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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
if we unwind a region of DNA: (2 possibilities - how to relieve tension for latter?)
- 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
one DNA supercoil forms in double helix for every __ bp opened (B-DNA)
10 bp! (complete turn)
27
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)
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
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)
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
supercoiling calculations - twisting #
twisting # (T): crossing of one strand of dsDNA over the other; measures how tightly helix is wound
30
twisting # (T) formula
T = (total # bp)/(# bp/turn) - normal B-DNA would be 10bp/turn convention: - right hand helix = positive - left hand helix = negative
31
writhing #?
writhing # (W) = number of superhelical turns; refers to twisting of dsDNA axis in space (how many times the duplex DNA crosses over itself)
32
writhing # formula
writhing # (W): relaxed dsDNA - W = 0 negative supercoils - W = -ve positive supercoils - W = +ve
33
linking #?
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
linking # formula
L = T + W
35
local unwinding only happens when _____ ____ occurs
negative supercoiling
36
favourable energy state when it comes to supercoiling?
W = 0 (relaxed)
37
supercoil runs faster/slower on electrophoresis gel?
smaller, so faster!
38
how can L be changed?
by breaking one or both DNA strands, winding them tighter or looser, and rejoining ends = change W
39
what kind of value is L?
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
most cell DNAs are ____ supercoiled
negatively
41
negative supercoils store ___; details
energy energy of negative supercoils can be converted into untwisting (unwinding) of double helix
42
DNA overwound - ___ supercoiling; meaning?
positive - reduced chance for DNA-protein interaction
43
DNA underwound - ___ supercoiling; meaning?
negative supercoiling - negative supercoils store energy that could help strand separation - untwisting favoured (important for replication and replication)
44
topoisomerases are?
enzymes that recognize and regulate supercoiling and play important role in replication & transcription
45
Topo I
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
Topo II
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
gyrase steps (5)
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
what drives gyrase reaction
ATP hydrolysis (1 ATP is hydrolyzed first to rotate GyrB, 1 ATP is hydrolyzed second to reset gyrase)
49
overview of gyrase steps
- G strand is grasped and cut - T strand is moved through cut G strand - G strand resealed - 2 negative supercoils added
50
prokaryotic supercoiling enzymes (2 types, 2 specific)
+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
eukaryotic supercoiling enzymes
- 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
diff types/functions of RNA:
- 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
conformations of RNA
- 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
Uracil vs thymine
thymine has an additional methyl
55
secondary structure in RNA:
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
G:U, G:A in RNA
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
modified bases
- facilitate non-watson crick base pairing - found in tRNA bases
58
tertiary structure in RNA
- 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
additional RNA conformations (tertiary) (5)
- 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

BIOL 308 Molecular Biology (14 decks)

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