1 Nucleic Acid Structure And Function 1

Nucleic Acid
Structure &
Function
Charlotte V. Bañes, MD
Department of Biochemistry

Introduction


ORGANISM






Store and preserve information
Pass information to future generations
Express information during life’s processes GENETIC INFORMATION


Coded along a polymeric molecule
(DNA)




the chemical basis of heredity

Organized into genes


units of DNA that encodes a protein or
DNA

Central Dogma of Molecular Biology

Gene Expression & DNA Replication
“The genetic information stored in the nucleotide sequence of DNA serves two purposes.” 

Source of information for synthesis of protein molecules of the cell




GENE EXPRESSION

Provides information inherited by daughter cells or offsprings


DNA REPLICATION

Gene Expression vs DNA Replication
GENE EXPRESSION

DNA REPLICATION

Produces all the proteins required by an organism

Duplicates the chromosomes before cell division

Transcription of DNA: RNA copy of a small section of a chromosome

DNA copy of the entire chromosome Average size of a human gene:
104 – 105 nucleotide pairs

Average size of human chromosome: 108 nucleotide pairs

Translation of RNA: protein synthesis Occurs throughout interphase
Transcription in nucleus; translation in cytoplasm

Occurs during S phase
Replication in nucleus

Semi-conservative Replication
“DNA replication involves separation of the 2 original strands and production of
2 new strands with the original ones as templates.” 


produce two copies each contained one of the original strands and one entirely new strand

The Meselson-Stahl experiment



Showed that DNA is replicated semiconservatively
Key to Meselson-Stahl experiment:
 DNA is made up of nitrogen bases
 Nitrogen has an isotope 15N (14N is the most common isotope)


DNA containing "heavy nitrogen" (15N) can be distinguished form DNA containing "light nitrogen"
(14N) by cesium chloride density-gradient centrifugation. Semi-conservative Replication
1. Bacterial

(E coli) DNA is placed in a media containing heavy nitrogen, which binds to the DNA, making it identifiable. 2. This DNA is then placed in a media with the presence of N14 and left to replicate only once.
“The new bases will contain

14

N while the originals will contain

15

N.”

3. The

DNA is placed in test tubes and centrifuged.
4. The DNA molecules will position at their corresponding level of density
“15N is more dense than 14N.”
5. These

test tubes are observed under ultraviolet rays.

“DNA appears as a fine layer in the test tubes at different heights according to their density.”



According to the semi-conservative theory:


After one replication of DNA, they obtained 2 hybrid molecules
(part 14N, part 15N) from each original strand of DNA.



After 2 generations in the 14N medium, half the DNA would be the 50-50 hybrid and half would be the lighter 14N DNA

Nucleotide
Structure & Nomenclature

Nucleic Acids


2 Kinds





DNA (deoxyribonucleic acid)
RNA (ribonucleic acid)

Assembled from nucleotides




Nitrogenous base
Five-carbon sugar (pentose)
Phosphate

Five-Carbon Sugars

Bases

PURINES

Bases

PYRIMIDINE
S

when a base bonds with ribose by an intermolecular dehydration, a nucleoside is formed. water molecule is formed and a bond is formed between a nitrogen atom in the base and a carbon atom in the ribose

nucleoside combines with phosphate to form a nucleotide water is released and a phosphate ester bond is formed

Nucleosides vs Nucleotides

Nomenclature of Ribonucleotides
Base

Nucleoside

Nucleotides

Adenine

Adenosine

Adenylic acid
Adenosine
monophosphate
(AMP)

Guanine

Guanosine

Guanylic acid
Guanosine
monophosphate
(AMP)

Cytosine

Cytidine

Cytidylic acid
Cytidine
monophosphate
(CMP)

Uracil

Uridine

Uridylic acid
Uridine
monophosphate
(UMP)

Adenosine diphosphate (ADP)

Adenosine triphosphate (ATP)

Guanosine diphosphate (GDP)

Guanosine triphosphate (GTP)

Cytidine diphosphate (CDP)

Cytidine triphosphate (CTP)

Uridine diphosphate (UDP)

Uridine triphosphate (UTP)

Nomenclature of Deoxyribonucleotides
Base

Nucleoside

Nucleotides

Adenine

Deoxyadenosi ne Deoxyadenylic acid Adenosine diphosphate (dADP)

Adenosine triphosphate (dATP)

Guanosine diphosphate (dGDP)

Guanosine triphosphate (dGTP)

Cytidine diphosphate (dCDP)

Cytidine triphosphate (dCTP)

Uridine diphosphate (dTDP)

Uridine triphosphate (dTTP)

Deoxyadenosine monophosphate (dAMP)

Guanine

Deoxyguanosi ne Deoxyguanylic acid Deoxyguanosine monophosphate (dGMP)

Cytosine

Deoxycytidine

Deoxycytidylic acid Deoxycytidine monophosphate (dCMP)

Thymidine

Deoxythymidi ne Deoxythymidilic acid Deoxythymidine monophosphate Nucleic Acids



Polymers of nucleotides
Joined by phosphodiester bonds




Each strand has a polarity





Phosphate group links the 3’ carbon of a sugar to the 5’ carbon of the next sugar in the chain
5’ end and 3’ end
Phosphate group at the 5’ end, hydroxyl group at the 3’ end

Base sequence is written by convention in the 5’  3’ direction Nucleic Acids
DNA

RNA

Molecule is a double stranded helix Molecule is singlestranded

Sugar is deoxyribose Sugar is ribose

Contains thymine and no uracil Contains uracil, and no thymine

A=T
G=C

A =/≠U
G =/≠ C

DNA

DNA Structure


Two strands are antiparallel



Two strands are complimentary  A pairs with T (2 H-bonds)
 G pairs with C (3 H-bonds)



Chargaff’s rule:
 %A = %T
 %G = %C
 %purine = %pyrimidine

Chargaff’s rule

Example:
A sample of DNA has 10% C. What is the %T? %A? %G?

Answer:
% C = %G;
%A + %T = 100% 20%
%G = 10%
100% = (%C + %G) + (%A + %A + %T = 80%
%T)
%A = 40%
100% = (10% + 10%) + (%A + %T = 40%
%T)

DNA Structure


Watson-Crick DNA






B-DNA
Right handed double helical molecule Hydrophilic sugar-phosphate backbone on the outside
Hydrogen-bonded base pairs stacked in the center
10 base pairs per complete turn DNA Structure


Inside diameter: 11Å (1.1nm)



Distance bet points of attachment of AT and G-C: 11Å (1.1nm)
Other base pairings are possible but they don’t have the correct H-bonding pattern (G-T/A-C =
1H-bond); or right dimensions (2 purines overlap,
2 pyrimidines produce a gap)



Outside diameter: 20Å (2nm)



Length of one complete turn of the helix along its axis: 34Å (3.4nm), with 10 base pairs



Sites where drugs and polypeptides bind to DNA (histones, Na+, Mg2+), serve as receptors to DNA regulatory proteins


Major groove: ~22Å



Minor groove: ~12Å

Forms of DNA
A-DNA

B-DNA

Z-DNA

Bp per turn

11

10

12

Orientation of base pairs lie 20˚ to the perpendicular lie perpendicular to the helix axis

Helical sense

Right handed

Right handed

Left handed

Diameter

2.6nm

2.0nm

1.8nm

Length of one turn

28Å

34Å

45Å

Right-handed vs Left-handed Helix

Organization of DNA
“Large DNA molecules must be packaged in such a way they can fit inside the cell and still be functional.”

Supercoiling


Mitochondrial DNA; DNA of prokaryotes Results from strain on the molecule caused by under- or overwinding the double helix



Negative supercoiling







Twisting to the left of a right handed helix, DNA is wound more loosely

Positive supercoiling


Twisting to the right of a right handed helix, DNA is wound more tightly Topoisomerase










Enzymes that can change the amount of supercoiling in DNA molecule Make transient breaks in DNA strands Alternate breaking and resealing the sugar phosphate backbone
Hydrolyze phosphodiester linkage in one strand of the double helix, relax the supercoiling by rotating one strand around the other, and then reseal the break.
DNA gyrase – introduce negative supercoiling in relaxed, closed circular DNA

Packaging of DNA













Nuclear DNA in eukaryotes
Found in chromatin associated with histones and non-histones
Nucleosome – basic packaging unit of chromatin Histones – lysine and arginine rich
(confer a positive charge)
Octamer – 2 copies each of H2A, H2B,
H3, H4
DNA is wound around the octamer to form a nucleosome
H1associated with linker DNA found between nucleosomes  tight packaging DNA Denaturation & Renaturation


Disruption of hydrogen bonds and base stacking


Heat, alkaline pH, chemicals
(formamide, urea)



No covalent bonds are broken



Detected by increase absorbance of a DNA sol’n at a wavelength 260nm


Hyperchromic effect



Melting temperature (TM) temperature at which DNA is half denatured



TM (G-C rich DNA) > TM (A-T rich
DNA)



Renaturation (annealing) removal of denaturing condition

RNA

Characteristics of RNA


Polymer of purine and pyrimidine nucleotide linked by 3’5’ phosphodiester bridges analogous to those in DNA



Transcribed from DNA, translated to proteins 

Sugar moiety is a ribose (deoxyribose in
DNA)



Does not contain thymidine, contains uracil 

Exists as a single strand



Does not follow Chargaff’s rule



Can be hydrolyzed by alkali to 2’, 3’ cyclic diesters of the mononucleotides
(alkali acts on the 2’ hydroxyl group)

Characteristics of RNA




Information in the RNA is in its primary structure (sequence of nucleotides)
Complementary to the template strand of DNA
The same as that of the coding strand (except for U replacing T)

Types of RNA


mRNA (messenger RNA)
 Serve as template for protein synthesis 

rRNA (ribosomanl RNA)
 Structural role (machinery for protein synthesis)



tRNA (transfer RNA)
 Serve as adapter molecules for the translation of RNA information into specific sequences of amino acids



snRNA (small nuclear RNA)
 Role in RNA processing
 Not involved in protein synthesis

Messenger RNA (mRNA)



Function as messengers – convey information in a gene to protein synthesis
Serves as template  polymerization of amino acid sequence in protein formation

Messenger RNA (mRNA): Characteristics


5’ end is capped by 7methyguanosine triphosphate




Linked to 2’-O-methyl ribonucleoside at its 5’-OH through 3 phosphates
Cap serves as ribosome binding site
(transcription begins downstream of the 5’ capped terminal); protect RNA chain from degradation (5’ exonucleases) Messenger RNA (mRNA): Characteristics


3’ hydroxyl end is attached to a poly-A tail (20-250 nucleotides length polymer of adenylate residues)




Endonuclease cuts the RNA molecule on the 3’ side of the sequence AAUAAA (poly-A addition signal), then poly A polymerase adds the poly-A tail to the new 3’ end
Protect mRNA against degradation by 3’-exonucleases

Processing of mRNA


DNA  hnRNA (heterogenous nuclear RNA)  mRNA

Transfer RNA (tRNA)









74-95 nucleotides
Produced from nuclear processing of RNA
Adapters for translation of the information in the mRNA nucleotide sequence to amino acids At least 20 species of tRNA in every cell (at least one corresponding to each amino acid)
Nucleotide sequence allows extensive folding and interstrand complementarity  secondary structure (cloverleaf appearance)

Transfer RNA (tRNA)


4 arms


Acceptor arm terminates in the nucleotides CCA-OH
(added
posttranscriptionally by nucletidyl transferase)




Appropriate amino acid is attached (“charged) - 3’OH group of A moiety

D,


, extra arms

Define each tRNA

Ribosomal RNA (rRNA)


Ribosome






cytoplasmic nucleoprotein structure; machinery for protein synthesis from mRNA templates
Where mRNA and tRNA interact to translate into protein molecules

Polysome


Ribosome + mRNA

Ribosomal RNA (rRNA)






Mol Wt: 4.2 x 106, sedimentation velocity 80S
(Svedberg unit)
Function: not fully understood, ribosomal assembly, binding of mRNA to ribosomes
2 nucleoprotein subunits


60S: 2.8x106




5S rRNA, 5.8S rRNA, 28S rRNA; 50 polypeptide chains

40S: 1.4x106


18S rRNA; 30 polypeptide chains



45S precursor RNA (nucleolus)
 5.8S, 18S, 28S



5S transcribed independently

Small RNA


Small, stable RNA species usually complexed with proteins to form ribonucleoproteins 

snRNA (small nuclear RNA)




Involved in mRNA processing and gene regulation

miRNA (micro RNA); siRNA
(small interfering RNA)



Involved in gene regulation
Inhibit gene expression


decrease protein production

Nucleases

Nucleases: Classification
Nucleases are enzymes capable of degrading nucleic acids.
Classification:


Deoxyribonucleases




Ribonucleases




Cleave internal phosphodiester bonds to produce either 3’hydroxy and
5’phosphoryl; or 5’hydroxy and 3’phosphoryl

Exonucleases






Act specifically on ribonucleotides

Endonucleases




Act specifically on deoxyribonucleotides

Hydrolyze a nucleotide at the terminal of a molecule
Act in one direction (3’5’ or 5’3’ only)
Edit/proofread the most recently added nucleotide

Restriction endonucleases


Recognize specific sequences in DNA