CHAPTER 9: NUCLEIC ACIDS

9.1 Levels of Structure in Nucleic Acids

Primary (1o) Structure order of bases in the polynucleotide sequence
Secondary (2o) Structure 3D conformation of backbone
Tertiary (3o) Structure supercoiling of the molecule
Quaternary (4o) Structure interaction between DNA & proteins
Two principle types of nucleic acids
- DNA (deoxyribonucleic acid)
- RNA (ribonucleic acid)
How do DNA and RNA differ?
Ribosomes: polypeptide-generating machinery of the cell
Tobacco mosaic virus: nucleic acid strand winds through a cylinder of coat-protein subunits
9.2 The Covalent Structure of Polynucleotides
Nucleotides: monomers of nucleic acids
1. Nitrogenous base
2. Sugar
3. Phosphoric acid residue
Order of nucleic acids of DNA contains the information necessary to produce the correct amino acid sequence in
the cells proteins
What are the structures and components of the nucleotides?
Nucleic acid bases (nucleobases): one or two-ring nitrogenous aromatic compound
- Pyrimidines single-ring aromatic compounds
Cytosine DNA & RNA
Thymine substitute for Uracil in DNA (sometimes in RNA)
Uracil RNA only
- Purines double-ring aromatic compounds
Adenine DNA & RNA
Guanine DNA & RNA
Methylation can modify bases
Nucleoside
- base + sugar covalently bonded
- lacks phosphate group
- base forms a glycosidic linkage with sugar
Ribonucleoside: -D-ribose
Deoxyribonucleoside: -D-deoxyribose
The glycosidic linkage is from C-1 carbon of the sugar to the N-1 nitrogen of pyrimidines or N-9 of purines
The numbers of the sugars are primed
The sugar is linked to a nitrogen in both cases (N-glycosidic bond)
Nucleotide
- Phosphoric acid is esterified to one of the hydroxyl groups of the sugar portion of a nucleoside
- Named for the parent nucleoside; suffix monophosphate
- Position of the phosphate ester is specified by the number of the carbon atom at the hydroxyl group to
which it is esterified
- 5 nucleotides are the most common in nature
- Additional phosphate groups form anhydride linkages to the first phosphate; diphosphates,
triphosphates are formed

How do the nucleotides combine to give nucleic acids?

Polymerization of nucleotides give rise to nucleic acids
Linkage between monomers in nucleic acids involves formation of two ester bonds by phosphoric acid
3, 5-phosphodiester bond: Hydroxyl groups to which the phosphoric acid is esterified are those bonded to the
3 and 5 carbons on adjacent residues
Nucleotide residues normally carry a phosphate group to the 3 end, has a free hydroxyl group
Sugar phosphate backbone
- Repeats itself down the length of the chain
The most important structure of nucleic acids are the identities of the bases
One system notation, single letters, A,G, C, U, and T
Vertical lines show the positions of the sugar moieties where the bases are attached; a diagonal line through the
letter P is a phosphodiester bond
Single letters show only the order of the bases
The letter p is written to the left of the single-letter code for the base to represent a 5 nucleotide and to the
right to represent a 3 nucleotide
Sometimes a letter d is added to indicate a deoxyribonucleotide residue
- E.g. d(GACAT)
9.3 The Structure of DNA
Double helix: Watson & Crick (1953)
What is the nature of the DNA double helix?
Determination of the double-helical structure was based primarily on model building and X-ray diffraction
patterns
Amount of A = amount of T
Amount of G = amount of C
Hydrogen bond between bases on opposite chains determine the alignment of the helix; paired bases lying in
planes perpendicular to the axis
Outer part of helix sugar-phosphate backbone
Chains run in antiparallel directions, one 3 to 5 and other 5 to 3
Base pairing is complementary (adenine-thymine; guanine-cytosine)
Complementary strands: two chains
Adenine-thymine (A-T) base pair: 2 hydrogen bonds
Guanine-cytosine (G-C) base pair: 3 hydrogen bonds
Sugar-phosphate backbone measurements of the double helix
- Inside diameter = 11 (1.1 nm)
- Distance between the points of attachment of bases (same for two base pairs) = 11
Makes a smooth backbone with no bulges
- Outside diameter = 20 (2nm)
- One complete turn along its axis = 34 (3.4 nm) ; 10 base pairs
Base pairs other than A-T & G-C are possible but they do not have a smooth backbone and the correct hydrogen
pair bonding
Grooves: empty spaces of the double helix
- Minor groove: large
- Major groove: small
At neutral pH, each phosphate group of the backbone carries a negative charge
Positively charged ions (Na+ or Mg+) must be associated with the DNA to neutralize its charges
- Eukaryotic DNA has histones (positively charged proteins) in the cell nucleus

Are there other possible conformations of the double helix?

B-DNA: principal form that occurs in nature
A-DNA
- Has 11 base pairs for each turn
- Base pairs are not perpendicular to the axis (20o)
- Originally found in dehydrated DNA samples; artifact of DNA preparation
A- and B-DNA are both right-handed helices
DNA:RNA hybrids can adopt an A formation because the2-hydroxyl on the ribose prevents an RNA helix from
adopting the B form
RNA:RNA hybrids are found in the A form
Z-DNA
- Left-handed
- Occurs when there is an alternating of sequence of purine & pyrimidine
- Play a role in the regulation of gene expression
- Can be considered as a derivative of the B form; produced by flipping one side of the backbone 180o
without breaking the hydrogen bonding of the complementary bases
- Gets its name from its zigzag look of the phosphodiester backbone
Base stacking: ring portions of the DNA bases are very hydrophobic and interact with each other via hydrophobic
bonding of their pi-cloud electrons
In standard B-DNA, each base pair is rotated 32o with respect to the preceding one perfect for base pairing but
not optimal for maximal overlap of bases
Propeller twist: edges of the bases that are exposed to the minor groove twist so that they can come in contact
with water; base-pairing distances are less optimal but base stacking is more optimal
Bases also slide sideways to allow them to interact better with the bases above and below them; depends on
the bases present
Step: Basic unit for studying DNA is a dinucleotide with its complementary pairs
Many DNA-binding proteins recognize the overall structure of a sequence of DNA, which depends upon the
sequence but not the DNA sequence itself
DNA molecule length < diameter; not completely stiff and can fold back on itself in a manner similar to that of
proteins (tertiary structures)
Double helix is relaxed, no twists in it aside from the helical twists themselves
Supercoils: extra twists in DNA
How does prokaryotic DNA supercoil into its tertiary structure?
Negative supercoils
- Underwound, fewer than normal number of turns
- Has torsional stress that favors unwinding of the right-handed B-DNA
- Naturally occurring circular DNA
Positive supercoils
- Overwound, more than normal number of turns
- DNA during replication
Both supercoiling compacts the DNA making it like a rubber band wound up into a ball
Topoisomerases: enzymes involved in changing the state of supercoiled DNA
- Class I Topoisomerase
Cut the phosphodiester bond backbone of one strand of DNA
Passes the other end through, reseals the backbone
- Class II Topoisomerase
Cut both strands of DNA
Pass some of the remaining DNA helix between cut ends, reseals backbone
o E.g. DNA gyrase
Supercoils can be added or removed; enzymes play an important role in replication and transcription
separation of the helix causes supercoiling

DNA gyrase: bacterial topoisomerase; introduces negative supercoiling; tetramer enzyme

Ultracentrifugation can be used to detect supercoiled DNA because it sediments more rapidly than the relaxed
form

How does supercoiling take place in eukaryotic DNA?

Super coiling of eukaryotic nuclear DNA is more complicated
Eukaryotic DNA is complexed with a number of basic proteins with positively charged side-chains at neutral pH
Chromatin: Electrostatic attraction between the negatively charged phosphate groups on the DNA and the
positively charged groups on the proteins favors the formation of complexes of this sort
Topological changes induced by supercoiling must be accommodated by the histone-protein component of
chromatin
Histones
- Principal proteins in chromatin
- H1, H2A, H2B, H3, H4
- Contain large numbers of lysine & arginine
- DNA is tightly bound to all types of histones except H1
- H1: easy to remove from chromatin
Nucleosome
- bead consisting of DNA wrapped around a histone core
- Protein core is an octamer; 2 molecules of each type except H1 [(H2A)2(H2B)2(H3)2(H4)2]
- 150 base pairs are in contact with the proteins
Spacer regions
- consist of DNA complexed to some H1 histone & non histone proteins
- 30 50 base pairs long
Histones can be modified by acetylation, methylation, phosphorylation, & ubiquitinylation
Ubiquitin: protein that degrades other proteins
9.4 Denaturation of DNA
Hydrogen bonds between base pairs are an important factor in holding the double helix together
Stacking of the bases in the native conformation of DNA contributes to the largest part of stabilization energy
Energy must be added to a sample of DNA to break the hydrogen bonds to disrupt the stacking interactions
- Usually carried out by heating solution
How can we monitor DNA denaturation?
Melting: heat denaturation of DNA; monitored experimentally by observing the absorption of ultraviolet light
Bases absorb light at 240nm wavelength
Hyperchromicity: As DNA is heated, strands separate; the wavelength of absorption doesnt change, absorbed
light increases
Heat denaturation is a way to obtain single-stranded DNA
When DNA is replicated, it first becomes single-stranded so that the complementary bases can be aligned
Same principle is seen during a chemical reaction to determine the DNA sequence
Characteristic midpoint of the melting curve for DNA from each distinct source
G-C has three hydrogen bonds; A-T has only two
Higher percentage of G-C base pairs, higher melting temperature
G-C pairs are more hydrophobic so they stack better
Renaturation of denatured DNA is possible on slow cooling
9.5 The Principal Kinds of RNA and Their Structures
What kinds of RNA play a role in life processes?
1. Transfer RNA (tRNA)
Transports amino acids to site of protein synthesis
2. Ribosomal RNA (rRNA)
Combines with proteins to form ribosomes

3. Messanger RNA (mRNA)

Directs amino acid sequence of proteins
4. Small nuclear RNA (snRNA)
Found only in the nucleus of eukaryotic cells
Involved in processing of initial mRNA transcription products to a mature form for export from
the nucleus to the cytoplasm for translation
5. Micro RNA (miRNA)
Part of one of the oldest evolutionary relationships
Produced by bacteria; bind to sequences of phage bacteria, preventing their infection;
important in repairing nerve damage in muscles
6. Small interfering RNA (siRNA)
Main players in RNA interference (RNAi)
RNA participate in the synthesis of proteins in a series of reactions ultimately directed by the base sequence of
the cells DNA
Base sequences of all types of RNA are determined by that of DNA
Transcription: Order of bases is passed from DNA to RNA
Ribosomes
- Sites for assembly of the growing polypeptide chain in protein synthesis
aminoacyltRNAs: amino acids that are brought to the assembly site and bonded covalently to tRNA
Translation: order of bases in mRNA specifies the order of amino acids in the growing protein
Sequence of 3 bases in mRNA directs the incorporation of a particular amino acid into the growing protein chain
Prokaryotes no nuclear membrane, mRNA directs the synthesis of proteins
Eukaryotes splicing out intervening sequences (introns); parts of mRNA that will be expressed (exons) are
contiguous to each other
RNAi is used to eliminate the effect of a gene to discover its function

What is the role of transfer RNA in protein synthesis?

tRNA
- Smallest of the three important kinds of RNA
- Can be found in every living cell because at least one tRNA bonds specifically to each of the amino acids
- Single-stranded polynucleotide chain (73-94 residues); molecular mass of about 25,000 Da
- Intrachain hydrogen bonding occurs, forming A-U & G-C base pairs; except in DNA except for the
substitution of uracil for thymine
Molecules can be drawn as a cloverleaf structure considered the secondary structure of tRNA because it shows
hydrogen bonding between certain bases
Stems: hydrogen-bonded portions of themolecule
Loops: non-hydrogen-bonded portions
During protein synthesis, both tRNA & mRNA are bound to the ribosome in a definite spatial arrangement
ensures the correct order of the amino acids in the growing peptide chain
A particular tertiary structure is necessary for tRNA to interact with the enzyme that covalently attaches the
amino acid to the 2 or 3 end
tRNA folds into and L-shaped conformation determined by X-ray diffraction
How does ribosomal RNA combine with proteins to form the site of protein synthesis?
rRNA
- large, only a few types exist in a cell
- RNA portion: 60-65%; protein portion: 35-40%
- Ribosomes consists of 2 subunits
Smaller 1 large RNA molecule & about 20 different proteins
Larger 2 RNA molecules & 35 different proteins (prokaryotes); 3 RNA molecules & 50 different
proteins (eukaryotes)
Subunits are easily dissociated from one another by lowering the Mg2+ concentration
Analytical ultracentrifugation: monitors the dissociation & reassociation of ribosomes

 Sedimentation coefficient: characterization of motion of the particle in Svedberg units (S)

Self-assembly of ribosomes takes place in the living cell
rRNA and not protein is the part of a ribosome that catalyzes the formation of peptide bonds in bacteria
How does messenger RNA direct protein synthesis?
mRNA
- least abundant of the main types of RNA
- no more than 5-10% of the total cellular RNA
- directs the synthesis of proteins then degraded so that the nucleotides can be recycled
- turns over the most rapidly in a cell
- sequence of bases directs the synthesis of a protein reflects the sequence of DNA bases in the gene that
codes for protein
- mRNA sequence is often altered after it is produced from the DNA
- heterogeneous in size
- protective units (5-caps and 3 poly(A) tails) are added before mRNA is complete
Heterogeneous nuclear RNA (hnRNA): initially formed as a larger precursor molecule in eukaryotes
Introns
- Lengthy portions of intervening sequences; does not encode protein
- Removed by posttranscriptional splicing
How does small nuclear RNA help with the processing of RNA?
Small nuclear ribonucleoprotein particles (snRNPs)
- Help with the processing of the initial mRNA transcribed from DNA into a mature form that is ready for
export out of the nucleus
In eukaryotes, transcription occurs in the nucleus; most protein synthesis in the cytosol, mRNA must first be
exported
What is RNA interference, and why is it important?
Short stretches of RNA (20-30 nucleotides long) have been found to have an enormous control over gene
expression
Protection mechanism in many species with siRNAs are used to eliminate expression of an undesirable gene