Biol/Chem 5310                 

Lecture: 23

November 19, 2002

Nucleotides

Ch. 3 and Ch.23

Nucleic Acid Structure

Nucleotides are the building blocks of nucleic acids. Nucleic acids are linear polymers of nucleotides-with phosphodiester linkages. There are 2 types of nucleic acids:

RNA: ribonucleic acid

DNA: deoxyribonucleic acid

Roles: DNA is the genetic material of all cells and some viruses. Some viruses use RNA as genetic material. All cells use various types of RNA molecules for important functions. Examples: ATP is a single ribonucleotide.

large RNA's are used in protein synthesis by ribosomes.

Definitions:

Nucleotide is a phospho-ester of a nucleoside.

Nucleoside is an N-glycosidic linked "base" with a sugar (ribose or deoxyribose).   Various examples:NADH, CoA, FAD

Ribose or deoxyribose are b-anomers

Ribose (b-D-ribofuranose)	N-linked Ribose
Deoxyribose (b-D-deoxyribofuranose)	N-linked Deoxyribose

Primes signify sugar positions in nucleosides and nucleotides. Non-primed numbers signify base positions.

5 bases are found primarily in RNA and DNA (3 are common to both)

Base
Adenine
Guanine
Cytosine
Thymine
Uracil

Base	Single letter	Nucleoside	Classification
Adenine	A	Adenosine	Purine
Cytosine	C	Cytidine	Pyrimidine
Guanine	G	Guanosine	Purine
Thymine (DNA)	T	Thymidine	Pyrimidine
Uracil (RNA)	U	Uridine	Pyrimidine

The nucleotides found in DNA and RNA are nucleoside-monophosphates. The phosphodiesters connect a phosphate to the 5 ' OH of one sugar to the 3 ' OH of another sugar.

Deoxyribose is 2 '-deoxy. Both ribose and deoxyribose have 3 ' and 5 ' OH groups.

So a linear nucleic acid has one free 5 ' OH at one end, and one 3 ' OH at the other end, just as a polypeptide has an amino terminus and a carboxy terminus, one at each end. By convention, the 5 ' end is written at the left, and the 3 ' end at the right.

A nucleic acid can be written:

pApCpGpT

or simply    ACGT

Several Points:

• Some bases are modified
  • in DNA  

N6 methyl dA	5 methyl dC

• in RNA, many modified bases are found in transfer RNA
• DNA is less susceptible to hydrolysis than is RNA, because the 2 ' OH can act as a nucleophile in the hydrolytic reaction at the 3 ' position.
• Bases can undergo tautomerization, keto forms are more stable

  Base	Amino	Imino
  Adenosine
  Cytosine
  Base	Keto	Enol
  Thymidine
  Guanosine

• At neutral pH nucleic acids are negatively charged because of phosphates. In the diester form, one OH remains and the ionization has a pK of about 1.
• Some bases can ionize at low or high pH

• Base	Ionized	pKa
  Adenosine		3.8
  Cytidine		4.5
  Guanosine		2.4
  Guanosine		9.4

How stable is DNA?

• Thermodynamically-DNA is unstable. For hydrolysis DG = -25 kJ/mol
• Kinetically-DNA is quite stable. At pH 7 the uncatalyzed rate of hydrolysis is slow.

Base Pairing

Erwin Chargaff (1905-2002) (Bio) is credited with establishing that the base composition of DNA from any organism is fixed (for that organism) and that

% C	=	% G
% A	=	% T

At that time (~1940's) Hydrogen bonding was not seen as a key element of structure in molecular biology. Also, the predominant tautomers had not been established, so many possible pairings of the bases existed.

{To examine this problem see my Chime exercise.}

Even the X-ray diffraction data that Watson and Crick analyzed in the early 1950's did not reveal the hydrogen bonding of base-pairing. That is because the DNA analyzed was only a fiber of mixed composition DNA, not a crystal of a single molecular type. So it revealed only average structure.

Nevertheless, Watson and Crick were able to deduce, correctly, the structure of DNA in 1953.

It took another 25 years before true crystals of DNA (short pieces of a defined sequence) were solved by x-ray diffraction.

2 forms of DNA fibers were initially analyzed by Watson, Crick, Franklin, and Wilkins et al.Some of the samples were prepared by Rosalind Franklin, but she died of cancer in 1958 at the age of 37, before the Nobbel Prize was awarded.

Rosalind Franklin

The Nobel Prize in Medicine or Physiology was awarded to Watson, Crick & Wilkins in 1962 (Link)

James Watson	Francis Crick	Maurice Wilkins

A-form (dehydrated)

B-form (hydrated)

B-form represents the structure generally found in living organisms.

Features of the B-form {see Chime link}

1) 2 polynucleotide strands are antiparallel, and wind around each other with a right-hand twist. End view is circular, with a 20 Å diameter.

2) Phosphates (and sugars) are on the outside-to minimize charge repulsion. Charge is further shielded by water and cations.

3) Bases are inside the structure, nearly perpendicular to the helix axis. Bases in one strand hydrogen bond to bases in the other strand, according to the rule: G to C, and A to T.

4) Dimensions: ~10 base pairs per turn, bases are ~3.4 Å thick and are closely packed (stacked). This yields a pitch of ~34 Å (rise per turn).

5) There are 2 surface grooves: Major and minor (by size). The ridges are formed by the deoxyribose-phosphates, and the bottoms of the grooves are formed by the edges of the bases.

The grooves are not defined by looking at the spacefilling models of the helices, but rather by considering a single base pair. Draw a line from the helix axis (the center) to each C-1' of the sugars. The face corresponding to the angle < 180 ° is in the minor groove.

2 additional points

1) the original model was made on average DNA. Now we have true crystals of short segments of DNA, 12 bp or 18 bp. {see link}. It is now known that details of the structure depend upon the actual DNA base sequence. e.g. TATATA looks different from GCGCGC. Proteins that bind to DNA can also distort the DNA.

2) The structure explains why %A=%T, and %G=%C in double-stranded DNA. Of all possible hydrogen bonding pairs, only these fit into this structure.

Other forms of DNA

1) A-form (dehydrated)

Bases are tilted ~20 degrees from the perpendicular to the helix axis. It is also a right-handed double helix. The pitch is ~28 Å., with 11 bp per turn. Major groove is shallow, and minor groove is deep. A-form DNA is similar to double-stranded RNA, e.g.transfer RNA., and RNA-DNA hybrids.

{see Chime link}

2) Z-form DNA

This is a left-handed double helix, that requires a repeating sequence of Pyrimidine-Purine-Pyrimidine-Purine......

Py = C or T, Pu = A or G

12 bp per turn, pitch = 45 Å.

Deep minor groove, with flat major groove

Purines are rotated relative to sugars (syn)

Pyrimidines are (anti) as in B or A-form DNA

syn-Adenosine	anti-Adenosine

Phosphates have a zig-zag pattern

{see Chime link}

Evidence indicates that short segments of Z-form exist in E.coli.

(See also the H-DNA)

Comparison of these forms of DNA {See Table 23-1}

See the Guided Exploration 21 on the CD-ROM for more images.

Last updated Friday, November 22, 2002

Comments/questions: svik@mail.smu.edu

Copyright 2002, Steven B. Vik, Southern Methodist University