Lecture 20

Question 1

Central Dogma

Answer 1

• DNA is the genetic material of all free living organisms
• the part of a DNA molecule that encodes the information required for producing a functional product is called a gene

Question 2

examples of DNA and RNA structure

Answer 2

primary structure - the nucleotide sequence
secondary structure - any regular, stable structure adopted by a segment of DNA, typically a base-paired double helix
tertiary structure - the 3D fold, the complex folding of DNA into bacterial nucleotides (supercoiled) or eukaryotic chromatin, or RNA into tRNA and other molecules
quaternary structure - ribosomes, spliceosomes, RISC

Question 3

DNA primary structure

Answer 3

• the nucleotide building block
• comprised of Phosphate + pentose sugar + base (purine or pyrimidine)
• nucleotide sequence
• phosphate is always on 5’
• since it only has a few residues (nucleotides) it is an oligonucleotide

Question 4

Explain the characteristics of the pentose sugar in ribose RNA and deoxyribose in DNA

Answer 4

• has a 5’ and 3’ end
• in RNA the 2’ carbon has a hydroxyl group (2’-OH) - important in RNA splicing reactions
• in DNA the 2’ carbon has a hydrogen
• the pentose in each nucleotide is attached to the base via its 1’ carbon
• the phosphate is attached to the 5’ carbon of the pentose
• the 3’OH of one nucleotide is linked to the 5’PO4 of an adjacent nucleotide to form the repeating sugar-phosphate backbone of DNA and RNA

Question 5

How can you distinguish between purines and pyrimidines

Answer 5

• purines are longer
• pyrimidines are smaller
• bases are highly conjugated aromatic ring structures
• called bases because some of their ring nitrogen’s can be protonated

Question 6

What type of bond does the pentose form with the base

Answer 6

• covalent bond via a beta-glycosidic linkage

Question 7

Deoxyribonucleotides

Answer 7

purine - adenine (base) - adenosine (with sugar)
- guanine (base) - guanosine (with sugar)
pyrimidine - cytosine (base) - cytidine (with sugar)
- thymine (base) - thymidine (with sugar)
- base + sugar = nucleoside
- base + sugar + phosphate = nucleotide

Question 8

Ribonucleotides

Answer 8

• differ from the deoxyribonucleotides
• have a hydroxyl at the ribose 2’ carbon (are not deoxy)
• have uracil base instead of thymine (uracil is like thymine but without the methyl group)
• uridine = uracyl + ribose (nucleoside)

Question 9

Characteristics of DNA and RNA primary structures

Answer 9

• phosphate groups link the pentoses in both DNA and RNA strands via a phosphodiester linkage/bond
• DNA/RNA strands are said to be asymmetric or polar, with a free 5’-phosphate at one end (5’ end) and a 3’ hydroxyl at the other end (3’ end)
• the phosphate group is an acid
• at physiological pH the phosphate group of every nucleotide within the DNA/RNA strand is deprotonated and carries a net negative charge
• because of these acidic phosphate groups (and despite having nitrogenous bases) DNA and RNA polymers are called nucleic acids

Question 10

How do bases interact and where

Answer 10

• interact by H-bond
• interact in the interior/core of double helix
• two anti-parallel strand can form base pairs

Question 11

DNA secondary structure - the double helix

Answer 11

• two DNA strands interact via hydrogen bonds between the bases: base-pairing (bp)
• together the strands form a twisted ladder, with a sugar-phosphate backbone forming the side of the ladder and the base pairs forming the rungs
• the strands run in opposite directions, one 3’ to 5’ and the other 5’ to 3’ antiparallel

Question 12

DNA base-pairing

Answer 12

• H-bond between the bases in the two DNA strands
• pairing is specific based on H-bond complementarity: guanine forms 3 H-bonds with cytosine, adenine forms 2 H-bonds with thymine
• high GC% is more stable 3 vs 2 H-bonds, more stable base interactions = more difficult to separate N strands
• these 2 types of base pairs predominate in double stranded DNA and RNA because they are the most complementary
• base pairing specificity - watson-crick base pairing (based on x-ray diffraction data)
• specific base pairing permits the duplication of genetic information because each strand is a template for its complementary strand

Question 13

G-C vs A-T

Answer 13

-GC interaction is stronger
- GC = 10.8
- AT = 11.1

Question 14

How are double helices held together

Answer 14

• base stacking
• base stacking between the hydrophobic bases minimizes their contact with water and stabilizes the double helix
• base stacking is a form of van der waals forces
• bases are slightly offset so they not directly on top of one another
• the bases lie in a plane almost perpendicular to the axis of the helix

Question 15

Major and minor groove

Answer 15

• the hydrophobic deoxyribose-phosphate backbones are exposed to the surrounding water and the hydrophobic bases are stacked on the inside of the helix nearly perpendicular to the backbone
• the offset pairing of the two strands forms a major groove (deep) and a minor groove (shallow) on the surface of the duplex
• base pairs more exposed to solvent on the major groove side than on the minor groove side

Question 16

Different forms of double helix

Answer 16

A form - dsRNA, DNA/RNA hybrid (right-handed) - bases more filled (more compact)
B form - dsDNA (right-handed), most common/dominant in all living forms
Z form - dsDNA, alternating pur/pyrim (left-handed)
- the structure of DNA proposed by W and C represents the sodium salt of DNA in a fiber produced at very high relative humidity
- if the relative humidity surrounding the DNA fiber is reduced to 75%, the sodium salt of DNA assumes the A form

Question 17

Nucleotide bases absorb UV light

Answer 17

• at 260 nm
• purines and pyrimidines are highly conjugated
• resonance among rings give most of the bonds a partial double-bond character, allows UV absorption
• nucleic acids are characterized by strong absorbance at a wavelength near 260nm
• aromatic aa absorb at 280 nm

Question 18

How can you distinguish between single-stranded and double-stranded DNA

Answer 18

• can use A260
• dsDNA helix can be disrupted by heating
• ssDNA can be disrupted by melting, base pairing and base stacking are disrupted (adding energy to disrupt weak bonds)
• melting occurs at a specific temp, Tm, which depends on the nucleotide sequence
• when the DNA is cooled, the strands reanneal (come back together) due to base complementarity (ssDNA -> dsDNA)
• stacked bases in nucleic acids (in a double helix) absorb less UV light than unstacked bases - absorbance is “quenched” when strands come together
• melting and annealing can be followed by A260

Question 19

Hyperchromic shift

Answer 19

• DNA melting (denaturation) and re-annealing (renaturation) can be followed by studying the hyperchromic shift
• dsDNA is held together by hydrogen bonds and base stacking interactions
• DNA strands can be melted apart by raising the temp or adding chaotropic (denaturing) agents like urea, and by removing salt
• both the formation and disruption of the DNA double helix are highly cooperative - the strands hold fast until the melting point Tm, and then rapidly let go

Question 20

What is the relationship between salt concentration and melting temp

Answer 20

• the stacking energy is more negative (more stable) for GC pairs, so Tm is higher
• the AT-rich regions melt first
• Tm is proportional to [salt] and sequence length
• high salt concentrations and longer sequences stabilize the duplex and increase the Tm
• salt ions shield the negatively-charged phosphates on the DNA backbones, which repel each other when unshielded, thereby stabilizing the ds structure
• [salt] increases, Tm increases