DNA Forms, Denaturation, Complexity, Supercoiling Flashcards
Cot analysis: rate of renaturation, re-association kinetics
- 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
Cot analysis variables
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)
units of complexity are measured in terms of ____
nucleotides
complexity formula
Complexity = # of unique nucleotides + total # of nucleotides from one copy of each repetitive sequence
if two DNA sequences don’t have repetitive seq (unique) and have similar GC content, their genome sizes are ____ to their Cot1/2
proportional
Cot analysis steps
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
0% reassociated (ds) DNA and 100% denatured (ss) DNA are ___?
the same! all still ss
Cot graph: how would organism A (some long unique, some identical bases) compare to organism B (shorter unique seq)
organism A: starts with faster renaturation rate, slows when unique seq are left
organism B: steady, moderate pace
general Cot curve: describe, identify purpose of seq types
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
reassociation is _____ _____ to genome size
inversely proportional
theoretically, if a genome does not contain repetitive seq, what is its complexity?
its genome size
Cot formula (2 steps)
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
complexity variables: N & C?
what are the values for humans?
what is C-value paradox?
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
prokaryotic genomes contain only ________ DNA.
how about eukaryotes?
non-repetitive
eukaryotic genomes vary in proportions of diff seq types
absolute content non-repetitive DNA ___ with genome size
increases
better definition of biological complexity
= size of functional and non-repetitive section of a genome
hypothesis: increase in size of unique part of genome is due to?
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)
circular DNA is composed of?
two strands of DNA that form a closed structure without free ends = “double circle”
circular DNA are used in what kind of organisms?
prokaryotic genomic DNAs, plasmids, many viral DNAs, chloroplast, mitochondrion = circular
endosymbiotic theory
anaerobe ancestral “eukaryotic” cell phagocytosed bacterium, (1) mitochondria (2) chloroplasts, transformed into organelle
denaturation of circular DNA
- two strands cannot unwind and separate like linear DNA
- in vivo, NICKING occurs naturally during DNA replication
- can be induced experimentally by using enzyme
structural similarities of circular and linear DNA, across structure order levels
- 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)
supercoiling - general intro, term - topological isomer, uses (3)
- 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
a circular DNA without any superhelical turn =?
relaxed
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)
one DNA supercoil forms in double helix for every __ bp opened (B-DNA)
10 bp! (complete turn)
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
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
supercoiling calculations - twisting #
twisting # (T): crossing of one strand of dsDNA over the other; measures how tightly helix is wound
twisting # (T) formula
T = (total # bp)/(# bp/turn)
- normal B-DNA would be 10bp/turn
convention:
- right hand helix = positive
- left hand helix = negative
writhing #?
writhing # (W) = number of superhelical turns; refers to twisting of dsDNA axis in space (how many times the duplex DNA crosses over itself)
writhing # formula
writhing # (W):
relaxed dsDNA - W = 0
negative supercoils - W = -ve
positive supercoils - W = +ve
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)
linking # formula
L = T + W
local unwinding only happens when _____ ____ occurs
negative supercoiling
favourable energy state when it comes to supercoiling?
W = 0 (relaxed)
supercoil runs faster/slower on electrophoresis gel?
smaller, so faster!
how can L be changed?
by breaking one or both DNA strands, winding them tighter or looser, and rejoining ends
= change W
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)
most cell DNAs are ____ supercoiled
negatively
negative supercoils store ___; details
energy
energy of negative supercoils can be converted into untwisting (unwinding) of double helix
DNA overwound - ___ supercoiling; meaning?
positive
- reduced chance for DNA-protein interaction
DNA underwound - ___ supercoiling; meaning?
negative supercoiling
- negative supercoils store energy that could help strand separation - untwisting favoured (important for replication and replication)
topoisomerases are?
enzymes that recognize and regulate supercoiling and play important role in replication & transcription
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
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
gyrase steps (5)
- free DNA and gyrase subunits
- gyrase subunits join and DNA wraps around enzyme. The DNA T segment is placed over the G segment
- upon ATP binding, GyrB forms a dimer and captures T segment. G segment is cut
- hydrolysis of one ATP allows GyrB to rotate. GyrA subunit opens wide. T segment is transported through cleaved G segment
- religation of G segment introduces 2 negative supercoils in DNA. T segment is released and hydrolysis of second ATP resets gyrase
what drives gyrase reaction
ATP hydrolysis
(1 ATP is hydrolyzed first to rotate GyrB, 1 ATP is hydrolyzed second to reset gyrase)
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
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
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
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)
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
Uracil vs thymine
thymine has an additional methyl
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)
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
modified bases
- facilitate non-watson crick base pairing
- found in tRNA bases
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”
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)
