lec 1.3 - review of DNA and RNA

DNA/RNA nucleotide composition

• sugar molecule - ribose (RNA) or deoxyribose (DNA)

• phosphate group

• nitrogen containing base

  • adenine - A

  • thymine - T

  • guanine - G

  • cytosine - C

  • uracil - U (RNA)

nucleoside

no phosphate (just sugar and base)

DNA/RNA bonds

phosphodiester bonds btwn 3’ OH from one n-tide and 5’ phosphate on next n-tide

DNA vs RNA strands

DNA = double strand

RNA = single stranded

• not always bcs complementary RNA strands can form double strand

backbone of nucleic acids

phosphate-sugar-phosphate-sugar, etc

how can the number of base pair types (A-T vs C-G) on a DNA molecule be determined?

• fewer H-bonds btwn A-T base pairs

  • will melt quicker as a result

• can start by using lower annealing temps to see how much of DNA molecule disintegrates at a lower temp

grooves of DNA

• major groove

  • where specific-base DNA binding proteins bind (because only place they can fit)

• minor groove

  • only general DNA binding proteins ine because they cannot see the bases (since groove is too small) so they must be general

how many angstroms is each turn in the DNA double helix?

34 angstroms

what bonds hold the two DNA strands together?

hydrogen bonds

three forms of DNA

• base pairs per turn of each

• B-DNA: most common, right handed

  • 10.5

• A-DNA: favoured in solutions devoid of water, not sure if it exists in cells

  • 11

• Z-DNA: left handed, evidence that it is found in small stretches of DNA

  • 12

benefit to Z-DNA’s structure

• grooves are bigger (even minor groove) therefore exposes bases better → easier protein binding

what is supercoiling and what does it do to DNA structure

• supercoiling - coiling of closed duplex DNA in space so it crosses over its own axis (twists back on itself)

  • affects the structure

• closed DNA is either circular DNA or linear DNA

  • ends are anchored so that they are not free to rotate

linking number

• closed DNA molecule has a linking number (L), which is the sum of twist (t) and writhe (W)

• L can be changed only by breaking and reforming bonds in the DNA backbone

twist and writhe

• t - property of the double helical structure itself, representing the rotation of one strand about the other

  • represents total number of turns of duplex and is determined by number of base pairs per turn

• w - in addition to this twisting, there can be extra twists or coils in the DNA called writhe

  • way to describe how much the DNA is "twisted" beyond its basic double helix structure

variation in DNA structure - backbone

• rotation around contiguous bonds that make up sugar-phosphate backbone

• nucleotides can either be linear (stacked on stop of each other) or puckered

sugar pucker of DNA

• A and B DNA differ bcs of sugar pucker

  • no water in btwn nucleotides in A-DNA allowing it to puker/sqeeze together

meselson-stahl experiment

• purpose

• what theories did they test

• used radioactive N to label parental DNA molecules and proposed what they would see in each of the scenario

  • N15 - heavy

  • N14 - light

• 3 theories about DNA composition

  • conservative

  • semi-conservative

  • dispersive

meselson-stahl experiment results

results show DNA exhibits

• showed one hybrid, and one N14 band therefore its not conservative or dispersive, must be semiconservative

semiconservative replication

• DNA replication accomplished by separation of the strands of a parental duplex, each strand then acting as a template for synthesis of a complementary strand

• sequences of daughter strands determined by complementary base pairing w/ separated parental strands

• replication fork

replication fork- point at which the parental strands are separated

nucleases

enzymes that degrade nucleic acids; they include DNases and RNases

2 categories:

• endonuclease - cleaves a bond within a nucleic acid (chews from within a strand)

  • example shows an enzyme that attacks one strand of a DNA duplex

• exonuclease - removes bases one at a time by cleaving the last bond in a polynucleotide chain (chews from the ends)

  • can be 3’ or 5’ end

RNA polymerase

• enzyme synthesizes RNA using a DNA template

  • formally known as DNA-dependent RNA polymerase

central dogma

• info cannot be transferred from protein to protein or protein to nucleic acid, but can be transferred between nucleic acids and from nucleic acid to protein

• translation of RNA into protein is unidirectional, can't be reversed (cannot go from protein to nucleic acid)

DNA can be unzipped and then re-zipped

• explain

• denaturation - involves the separation of the two strands due to breaking of hydrogen bonds between bases

• renaturation - re-association of denatured complementary single strands of a DNA double helix

  • aka: annealing

  • happens when temperature is reduced after being risen

melting temperature (Tm)

• is the midpoint of the temperature range for denaturation

• 50% of DNA has separated to 2 strands

• depends on the G-C content → can be used to characterize genomes

nucleic acid denaturation and renaturation/hybridization

• what molecules can be involved?

• condition?

• intramolecular or intermolecular?

DNA-DNA, DNA-RNA, or RNA-RNA combos

• they just need have complementary base pairing

• base pairing occurs in duplex DNA and also in intra- and intermolecular interactions in single-stranded RNA (or DNA)

complementarity test - filter hybridization

• ability of two single stranded nucleic acids to hybridize is a measure of their complementarity

  • can compare 2 genomes to look at overlap

c-value paradox

• observation that more complex organisms will not always need more genes than simple organisms

  • most likely explanation for the paradox is that less complex organisms have more DNA that does not code for genes

  • more complex organisms means more complex gene regulation (because our genes can make multiple proteins)