Chromosome of _______ are long, linear DNA molecules that are associated with ______.
Chromosome of most prokaryotes are ______ also genetic materials of some viruses and DNA of ______.
Plasmids are independent ________ DNA mostly found in ______, but plasmids are not considered to be part of their ______.
cannot very freely rotate
A circular DNA can be generated by ______ joining both ends of a DNA molecule. This is called _____ _____ ____ DNA. Circular DNA is also in ______ _____.
- topological constrict
- covalently closed circular DNA (cccDNA)
Twist number (Tw)
number of turns of one strand around the other strand, one twist per ~10base. (positive number)
Writhe number (Wr)
the number of turns of the double strand around itself
Opening the two strands of DNA, for example during replication causes _______ aka _____.
supercoil aka writhe
Supercoil can be positive or negative. How does each affect torsion?
- Positive supercoil increases the torsion
- Negative supercoil decreases the torsion
Linking number (LK)=
- If there is no writhe the cccDNA is called ______.
- LK= Tw + Wr
- relaxed (LK°=Tw+0)
If LK=Tw-Wr= an LK that is < LK° then supercoil is ______
- If LK=Tw+Wr= an LK that is >LK° then supercoil is ______
- ΔLK (linking difference)=
- ΔLK = LK - LK°
DNA can be long or short which changes he ___value to normalize it. In order to do this, we calculate the _____ _____ ____ (σ)
DNA in negative supercoil can be unwound (separated) easier than ______ DNA. Organisms keep the DNA in ______ ______. The _______ ______ living in hot springs have ______ supercoil, this prevents the ______ of DNA in they very high temp of the environment
- relaxed DNA
- negative supercoil
- thermophile bacteria
_____ _________ provide a solution to the topological problem.
The supercoil can be resolved by making temporary break in ________ ______ of one or both DNA strands. This is usually done by ________ _______. After the release of the supercoil (decreasing ___) the ________ _____ is restored.
- phosphodiester bonds
- topoisomerase enzymes
- decreasing LK
- phosphodiester bonds
Type I topoisomerase
introduce a break in one strand’s phosphodiester bond (a nick) and pass the second strand through the generated gap. The two ends of the broken strand are then religated.
Type II topoisomerase
breaks both strands of the double helix, creating a “gate” through which a second segment of the double helix is passed. Unlike topoisomerase I, activity of topoisomerase II requires the use of ATP.
Bacteria also have the enzyme ______ which is a type of topoisomerase. Instead of relaxing the DNA, _____ induces negative supercoils which is favorable for ____ ______ and _______.
- DNA replication & transcription
Two circular DNA can ______ with each other especially during the replication of circular DNA. Topoisomerase II can _______ (separate) them. If there is already a nick in one DNA, topoisomerase I also can _____ them
Linear chromosomes of ________ also can be entangled especially during ____ _______. It is important that ________ untangle the two newly made DNA from each other
- DNA replication
- topoisomerase II
In some cases, DNA can get tangled in knots, ________ can undo them. If DNA develops nicks, ________ takes care of it
- Topoisomerase II
- Topoisomerase I
Story #1 on how topoisomerase works
- One end of each cut polynucleotide becomes covalently attached to a tyrosine amino acid at the active site of the enzyme (held tightly in place) while the free end is being manipulated.
- This assures to rejoin the strand and prevents a permanent cut in the DNA.
- Then open hydroxyl on the other side of the cut attacks the phosphotyrosine
- This restores the bond and religating the two pieces of
- DNA together.
Story #2 on topoisomerase enzyme activity (lead with the three most basic steps then flesh them out)
- cleavage of DNA
- Strand passage
- DNA rejoining
- Topo binds to the melted cleavage of DNA
- phosphotyrosine formation (covalent bond of enzyme & DNA) and holding the other side of broken DNA strand.
- Change of enzyme conformation and moving the intact strand through the broken one.
- Change of conformation again and rejoining the two sides of broken DNA (no change in DNA sequence but change in supercoiling)
Topo I and Topo II have ______ mechanisms. However, in Topo II, the enzyme is a ______ or a ______ and makes the cuts on _____ strands
- the same
- dimer or a tetramer
circular DNA of the same size but different LK
______ _____ DNA moves slower in gel electrophoresis than ______ DNA. While _______ DNA moves the fastest
- Relaxed circular DNA
- linear DNA
- supercoiled DNA (the more supercoiled the faster)
EtBr _____ between bases and ______ them. EtBr _____ the twist between DNA. What is the approx value per base pair?
- ~36° to 10° per base pair
The value of Tw is _______ by EtBr
If EtBr is decreased we will have a ______ supercoil (Wr is ______) making it closer to ______. If EtBr is added, DNA becomes ______ supercoiled.
How does EtBr affect movement of cccDNA in the gel
- Negative supercoiled + EtBr =
- Less negative supercoiled + EtBr=
- Negative supercoiled + EtBr = more relaxed and slower movement
- Less negative supercoiled + EtBr = going
- beyond the relaxed to become positive
- supercoiled & fast movement
The building blocks of RNA are ______ to DNA. What can we say about their 2' carbons and methyls?
- Oh at 2' of sugar in RNA but just H in DNA's 2'
- There is an absence of a methyl on one base of the RNA structure
RNA is _____ stranded, but it can fold on itself and make ______ ______ _____ and ____ ______ ______. RNA conveys info from DNA to the _______ ______ (____)
- single stranded
- internal double strands & three dimensional structures
- translation machinery (mRNA)
RNA works as an ______ for amino acids in _______ _______ (___). RNA is the crucial part of ______, the translation machinery.
- protein synthesis (tRNA)
RNA contains modified _____ and is involved in unusual _____ ____
Some RNAs carry on _______ activities and some RNAs have ______ roles and control the _____ and _______ of some _____.
- regulatory roles
- level & translation
Three main differences between RNA and DNA
- Use of ribose instead of deoxyribose in the backbone (OH in the 2' position)
- Use of uracil instead of thymine. Lack of the methyl in position 5.
- RNA is single stranded but can fold on itself and make double strand structures within the molecule or it can make double strand structures with parts of other RNA molecules (important for many functions).
In RNA, _____ still base pairs with adenine the same way ______ does in DNA.
Only some ____ _____ have the double strand as their genetic material
RNA can fold back on itself and generate structures such as _____ & _____ (______), ______ _____, _______and _______ between few double stranded regions.
- stem and loop aka the (hairpin)
- internal loops
RNA base pairings between distant bases and involving more than one structure
What is the non-standard base pairing in RNA. How does it work?
- two hydrogen bonds between G:U
____ forms of base pairings can happen in RNA, but ___ & ___ are more common. There are local regions of self base pairing, but no long stretches of _____ _____ as seen in DNA
- G:U & G:A
- double helices
Why are RNA double helices most similar to A-DNA
The 2' OH causes wide and shallow minor grooves and narrow deep major grooves.
In the case of RNA double helices, the ______ groove is accessible to protein but not the _____ groove, and the ______ groove does not have much chemical information.
RNA double helix conformations are not well suited for ______ specific ______ of DNA.
sequence specific recognition
How is sequence recognition achieved in RNA double helices
the sequence recognition of RNA is achieved by relaying on the structure of hairpins loops, bulges, and distortions of non-canonical base pairs (e.g. tRNA)
State an example of how the sequence recognition achieved in RNA double helices
- A virulent gene of a pathogenic bacteria is not expressed outside of the body in low temperature (30⁰C)
- But upon infection, the gene (prfA) is expressed (in 37⁰C).
- The reason: at low temperature mRNA of the gene makes a loop preventing ribosome binding to its site on mRNA
- This leads to no translation
- However, at 37C the loop melts and ribosome binds to its site
- This leads to translation of mRNA and production of virulent protein.
In the unpaired regions, RNA backbone is very _____. There is a high ______ in rotation of backbone
Making three dimensional foldings usually involving ______ _____ ______ such as _____ ____ (_____) and ______ ______ interactions.
- unusual base pairings
- base triple (U:A:U)
In some cases ______ help for formation or stabilization of these 3D structures by shielding the ______ charges of the RNA ______. These 3D structures may have different _______ in equilibrium with each other
________ are short sequences of RNA that change their conformation upon binding with small molecules such as _______ (mostly in ______.)
Changes in the conformation of RNA causes the termination of ______. State an example (4-piece ans):
- In the absence of purines the genes for purine biosynthesis are expressed.
- In the presence of high levels of guanine, this base binds to the riboswitch and changes the conformation of the RNA
- This leads to termination of transcription
- As a result, purines will not be made.
RNA molecules that act as biological catalysts
State 4 characteristics of ribozymes
- similar to other enzymes
- have an active site
- substrate interacting domains
- binding sites for co-factors
Example of ribozymes
RNaseP, cleaving 5' end of pre-tRNA to generate mature tRNA
RNase P is a ______ but the _____ activity is carried out by the RNA part. The protein part only shields the ________ charges of the ____ ____ _____
- two RNA molecules
Systematic evolution of ligands by exponential enrichment: An evolution based system to generate small RNA molecules that can bind to a particular molecule or protein.
SELEX's generated RNAs are known as ______
- 1- generating a pool of 20 base RNAs with random sequences (4^20 permutations)
- 2- Selection of RNAs that interact with the molecule of interest by affinity chromatography
- 3- Recovery of the selected RNAs
- 4- Amplifying the selected RNAs and mutating them (by PCR)
- 5- Repeating the whole process with this amplified and mutated RNAs.
- The purpose of mutation is to improve the binding with the ligand (substituting some bases may improve the binding).
- After several rounds of SELEX the selected aptamers should have very high affinities for the ligand.
By SELEX method, it was possible to select RNAs, aka _____ that can bind to different ______. Each one would make a different _____ _____ upon interaction.
- color fluorophores
Give a more advanced example of a SELEX design (story)
- RNA can be designed that binds to a metabolite.
- Upon interaction of RNA with the metabolite A change in configuration of
- RNA occurs
- This leads to our aptamer gaining the ability to bind a fluorophore
- We achieve fluorescence (only if both molecules are attached to the RNA).
- This system can be used to monitor real time production of that metabolite within the cell.