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BIOL 308 Molecular Biology > DNA Forms, Denaturation, Complexity, Supercoiling > Flashcards

DNA Forms, Denaturation, Complexity, Supercoiling Flashcards

1
Q

Cot analysis: rate of renaturation, re-association kinetics

A
  • rate of renaturation = measure of DNA/genome complexity
  • re-association kinetics = speed at which a ss seq is able to find a complementary seq + base pair

expect: increase in genome size = increase in complexity

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2
Q

Cot analysis variables

A

Co = starting conc (moles of nucleotide per liter)
t = reaction time (sec)
Cot(1/2) commonly used (50% done)

Note: Co is for ssDNA (since we start with ssDNA)

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3
Q

units of complexity are measured in terms of ____

A

nucleotides

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4
Q

complexity formula

A

Complexity = # of unique nucleotides + total # of nucleotides from one copy of each repetitive sequence

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5
Q

if two DNA sequences don’t have repetitive seq (unique) and have similar GC content, their genome sizes are ____ to their Cot1/2

A

proportional

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6
Q

Cot analysis steps

A

control DNA (known unique 100% complementarity) and unknown DNA
-> sheared (~200 bp)
-> denatured (w/ heat)
-> allowed to cool slowly (re-anneal)
-> sub-samples removed - ds & ssDNA measured (absorbance at 260 nm measured over time decrease during renaturation)
-> data plots plotted as a proportion of ssDNA (or %dsDNA) out of total DNA

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7
Q

0% reassociated (ds) DNA and 100% denatured (ss) DNA are ___?

A

the same! all still ss

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8
Q

Cot graph: how would organism A (some long unique, some identical bases) compare to organism B (shorter unique seq)

A

organism A: starts with faster renaturation rate, slows when unique seq are left
organism B: steady, moderate pace

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9
Q

general Cot curve: describe, identify purpose of seq types

A

highly repetitive: fast renaturation
- role unknown (centromeres, telomeres?)
moderately repetitive: middle renaturation
- some lack coding function, some code for diff gene families: globin genes, immunoglobulin genes, genes for tRNA and rRNA, etc (KNOW)
unique: slow renaturation
- mostly protein coding seq

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10
Q

reassociation is _____ _____ to genome size

A

inversely proportional

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11
Q

theoretically, if a genome does not contain repetitive seq, what is its complexity?

A

its genome size

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12
Q

Cot formula (2 steps)

A

Step 1: finding unique seq for test genome
- Cot1/2(known) / Cot1/2(test)
= size of known genome / x
- Note: Cot1/2 (test) value here is %genome that is unique

  • x = size of unique seq of unknown genome, which represents n% of whole unknown genome

Step 2: finding total genome size
- y/100 = x/n%
- y = total genome size

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13
Q

complexity variables: N & C?
what are the values for humans?
what is C-value paradox?

A

N = haploid chromosome #
C = DNA mass/haploid cell

humans are 2C and 2N

C-value paradox = no correlation between amount of DNA (genome size) and apparent complexity of organisms

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14
Q

prokaryotic genomes contain only ________ DNA.
how about eukaryotes?

A

non-repetitive
eukaryotic genomes vary in proportions of diff seq types

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15
Q

absolute content non-repetitive DNA ___ with genome size

A

increases

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16
Q

better definition of biological complexity

A

= size of functional and non-repetitive section of a genome

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17
Q

hypothesis: increase in size of unique part of genome is due to?

A

positive feedback mechanisms in evol
- already present genes (ex. proof reading) help establishment (survival) of new genes (bigger genomes grow faster)
- big genomes provide more options for recombinations and duplications, leading to new gene creation (bigger genomes grow faster)
- complex metabolic pathways and complex body structure (found in higher organisms) require more protein coding genes - question of efficiency (bigger genomes grow faster)

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18
Q

circular DNA is composed of?

A

two strands of DNA that form a closed structure without free ends = “double circle”

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19
Q

circular DNA are used in what kind of organisms?

A

prokaryotic genomic DNAs, plasmids, many viral DNAs, chloroplast, mitochondrion = circular

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20
Q

endosymbiotic theory

A

anaerobe ancestral “eukaryotic” cell phagocytosed bacterium, (1) mitochondria (2) chloroplasts, transformed into organelle

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21
Q

denaturation of circular DNA

A
  • two strands cannot unwind and separate like linear DNA
  • in vivo, NICKING occurs naturally during DNA replication
  • can be induced experimentally by using enzyme
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22
Q

structural similarities of circular and linear DNA, across structure order levels

A
  • primary structure of DNA: sugar-phosphate “chain” with purine and pyrimidine bases as side chains (ss)
  • secondary structure of DNA: double helical structure (hydrogen bonding between bases; stacking interactions; phosphate backbone “outside”) (ds)
  • tertiary or higher structure: double stranded DNA (both circular and linear) makes complexes with proteins - SUPERCOIL (coiling of a coil)
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23
Q

supercoiling - general intro, term - topological isomer, uses (3)

A
  • reduces stress on DNA by twisting/untwisting
  • topological isomers - DNA differing only in their states of supercoiling
  • important for packing DNA (condense - circular/linear)
  • DNA helix becomes topographically linearized (locally uncoiled) during replication and transcription
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24
Q

a circular DNA without any superhelical turn =?

A

relaxed

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25
Q

if we unwind a region of DNA: (2 possibilities - how to relieve tension for latter?)

A
  • if one end is “free”, we can just “untwist”
  • if both ends are “fixed” (in circular DNA and locally in long linear DNA)
    – strain is released by writhing into superhelical turns (supercoils)
26
Q

one DNA supercoil forms in double helix for every __ bp opened (B-DNA)

A

10 bp! (complete turn)

27
Q

situation: what happens if protein “opens” dsDNA -> two DNA strands behind opening become wrapped around each other LESS than every 10bp -

UNDERWINDING (turns are longer - more bases/new turn)

A

result: formation of NEGATIVE supercoils behind opening, loosening tension, causing B-DNA to start un-twisting/un-winding, increasing stress leading to negative left-handed supercoils

28
Q

situation: what happens if protein “opens” dsDNA -> two DNA strands in front of opening become wrapped around each other MORE than every 10bp -

OVERWINDING (turns are shorter - fewer bases/new turn)

A

result: formation of POSITIVE supercoils in front of opening; occurs when right-handed B-DNA is twisted really tightly about its axis, the double helix starts to distort & knot into positive right-handed supercoils

29
Q

supercoiling calculations - twisting #

A

twisting # (T): crossing of one strand of dsDNA over the other; measures how tightly helix is wound

30
Q

twisting # (T) formula

A

T = (total # bp)/(# bp/turn)

  • normal B-DNA would be 10bp/turn

convention:
- right hand helix = positive
- left hand helix = negative

31
Q

writhing #?

A

writhing # (W) = number of superhelical turns; refers to twisting of dsDNA axis in space (how many times the duplex DNA crosses over itself)

32
Q

writhing # formula

A

writhing # (W):

relaxed dsDNA - W = 0
negative supercoils - W = -ve
positive supercoils - W = +ve

33
Q

linking #?

A

linking # (L) = total # of times one strand of closed molecule of dsDNA encircles the other strand (integer)
- reflects BOTH T of native (secondary structure) DNA helix and W (presence of any supercoiling)

34
Q

linking # formula

A

L = T + W

35
Q

local unwinding only happens when _____ ____ occurs

A

negative supercoiling

36
Q

favourable energy state when it comes to supercoiling?

A

W = 0 (relaxed)

37
Q

supercoil runs faster/slower on electrophoresis gel?

A

smaller, so faster!

38
Q

how can L be changed?

A

by breaking one or both DNA strands, winding them tighter or looser, and rejoining ends
= change W

39
Q

what kind of value is L?

A

L is a a constant in unbroken duplex DNA, so many change in T must be accompanied by equal&opposite change in W (supercoiling)

40
Q

most cell DNAs are ____ supercoiled

A

negatively

41
Q

negative supercoils store ___; details

A

energy
energy of negative supercoils can be converted into untwisting (unwinding) of double helix

42
Q

DNA overwound - ___ supercoiling; meaning?

A

positive
- reduced chance for DNA-protein interaction

43
Q

DNA underwound - ___ supercoiling; meaning?

A

negative supercoiling
- negative supercoils store energy that could help strand separation - untwisting favoured (important for replication and replication)

44
Q

topoisomerases are?

A

enzymes that recognize and regulate supercoiling and play important role in replication & transcription

45
Q

Topo I

A

single stranded transient cut
- breaks DNA strand (Tyr residue attacks phosphate backbone), formation of phosphotyrosine bond
- passes other strand through break and reseal strand
- adds positive supercoil

46
Q

Topo II

A

double-stranded cut, pass a duplex DNA through it and re-seal cut
- G (gate) cut and T (Transfer), taken through gate, - segments are dsDNA

  • gyrase is a topo II enzyme
47
Q

gyrase steps (5)

A
  1. free DNA and gyrase subunits
  2. gyrase subunits join and DNA wraps around enzyme. The DNA T segment is placed over the G segment
  3. upon ATP binding, GyrB forms a dimer and captures T segment. G segment is cut
  4. hydrolysis of one ATP allows GyrB to rotate. GyrA subunit opens wide. T segment is transported through cleaved G segment
  5. religation of G segment introduces 2 negative supercoils in DNA. T segment is released and hydrolysis of second ATP resets gyrase
48
Q

what drives gyrase reaction

A

ATP hydrolysis
(1 ATP is hydrolyzed first to rotate GyrB, 1 ATP is hydrolyzed second to reset gyrase)

49
Q

overview of gyrase steps

A
  • G strand is grasped and cut
  • T strand is moved through cut G strand
  • G strand resealed
  • 2 negative supercoils added
50
Q

prokaryotic supercoiling enzymes (2 types, 2 specific)

A

+ve and -ve supercoiling essential in proks:
- Topo I - nicking-closing enzyme, makes transient cuts in one strand - relaxes negative supercoiling in prokaryotes (changes L# in steps of 1)

  • Topo II - relaxes positive supercoiling (ATP); makes ds-cut; pass a duplex DNA through it and reseals cut (changes L# in steps of 2)
    – works on ALREADY positive supercoiled DNA; doesn’t result in negative supercoils
  • Gyrase (one of bacterial Topo II) introduces negative supercoils
    – works on already relaxed DNA; does result in negative supercoils
  • Reverse Gyrase - discovered in hyperthermophilic archaea Sulfolobus; Topo I generating positive supercoils (ATP)
    – stabilizing genome at high temp (mutant is viable but significant growth defects at high temp)
    – protecting DNA strands to be together, promoted by exposing DNA to high temp
51
Q

eukaryotic supercoiling enzymes

A
  • TOPcc - active as topoisomerase cleavage complex
  • no seq preference
  • function in replication, transcription, repair, recombination - when topological problems arise
  • TOP1, TOP1mt: topoisomerase I action; relaxes both +ve and -ve supercoils; ss cleavage; TOP1 is found in nucleus; TOP1mt in mitochondria
  • TOP2alpha, TOP2beta: topoisomerase II action; relaxes both +ve and -ve supercoils (decatenate - break chains); nuclear and mitocondria
  • TOP3alpha, TOP3beta: topoisomerase I activity; only relaxes hypernegative supercoiling; requires Mg2+; TOP3beta can also act as RNA helicase
52
Q

diff types/functions of RNA:

A
  • mRNA - messenger RNA; specifies order of AAs during protein synthesis
  • tRNA - transfer RNA; during translation mRNA information is interpreted by tRNA (Aways in cytosol)
  • rRNA - ribosomal RNA, combined with proteins aids tRNA in translation
  • small RNAs - variety of regulatory functions
  • RNAs with enzymatic functions - ribozymes (in splicing and peptide bond formation during protein synthesis)
53
Q

conformations of RNA

A
  • primary structure of RNA similar to DNA
    – 2’ OH group prevents formation of B-helix; A HELIX is formed
  • RNA can be ss or ds, linear or circular
  • unlike DNA, can exhibit diff conformations
  • diff conformations (secondary and tertiary) permit diff RNAs to carry out variety of functions in cell
54
Q

Uracil vs thymine

A

thymine has an additional methyl

55
Q

secondary structure in RNA:

A

RNA molecules frequently fold back on themselves to form base-paired segments between short stretches of complementary seq (INTRA-strand hydrogen bonds)
- e.g., hairpin loop, internal loop, bulge
- secondary structures: areas of regular helices and discontinuous helices with stem loops or hairpins (more common in RNA)

56
Q

G:U, G:A in RNA

A

additional, non-Watson & Crick base pairing possible in RNA –>
- enhances potential for self-complementarity in RNA

G:A and G:U = noncanonical base pairs, permitted in RNA

57
Q

modified bases

A
  • facilitate non-watson crick base pairing
  • found in tRNA bases
58
Q

tertiary structure in RNA

A
  • formed through secondary structure interactions: lack of constraint by long-range regular helices
    – RNA has high degree of rotational freedom in backbone of non-base-paired regions -> can fold into complex tertiary structures
  • formation of TRIPLE base pairing is possible
  • PSEUDOKNOTS can form due to base-pairing between sequences that are not adjacent
  • telomerase RNA has “pseudoknots”
59
Q

additional RNA conformations (tertiary) (5)

A
  • A-minor motif (2 ssRNA interacting - 3 A’s that interact through minor grooves)
  • tetraloop motif (base-stacking interactions promote and stabilize tetraloop structure)
  • ribose zipper motif (H bonds between ribose sugars instead of bases - possible b/c donors and acceptors in sugars)
  • kink-turn motif
  • kissing hairpin loop motif (2 hairpin loops in RNA are “kissing” - hydrophobic interactions)
60
Q
A

BIOL 308 Molecular Biology (14 decks)

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