forms with
cleavage below the phosphate
Ring collapses, His-12 returns
proton to 2’-OH, bases restored
Nucleic Acid Chemistry
p.34 of 43
PDB
1KF8
13.6 kDa
monomer
bovine
01 May 2008
Restriction
endonucleases
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These are sequencespecific enzymes that
cleave phosphodiester
bonds in DNA
Found in bacteria, which
use them to cleave
foreign DNA
Nucleic Acid Chemistry
p.35 of 43
EcoRI with DNA
PDB 1ERI
61 kDa dimer +
13 bp
E.coli (obviously)
01 May 2008
The biology problem
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How does the bacterium mark its own DNA so
that it does replicate its own DNA but not the
foreign DNA?
Answer: by methylating specific bases in its
DNA prior to replication
Unmethylated DNA from foreign source gets
cleaved by restriction endonuclease
Only the methylated DNA survives to be
replicated
Most methylations are of A & G,
but sometimes C gets it too
Nucleic Acid Chemistry
p.36 of 43
01 May 2008
How it works
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When an unmethylated specific
sequence appears in the DNA, the
enzyme cleaves it
When the corresponding methylated
sequence appears, it doesn’t get
cleaved and remains available for
replication
The restriction endonucleases only
bind to palindromic sequences
Nucleic Acid Chemistry
p.37 of 43
01 May 2008
Palindromic DNA
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Sequences that read the same on one
strand from left to right as they do on the
opposite strand reading right to left
Example, found in EcoRI recognition
sequence:
5’-GAATTC-3’
3’-CTTAAG-5’
Most DNA isn’t palindromic, but
palindromic sequences are common
enough that we can frequently find them
Nucleic Acid Chemistry
p.38 of 43
01 May 2008
Nomenclature for restriction
endonucleases (table 19.4)
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Name has three pieces:
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3- or 4-character designation for organism, e.g.
Eco (E.coli), Kpn (Klebsiella pneoumoniae), Bam
(Bacillus amyloliquefaciens)
(optional) one-character designation for strain (R,
H) (e.g. R is strain R of E.coli)
Roman-numeral characters for enzyme
Thus :EcoRI, BamH1
Simpler: XbaI
Nucleic Acid Chemistry
p.39 of 43
01 May 2008
Generalizations about
restriction endonucleases
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Each binds a specific 4-7 base-pair
sequence
Always recognize palindromic sequences;
often local dimer within enzyme lines up on
the two identical strands of DNA
Cleavage site can be anywhere within the
sequence
Methylation site typically not on the cleaved
base
Nucleic Acid Chemistry
p.40 of 43
01 May 2008
Cf. table 19.4!
Common lab endonucleases
Nuclease Source
ApaI
BamHI
EcoRI
EcoRII
HinDIII
HpaII
Sequence
Acetobacter 5’GGGCCC
pasteurianus 3’CCCGGG
Bacillus amilo 5’GGATCC
-liquifaciens 3’CCTAGG
Escherichia 5’GAA*TTC
coli
3’CTT*AAG
E.coli
5’CC*WGG
3’GG*WCC
Haemophilus 5’A*AGCTT
influenzae 3’T*TCGAA
Haemophilus 5’CCGG
parainflu.
3’GGCC
Nucleic Acid Chemistry
Cut
5’-GGGCCC
C-3’
3’-C
CCGGG-5’
5’-G
GATCC-3’
3’-CCTAG
G-5’
5’-G
AATTC-3’
3’-CTTAA
G-5’
5’CCWGG-3’
5’-GGWCC
-5’
5’-A
AGCTT-3’
3’-TTCGA
A-5’
5’-C
CGG-3’
3’-GGC
C-5’
p.41 of 43
01 May 2008
More endonucleases
Nuclease Source
Sequence
KpnI
Klebsiella
5’GGTACC
pneumoniae 3’CCATGG
NotI
Nocardia
5’GCGGCCGC
otitidis
3’CGCCGGCG
PstI
Providencia 5’CTGCAG
stuartii 164 3’GACGTC
SmaI
Serratia
5’CCCGGG
marescens 3’GGGCCC
XbaI
Xanthomonas 5’TCTAGA
badrii
3’AGATCT
XhoI
Xanthomonas 5’CTCGAG
holcicola
3’GAGCTC
TaqI
Thermus
5’TCGA
aquaticus
3’AGCT
Nucleic Acid Chemistry
Cut
5’-GGTAC
C-3’
3’-C
CATGG-5’
5’-GC
GGCCGC-3’
3’-CGCCGG
CG-5’
5’-CTGCA
G-3’
3’-G
ACGTC-5’
5’-CCC GGG-3’
3’-GGG CCC-5’
5’-T
CTAGA-3’
3’-AGATC
T-5’
5’-C
TCGAG-3’
3’-GAGCT
C-5’
5’-T
CGA-3’
3’-AGC
T-5’
p.42 of 43
01 May 2008
Applications
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Cleaving DNA at the restriction sites
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Building cleavable constructs in plasmids
Recombinant DNA depends on identifying
restriction sites and cleaving them
Identifying mutations in a population
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That allows studies of genetic drift
DNA fingerprinting in forensics
Can be combined with PCR so the starting
DNA sample can be very small
Nucleic Acid Chemistry
p.43 of 43
01 May 2008