UCLA

Chemistry and Biochemistry 153B

DNA, RNA, and Protein Synthesis
Winter 2016 - Albert Courey

Powerpoint #1: Nucleotides and Nucleic Acids

1.2) Organizing Principle of the Course=Central Dogma of Molecular Biology

processing
replication
repair

DNA

dNTPs

transcription

rNTPs

RNA

First ~23 Lectures

translation

Protein

salvage
synthesis
catabolism

Breakdown products

De novo
synthesis

(waste products, useful metabolites)

Metabolites
(amino acids, HCOO-, etc.)

Last ~3 Lectures

Why we study this stuff:

#1 reason: the universe enjoys being observed* and so the universe
created creatures like you and me who enjoy observing the universe!
Other reasons: the mechanisms by which genetic information is encoded
and decoded are central to our understanding of who we are as human
beings, and to our understanding of human physiology and human disease.

1.3) Covalent Structure of

Nucleic Acids

Voet, Voet, and Pratt: Figure 3-3

iClicker Question
To which of the following things is the
red arrow NOT pointing?
A) A purine residue
B) An adenine residue
C) A 5 end residue
D) A deoxyadenosine residue
E) None of the above

1.4) Nucleotide Structure

Base

Phosphates

Purine or Pyrimidine

1, 2, or 3

Sugar

Ribose or Deoxyribose

Nucleoside
Nucleotide

1.5) The Sugars

-D-ribose
(this anomer found in
ribonucleotides and RNA)

C
Furanose rings
Fischer
Projection
D-ribose

-D-ribose
(this anomer found in some
nucleotide precursors)

Linear aldehyde

-D-2-deoxyribose
(found in deoxynucleotides
and DNA)

1.6) The Bases

The bases in nucleotides are
substituted derivatives of two
different heterocycles.

:
:
pyrimidine

:
:
purine

They are classical Bronsted bases, because N1 and N3 of pyrimidine, and N1, N3, and
N7 of purine have pairs of non-bonding electrons that can be used to form covalent bonds to
protons. pKas are in the 4-5 range.

Questions:
1) Will the nitrogens be protonated at physiological pH? Why or why not?
2) Why isnt N9 of purine basic?

iClicker Question
Which of the following statements describes the
state of the N1, N3, and N7 nitrogen atoms in purine
and pyrimidine at physiological pH?
A. They will not be protonated because their pKas are too high.
B. They will not be protonated because their pKas are too low.
C. They will all be protonated.
D. Only N7 will be protonated.
E. I dont know.

iClicker Question
Why isnt N9 of the purine ring basic?
A. N9 is sp3 hybridized and therefore cannot accept a proton
B. The non-bonding electrons on N9 are delocalized
C. Protonation of N9 would lead to a loss of resonance
stabilization
D. Both B and C
E. All of the above

:
:

1.8) Purine and Pyrimidine Derivatives in DNA and RNA

NH2

NH2

3
2

5
6

1
2

8
4

N
X

X
O
H

O
H

O
N

CH3

O
N
X

N
X

H2N

N
N

Idiosyncratic nomenclature
Bases

Nucleosides Nucleotides

Cytosine
Uracil
Thymine
Adenine
Guanine

Cytidine
Uridine
Thymidine
Adenosine
Guanosine

Cytidylic acid
Uridylic acid
Thymidylic acid
Adenylic acid
Guanylic acid

1.9) Base Aromaticity

Planarity of aromatic bases allows efficient
stacking.
Because of delocalized orbital electrons, adjacently
stacked bases attract one another strongly through
favorable induced dipole interactions.
This stacking makes a significant contribution to the 3dimensional structure of nucleic acids.

Aromatic bases absorb UV light.

UV spectroscopy can be used to probe structural transitions
in nucleic acids.

1.10) Tautomerism
The bases can exist in multiple tautomeric forms.
H

O
H

H2N

Keto tautomer
(>99.9%)

N
X

H2N

Enol tautomer X
<0.1%)

O
H

keto
H2N

enol
N
X

H2N

N
X

iClicker Question
In the above equilibrium, why does the keto
tautomer predominate over the enol tautomer?
A. The standard free energy of the keto tautomer is greater than
the standard free energy of the enol tautomer.
B. The standard free energy of the keto tautomer is less than the
standard free energy of the enol tautomer.
C. The O-H bond is more polarized than the N-H bond.
D. The keto tautomer is better able to form a Watson-Crick
basepair than is the enol tautomer.
E. Both B and C.

1.11) The Phosphates

Phosphoric acid contains three ionizable oxygens
pKa = 2

pKa = 7

pKa = 12

Phosphates polymerize by formation of anhydride linkages

pyrophosphate
pKas of pyrophosphoric acid = 1, 2.5, 6.1, 8.5

Phosphates join to sugars by formation of phosphoester linkages

(e.g., 2, 3, or 5 O of ribose)

Monoester pKas 1, 6
Diester pKa 1

1.12) What you should know from slides 1.3-1.11

I.

II.

III.

Structures and nomenclature to know from memory:

A.

The bases shown on slide 1.8, along with their

nucleoside and nucleotide derivatives, including atom
numbering, stereochemistry, and ionization states.

B.

Primary structure of RNA and DNA strands, including

correct stereochemistry and ionization states, and the
meaning of strand polarity. You should be able to
recognize the 3 and 5 ends.

The implications of base aromaticity on:

A.

Nitrogen basicity

B.

The potential for stacking

Keto-enol tautomerism - the relative stability of the

tautomers and the significance of this to basepairing

1.13) Watson and Crick Model

Right Handed anti-parallel
double helix
Helical parameters*
Helical rise 3.4
Helical twist 36
Diameter
20
Q1: Based on above data, how many
basepairs are there per turn?
Q2: Based on the above data, what is the
helical pitch?
*These are the values that W/C deduced from Franklins fiber
diffraction data. The actual values for DNA in solution are a little
different. For example, the average helical twist in solution is
more like 34.

Watson, J.D. and Crick, F.H.C. (1953) Nature

171, 737-738,

1.14) Rosalind Franklins X-ray diffraction data

Franklin, R.H. and Gosling,

R.G. (1953) Nature 171,
740-741

1.15) Use of X-ray diffraction to determine molecular dimensions

Braggs Law*: n = 2dsin B

x
n = an integer
= wavelength
d = distance between rows of atoms
2(B) = angle of diffraction that gives rise to constructive interference
*http://www.matter.org.uk/diffraction/geometry/geometry_of_diffraction_braggs_law_1.htm

Left picture: constructive interference

Right picture: destructive interference

In phase

Out of phase

1.16) Why do we use X-rays to determine

molecular structure?
2d sin B n
d

n
2sin B

Smallest possiblevalue for d

So the smallest distance that can be measured

by probing with electromagnetic radiation is half
the wavelength.
Covalent bonds are typically 1-2 in length.
So in order to determine molecular structures at
atomic resolution, we need X-rays.

X-ray crystallographers typically use copper X-ray tube ( = 1.5418 )

1.17a) Information used by Watson and Crick to build their model

1)

R. Franklin DNA fiber X-ray diffraction data

Cross suggests helix - angle gives helical diameter of 20

Helical Pitch = 34

Helical Rise = 3.4

2)
3)
4)

Franklin, R.H. and Gosling, R.G.

(1953) Nature 171, 740-741

Franklins density measurements suggested a 2 or 3-stranded helix.

Chargaffs rules
T & G in keto tautomeric forms

1.17b) Importance of keto tautomers

H

O
H

H2N

N
X

H2N

N
X

Slide 1.10 implies that keto tautomers predominate over enol

tautomers by a ratio of >1000 to 1. What is the evidence?

Presumably you could measure the equilibrium ratio by NMR or some other
spectroscopic technique, but I was unable to find such measurements in the
literature.

Watson and Crick relied on quantum mechanical calculations to which they were
referred by Cavendish laboratory colleague Jerry Donahue.

The keto tautomeric state has since been verified by countless crystallographic
and NMR studies.

1.17c) Evidence that keto tautomers predominate in vivo

If T or G assume enol form then GT basepairs become possible:

This could result in mismatches during DNA replication

and therefore in mutations.
The fact that such mutations rarely occur verifies that the
enol tautomers rarely form in vivo.

iClicker Question
To calculate the distance (d) associated with a
spot in a fiber diffraction pattern, which of the
following things do we NOT need to know?
A. The distance of the spot from the center of the diffraction
pattern.
B. The distance of the X-ray source from the specimen.
C. The distance of the film from the specimen.
D. The wavelength of the radiation.
E. None of the above.

1.18) Watson and Crick Model

Essential features of the model that proved correct
1.

Antiparallel right-handed double helix

2.

Strands are linked by complementary sets of Hbond donors and acceptors on the bases!!
Watson and Crick (1953): It has not escaped our
notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism
for the genetic material.

Watson, J.D. and Crick, F.H.C. (1953) Nature

171, 737-738,

1.19) Watson-Crick basepairs

I want you to have a solid grasp of these
structures.
Why? Because an understanding of the
specificity of basepairing is critical to an
understanding of:
Nucleic acid structure and chemistry
DNA replication
DNA repair
Transcription
RNA processing
Translation
In other words, almost everything we
study in this course!
As we discuss topics like DNA replication,
RNA processing, and translation, well see
that the basepair rules can sometimes be
relaxed. To understand how they can be
relaxed, you first need to understand what
dictates this specificity in the first place.
Voet, Voet, and Pratt:
Figure 24-1

Hence, next weeks quiz!!!

iClicker Question
Why does the DNA double helix only
contain AT and GC basepairs?
A. These are the only pairs of bases that have
complementary arrays of H-bonding groups.
B. These are the only pairs of bases that fit into the double
helix without leaving any unsatisfied H-bond donors or
acceptors.
C. These are the only basepairs that exhibit perfect symmetry.
D. Both A and B.
E. All of the above.

1.20) The pseudodyad axis

The glycosidic bonds are
symmetrically arranged
around the pseudodyad axis.
So the double helix smoothly
accommodates AT, TA, GC,
and CG basepairs

Voet, Voet, and Pratt:

Figure 24-1

1.21) What accounts for the absence of nonWatson-Crick basepairs in DNA?

1.22) What you should know from slides 1.13-1.21

I.

The Watson/Crick model

A.

B.
II.

What was the significance of the following in formulating the

model?
1.

Rosalind Franklins data and Braggs law

2.

Chargaffs rules

3.

Knowledge of the correct tautomeric states

What are the most important features of the model?

Watson/Crick basepairs
A.

You should be able to draw the AT and GC basepairs with

good geometry.

B.

What is the meaning of the pseudodyad axis?

C.

Why does the double helix only accommodate these two

basepairs?

1.23) Two Forms of DNA

H2O

A-DNA

Reversible transition

B-DNA

H2O
Implies that B-form is stabilized by an interaction with water.

Rosalind Franklin was the first to recognize that there were two different
forms of DNA and to coin the terms A- and B-DNA.
Because she recognized this, she was able to look at one form at a time
explaining the high quality of her fiber diffraction data

1.24) Protein Data Bank (PDB)

Molecular graphics programs (such as Jmol and Pymol) draw structures of proteins and
nucleic acids using data in the Protein Data Bank (http://www.rcsb.org).
- Each structure exists as a PDB file, a list of all the atoms with their coordinates.
- As of 12/28/2015, 114,569 structures had been deposited in the PDB.

Experimental Method
X-ray structures 102,151
NMR structures 11,214
EM structures 922
Hybrid 89
Other 193

1.25) Examples of B-DNA and A-DNA

structures in the PDB

PDB ID (year)

Type of helix (length) (resolution)

Sequence

1BNA (1981)
3BSE (2009)
3V9D (2012)
1ZF1 (2005)

B-form (12mer) (1.9 )

B-form (16mer) (1.6 )
A-form (14mer) (2.5 )
A-form (10mer) (1.35 )

CGCGAATTCGCG*
ACTACAATGTTGCAAT
CCCCGGTACCGGGG
CCGGGCCCGG

*The Dickerson Dodecamer the first right-handed double helix to have

its structure determined at near atomic resolution

What does a pdb file look like? Lets go to the pdb to

find out.

1.26) Lets use Pymol to look at BDNA and A-DNA structures

Well address the following questions:
1) What are the major and minor grooves?
2) What do we see in the grooves?
3) What is the position of the helical axis relative to the
basepairs?
4) What is the sugar pucker?
5) What do we mean by basepair tilt?
6) What do we mean by propeller twist?
7) What are the major differences between B-DNA and ADNA?
Well also practice reading the sequence of DNA by looking into
the grooves

1.27) Why are the grooves of different widths?

210

Helical
axis

150

1.28) Differences between A and B-DNA

B-DNA
Diameter
20
Helical rise
3.4
Helical twist
36*
Helical repeat (h) 10* bp
Helical pitch
34 *
Tilt
6
Propellor twist
11
Sugar Pucker
C2-endo

A-DNA
26
2.9
31
11.6 bp
34
20
12
C3-endo

These averages mask a lot of variability:

e.g., in the Dickerson Dodecamer, helical
twist varies from 26 to 43, while helical
rise varies from 2.8 to 4.4 .

*While these values are based on fiber diffraction

studies, solution studies demonstrate that h for BDNA in solution is ~10.5 bp, corresponding to a
helical twist of 34 and a helical pitch of 35.7 .

Important differences
1)

A-DNA has less rise than B-DNA due, in part, to the different sugar puckers

2)

The bases are shifted away from helical axis in A-DNA:

3)

a)

Results in cavernous major groove and shallow minor groove

b)

Results in 6 hole

Base pairs dramatically tilted in A-DNA

1.29) Effect of sugar pucker on backbone extension

150 (close to
anti)

80 (close to g-)

Butane
(Newman projection)
Voet, Voet, and Pratt: Figures 24-5 and 24-7

3BSE

iClicker Question
What is this basepair?

5-me O4 N6 N7
N7

O6 N4

A) TA
B) AT
C) CG
D) GC
E) None of the above

3BSE

iClicker Question
What is this basepair?

N3
O2

N2 O2
N3

A) TA
B) AT
C) CG
D) GC
E) None of the above

1.30) Relative stability of A- vs. B-form helices

DNA
1. In aqueous solution (i.e. in cells), B-DNA is
favored over A-DNA, apparently due to B-DNAs
spine of hydration.
2. Reduced water activity in solutions containing high
concentrations of organic solvents (or in partially
dried out DNA fibers) favors A-DNA.
RNA - Steric crowding with 2-OH group in the C2 endo
sugar pucker forces RNA double helices into Aconformation

1.31) Watery spine in B-DNA

G
N
NH2
2

HN
O

NH2
2

HN

HN

HN

T
Dickerson, R.E. (1983)
Scientific American 249,
94-111

2 Layers of water in the minor groove

Layer 1 (W) bridges adjacent basepairs on opposite strands

- O2 of T and C
- N3 of G and A
- N2 of G
Layer 2 (W) bridges layer 1 water molecules

NH2

HN

N
W

NH2

NHW
2

1.32) 2-OH group results in steric crowding

Crowding is worse in C2 endo sugar pucker

iClicker Question
Why does water stablize
B-DNA relative to A-DNA?
A) Only the B-DNA minor groove has appropriate H-bonding
groups for H-bonding to water.
B) The narrow minor groove of A-DNA excludes water while the
broader minor groove of B-DNA admits water.
C) Both A and B.
D) None of the above.

1.33) The Watson-Crick Model

Watson, J.D. and Crick, F.H.C. (1953) Nature

171, 737-738,

1.34) B- vs. Z-DNA

Z-DNA

B-DNA

2DCG

3BSE

1.35) Z- vs. B-DNA basepair

C2 endo
C3 endo

anti

syn
?
C2 endo

anti

anti

2DCG

1BNA

estions:
Why do bulky substituents (e.g., Br) at 8 position of purine ring favor Z-DNA?
Why does high salt favor Z-DNA?

If you can answer question 7 on PS1 you understand everything you

need to know about Z-DNA!

1.36) What you should know from slides 1.23-1.35

I.

II.

III.

A- vs. B-form helices

A.

What is the meaning of each of the parameters on slide 1.28?

B.

How do the overall shape of A- and B-form helices differ and how
do the different sugar puckers contribute to this?

C.

What is the role of water in stabilizing B- relative to A-DNA?

D.

What is the role of the 2OH group in stabilizing A- relative to BRNA?

E.

How is the Watson-Crick model a hybrid between A- and B-DNA?

Z-form helices
A.

Why are Z-form helices restricted to sequences that alternate

between purine and pyrimidine residues?

B.

What factors influence the stability of Z-form helices?

You should be able to read the sequence of a piece of DNA by

looking into the grooves.