Chapter 9: Nucleic Acids

Primary structure

order of bases in the polynucleotide sequence

Secondary structure

three-dimensional conformation of the backbone

Tertiary structure

super coiling of the molecule

Quaternary structure

interaction with other macromolecules

Nucleic acid bases

Nitrogen-containing aromatic compounds that make up the coding portion nucleic acids

Pyrimidine bases

• Compounds that contain a six-membered ring

• Parent compounds of several nucleobases

• Cytosine, Thymine, Uracil

Purine bases

• Compounds that contain a six-membered ring fused to a five-membered ring

• Parent compounds of adenine and guanine

Nucleoside

• Purine or pyrimidine base bonded to a sugar (ribose or deoxyribose)

• Lacks phosphate group

• Forms a glycosidic linkage with the sugar

Nucleotide

• Monomers of nucleic acids

• 3 parts: nitrogenous base, sugar, phosphoric acid residue

• Naming: parent nucleoside, with the suffix -monophosphate added

3’, 5’-phosphodiester bond

Covalent linkage in which phosphoric acid is esterified to the 3′ hydroxyl of one nucleoside and the 5′ hydroxyl of another nucleoside

Dr. Rosalind Franklin

Created the famous Photo 51, an X-ray diffraction work demonstrating the double helix structure of DNA (the molecule containing the genetic instructions for the development of all living organisms

DNA Double Helix

Two polynucleotide chains wrapped around each other; the fundamental structural motif of DNA

DNA

Biopolymer that consists of a backbone of alternating units of 2-deoxy-D-ribose and phosphate

Phosphodiester bond

3′ —OH of one 2-deoxy-D-ribose is joined to the 5′ —OH of the next 2-deoxy-D-ribose by a _____

5’ end to 3’ end

Nucleotide residues of nucleic acids are numbered from the _____

Base pairing

Complementary joining of two nucleic acid bases making the two chains of the double helix as complementary strands

Sugar moieties

Vertical lines show the positions of the _____ to which the individual bases are attached

Phosphodiester bond

Diagonal lines through the letter P represents a _____

Deoxyribonucleotide residue

Letter “d” is added to indicate a _____

A-DNA

DNA Conformation

• Not found in in vivo techniques

• Right-handed helix (helix winds upward) but thicker than B-DNA

• 11 base pairs per turn of the helix

• Base pairs are not perpendicular to the helix axis; lie at an angle of about 20º to the perpendicular

• Originally found in dehydrated DNA samples

DNA:RNA and RNA:RNA

Hybrids that may be found in the A-DNA form

B-DNA

DNA Conformation

• Normal, physiological DNA form

• Right-handed helix

• 10 base pairs per turn of the helix

Z-DNA

DNA Conformation

• Usually consists of alternating purine-pyrimidine bases; regulation of gene expression

• Left-handed double helix

• Derivative of the B form

• Produced by flipping one side of the backbone 180 ̊ without disturbing the backbone covalent bonds or hydrogen bonds

• Zigzag look of the phosphodiester backbone when viewed from the side

hydrophobic

Bases are (hydrophobic/hydrophilic)?

Base stacking

The ring portions of the DNA bases interact with each other via hydrophobic bonding (van der Waals) of their pi-cloud electrons

• In standard B-DNA, each base pair is rotated 32º with respect to the preceding one (optimal for maximal base pairing but not optimal for maximal overlap of bases)

B-DNA

In standard _____, each base pair is rotated 32º with respect to the preceding one

Minor groove

Bases that are exposed to the _____ must come in contact with water

Propeller twist

• Base-pairing distances are less optimal, base stacking is more optimal, and water is eliminated from minor-groove contacts with bases

• H-bonds between bases are distorted by this motion, yet remain intact

• Bases slide sideways

Step

The dinucleotide with its complementary pairs is called a _____ in the nomenclature of DNA structure

Prokaryotic DNA

• Circular and forms supercoils

• Extra twists (over and above those of the double helix) in closed circular DNA

Circular DNA

The 5′ and 3′ ends of each strand are joined by phosphodiester bonds

Negative supercoils

Circular DNA with fewer than normal number of turns of the helix (underwound)

Positive supercoils

Circular DNA with more than normal number of turns of the helix (overwound)

Replication

Naturally occurring circular DNA is negatively supercoiled except during _____

Topoisomerases

Enzymes that relax supercoiling in closed circular DNA

Class I topoisomerases

Cut the phosphodiester backbone of one strand, pass the other end through, and reseal

Class II topoisomerases

Cut both strands, pass some of the remaining DNA helix between the cut ends, and reseal

DNA gyrase

(Class II topoisomerase) Bacterial topoisomerase that introduces negative supercoils into DNA

• tetramer

• cuts both strands of DNA

Class II

Which class of topoisomerase is gyrase?

chromatin

Supercoiling in eukaryotic DNA results in _____

Chromatin

• Complex of DNA and protein found in eukaryotic nuclei

• Resembles beads (nucleosome) on a string

• String portions are called spacer regions

• Structure and spacing of nucleosomes is important in its function

Spacer regions

• Consist of DNA complexed to some H1 histone and nonhistone proteins

• 30 to 50 base pairs long

• Further coiling of this region produces the compact form of chromatin found in the cell

Spacer regions

Further coiling of the _____ produces the compact form of chromatin found in the cell

Ubiquitin

A protein involved in the degradation of other proteins

histone-protein

Topological changes induced by supercoiling must be accommodated by _____ component of chromatin

Histones

• Principal proteins in chromatin

• Basic proteins found complexed to eukaryotic DNA

• Main types - H1, H2A, H2B, H3, and H4

• Rich in the basic amino acid residues, lysine and arginine

DNA Denaturation

• Energy must be added to a sample of DNA to break the hydrogen bonds and to disrupt the stacking interactions

• Carried out by heating the DNA in a solution

  • Melting: heat denaturation of DNA

  • Slow cooling: renaturation is possible

Slow cooling

DNA renaturation is possible through _____

260nm

Bases absorb light in the _____ wavelength region

does not change, increases

As the DNA is heated and the strands separate, the wavelength of absorption _____ change, but the amount of light absorbed _____

Hyperchromicity

As the DNA is heated and the strands separate, the wavelength of absorption does not change, but the amount of light absorbed increases

G—C

Melting temperature (Tm), midpoint of the melting curve, is higher when the percentage of _____ is higher

3.4 Å / 0.34 mm

Individual base pairs are _____ apart

RNA

• Single-stranded

• Consists of long, unbranched chains of nucleotides joined by phosphodiester bonds between the 3′ —OH of one pentose and the 5′ —OH of the next

• Pyrimidine bases are uracil and cytosine
  

β-D-ribose

Pentose unit of RNA

2-deoxy-D-ribose

Pentose unit of DNA

Replication

Yields 2 DNA molecules identical to the original one, ensuring transmission of genetic information to daughter cells with exceptional fidelity

Transcription

• The process by which the order of bases is passed from DNA to RNA

• The sequence of bases in DNA is recorded as a sequence of complementary bases in a single-stranded mRNA molecule

Translation

• The process by which the order of bases in mRNA specifies the order of amino acids in the growing protein

• 3-base codons on the mRNA corresponding to specific amino acids direct the sequence of building a protein

  • These codons are recognized by tRNAs carrying the appropriate amino acids

Splicing

The process of intron removal and exon joining

Transfer RNA (tRNA)

RNA Type

• Small, transports amino acids to site of protein synthesis

• Single-stranded polynucleotide chain between 73 and 94 nucleotide residues long

• Carries an amino acid at its 3′ end

• Where intrachain hydrogen bonding occurs

• Cloverleaf structure

  • Stems: H-bonded portion

  • Loops: Non-H-bonded portion (contain modified bases)

tRNA and mRNA

Both _____ and _____ bound to the ribosome ensures the correct order of the amino acids in the growing polypeptide chain

L-shaped conformation

tRNA folds into this conformation to produce the specific tertiary structure needed for tRNA to interact with the enzyme that attaches the amino acid

Ribosomal RNA (rRNA)

• Combines with proteins to form ribosomes

• Accounts for 60% to 65% of the total weight of a ribosome and the protein portion constitutes 35% to 40% of the weight

• Both prokaryotes and eukaryotes have ribosomes with two subunits (one larger than the other)

Analytical ultracentrifugation

Used to monitor the dissociation and reassociation of ribosomes

• Motion of particles is characterized by a sedimentation coefficient, expressed in Svedberg units (S)

Messenger RNA (mRNA)

• Least abundant type of RNA

• Directs amino acid sequence of proteins

• Initially formed as a larger precursor molecule called heterogeneous nuclear RNA (hnRNA)

• Carries coded genetic information from DNA to

  ribosomes for the synthesis of proteins

• Present in cells in relatively small amounts and is short-lived
  
  

Heterogenous nuclear RNA (hnRNA)

Precursor molecule of mRNA

3’

Complementary strand of mRNA is synthesized along one strand of an unwound DNA, starting from the _____ end

Introns

IxEntervening sequences that do not encode a protein

Exons

Protein-coding regions

Small nuclear RNA (snRNA)

• Recently discovered

• Found in nucleus of eukaryotic cells

• About 100 to 200 nucleotides long

• Complexes with proteins and forms small nuclear ribonucleoprotein particles (snRNPs)

Small nuclear ribonucleoprotein particles (snRNPs)

• Protein–RNA complexes found in the nucleus that aid in processing RNA molecules for export to the cytosol

• Help with processing (splicing) of the initial mRNA transcribed from DNA into a mature form

• Have a sedimentation coefficient of 10s

Cytosol

Most protein synthesis occurs in the _____

Small interfering RNAs (siRNAs)

• Short stretches of RNA have been found to have an enormous control over gene expression

• Used to eliminate expression of an undesirable gene

• Used in the study of gene expression

Micro RNAs (miRNAs)

• Control the production of many gene products

• Can lead to inhibition or activation depending on which mRNA is being bound

• Bind to mRNA and prevent its translation

• Used in the treatment of hepatitis C

Hepatitis C

Leading cause of liver cancer and liver failure that kills 350,000 people annually in the US

Miravirsen

RNA drug that binds to a liver miRNA (miR-122) designed to attack another miRNA

Small interfering RNAs (siRNAs)

• Bind to mRNA but lead to the cleavage of the RNA in question

• Used in the treatment of Ebola virus

Long noncoding RNA (lncRNA)

• Promising and controversial model for study for future therapies

• Ubiquitous in the cell

• Disruption of many of them lead to nonviable offspring in mouse models, and alterations in lncRNA are found in many cancer types

CRISPR (Cas)

• Clustered regularly interspaced short palindromic repeats

• Repetitive stretches of DNA found in bacteria and archaea

• Used to develop treatments for cystic fibrosis and sickle-cell anemia

Epigenetics

Refers to changes in DNA that are not reflected in the actual base sequence

Acetylation, methylation

_____ generally switches on gene expression, while _____ usually silences the expression