Study Questions Set 2 Flashcards
Thinking question re DNA/RNA structure: Certain chemical agents acting on DNA could convert cytosine to uracil through the process of deamination (chopping off the amino group). This mutation is routinely repaired by the existing repair mechanism (uracil is removed and it gets replaced by cytosine). Knowing this, how would you explain why DNA contains thymine and NOT uracil.
-it is important that DNA uses thymine because if uracil was used, the repair mechanism wouldn’t be able to differentiate between mutated cytosine and actual uracil that is important to the sequence of bases
How does complexity of bacterial genome differ from that of eukaryotic (calf) genome?
-E. coli genome has no repetitive sequences whole genome is one unique sequence
-calf genome has lots of repetitive sequences its unique sequences are more complex than E. coli however
A bacterial genome tends to have more complexity. This is because there are few non-repeating areas of the genome, while eukaryotic genome has many repeating parts.
Explain C value paradox.
-no correlation between the amount of DNA and the apparent complexity of organisms -> prokaryotic genomes contain only non-repetitive DNA
The C value paradox (where C = DNA/haploid) is the idea that there is no correlation between amount of DNA and complexity of an organism.
List and briefly explain factors that influence DNA renaturation kinetics.
- DNA concentration greater [DNA], greater chance of faster renaturation
- salt concentration ionic conditions mask the repulsion forces of phosphate backbone
- temperature melting temperature = temp. at which 50% of DNA is denatured high temperatures denature DNA
- time
- size of DNA fragment bigger the DNA fragment, more difficult is renaturation
- complexity simple sequences renature faster than complex sequences
You have found a new species of insects. To evaluate the complexity of the genome of this species, you isolate genomic DNA from, fragment the DNA to uniform 500 base pair pieces, denature the DNA and measure the rate of reassociation. Your data is represented in the curve below (sorry for the bad drawing): a) How many classes of DNA are found here? b) What can you say about the relative complexity?
a) 3 classes
b) fast -> not complex at all, very repetitive (25%)
medium -> somewhat complex (25%)
slow -> very complex
List 3 differences between prokaryotic topoisomerase I and gyrase
- Topoisomerase I relaxes negative supercoils; gyrase introduces negative supercoils
- Gyrase is a topoisomerase II, cuts double stranded DNA; topoisomerase I cuts one strand
- Topoisomerase I changes L in steps of 1, gyrase changes L in steps of 2
- Topo I passes ssDNA through a nick, topo II (gyrase) passes dsDNA through a cut
- Topo I uses ATP, Topo II does not
- Topo I uses tyrosine residues to break the bond
What are topological isomers of DNA?
TopoI – very similar to bacterial, can relax both a positive and negative supercoils.
TopoII – only relaxes supercoiled DNAs, cannot induce supercoils. Work on both positive or negative.
Explain the role of DNA supercoiling in cell survival?
i. important for packaging of DNA, it allows DNA to be more condensed. one use of this is in heterochromatin.
ii. RNA polymerase will transcribe anything it will get its lil hands on, so it’s important to hide the DNA so it can’t be transcribed
iii. coiled structure protects from damage
iv. negative supercoils release tension when DNA is coiled/uncoiled
The DNA must supercoil in order to allow for cell survival because there is tension created as the DNA unwinds. The DNA must be able to unwind to allow it to express genes, as well as replicate. In gene expression, where portions of the DNA are fixed, it allows for the relaxation.
There is also supercoiling of bacterial DNA, and this is slightly different, where there is supercoiling in “threads” around the a protein core, and a nick allows the supercoils to become relaxed
What is the difference between primary, secondary, and tertiary structures of DNA/RNA?
Primary = the linear bonding. Primary is just the formation of phosphodiester bonds with the nitrogenous bases. Primary RNA vs DNA:
• RNA = CGAU
• DNA = CGAT
Secondary for RNA:
• Triple bond
• Folding back on self
• GU = non canonical BP
• Helices, stem-loop structure
2o for DNA:
• Helical structure, H bonding between AT and GC, stacking interactions, salts
• Triple helix
• Cruciform, slipped structures
3o RNA vs DNA
• Interactions of 2o structures
• Lack of constraint through long-range structures means there is dynamic 3o structure in RNA
• RNA has pseudoknots, where there are loops that are looped. 2 stem-loop structures are intercalated between the halves of another stem.
• DNA has supercoiling. Introduced to reduce tension, and packing of DNA. It is impotant to condense the DNA. Exhibited during gene expression/DNA rep. it is genetically “cheaper”/more efficient to use bend long DNA than to untwist. It’s a situation in long circular DNA and locally in long DNA with fixed DNA. When the fuck does DNA have fixed ends? jOne DNA supercoil will form for every 10bp opened (the length of one DNA turn).There are 2 types of supercoils: positive and negative. Positive supercoils are less common, when the right-handed B DNA starts to twist more than it should, when there’s overwinding, the double helix will distort and knot into a positive right handed supercoils. This is different from negative supercoils, where a supercoil forms less than once every 10 bp. It is left handed, and the most common type of supercoil.
• DNA 3o structure includes hella proteins. Does RNA’s 3o tertiary structure include proteins and domains?
You have a small 4800 bp long circular DNA. It has a linking number of 450 (L=450). What are the twist (T) and the writhe (W) of this DNA? What assumptions about the structure of the DNA have you made in your answer? (Hint: what is the definition of the twist #?)
T = twisting number. It is the crossing of one strand of DNA over the other. It is a measure of how tightly the helix is wound. It is calculated through:
T = total # of BP/ # of BP per turn.
T can be positive or negative. Positive if right handed. Negative if left handed.
W = Writhing number. This is the number of superhelical turns, so how many times the duplex DNA crosses over itself. Can be negative or positive, reflecting positive or negative supercoiling.
L = linking number. It is the total number of times a close molecule of dsDNA encircles the other. Reflects both the T and the W. it can only be changed by breaking one or both strands of DNA, winding it tighter or looser, and then rejoining the ends. It is a constant unbroken DNA, so any change in W must reflect a change in T, and vice versa.
Okay, on to the question: L = 450. T and W = ? T = 4800/# of turns/bp. In order to solve this, the number of turns /bp must be assumed as 10 for a normal DNA. 4800/10 = 480. L = T + W. W = L – T = 450 – 480 = -30.
Thinking question 1: Many different mutations have been observed in almost all genes. However, only few have been isolated in histones. How would you explain this finding?
Let’s talk histones. We have to consider how the fuck we take a massive euk DNA and compact it into a tight little ball, right? So we know that each chromosome has a single, linear DNA molecule. Chromatin is DNA and the associated proteins involved in packaging. A chromatin fiber has a basic unit: the nucleosome. Each nucleosome has a core particle of histone proteins which are wrapped by DNA. We found this out by doing a partial digest by DNaseI. The size of the linker variable depends on DNAse I concentration. Aka, the less DNase I, the greater the size of the linker variable.
Okay, now let’s really talk histones. These are present in all eukaryotic nuclei. These are small proteins rich in argenine and lysine, and at normal pH, they are basic proteins. They will interact with DNA through electrostatic interactions. There are 5 major types: H1, H2A, H2B, H3, H4. Everything but H1 work in concert to form an octet that DNA will bind to. So, when I said nucleosome before – this is the smallest unit of a chromatin fiber, I was talking about a an octet of histones and wrapped eukaryotic DNA. So the eukaryotic DNA is wrapped around this octamer. Remember earlier we discussed that thing about supercoiling, how there’s restrained and unrestrained supercoils? Well this becomes an example of restrained supercoils, where you have supercoils wrapped around these basic histones (rich in lysine and argenine), thereby reducing tension of the supercoil. What happens to H1, you ask? Well it’s going to associate with that DNA and octet region in something called a “linker region”, which binds two distinct regions in the DNA duplex.
Okay, now that that’s established, let’s get back to the questions: why have there only been a few isolated mutations in histones? Well, histones are so fucking important. If there’s a mutation, it’s likely the cell would just rather go through apoptosis, rather than fuck around with the organism.
@ anoja please separate the previous answer into different questions, that answer is huge and overkill for the question
@ anoja please separate the previous answer into different questions, that answer is huge and overkill for the question
What are some of the distinctive features of eukaryotic chromosomes? (note: I expect you to first define chromosomes and after that you have to briefly explain nucleosomes/histone proteins/octet +H1/wrapped DNA, different levels of chromosome condensation, centromere and telomere regions)
Chromosomes = all the DNA and its associated proteins in a cell. Chromatin fibers are a form of condensation, where its most basic unit is the nucleosome, which consists of 4 histone proteins (H2A, H2B, H3, H4). These four histone proteins will allow for the wrapping of DNA around them. Euk DNA will wrap around an octet, as each histone forms a dimer, and then associate with each other. This means that the DNA can condense itself and have restrained supercoiling, which involves no tension. This is because histones are rich in lysine and argenine, and are basic molecules at a normal pH. The next level of chromatin condensation involves the 5th histone protein, H1. This attaches to the nucleosome, establishing a linker region. This allows for further condensation, promoting 25-100 fold condensation.
Chromatin, of course, can be uncondensed. Uncondensed chromatin is still wrapped around histones, but the histones are not packaged so tightly. These are euchromatin. heterochromatin is very tightly bound, and unable to undergo gene expression.
There 2 types of heterochromatin – facultative and constitutive. Constitutive is highly condensed, it consists of repetitive DNA, and there are very few different genes. Telomeres are an example of this. As are centromeres. Facultative heterochromatin is different in that it is how the genes are regulated between cells. Like, a liver cell would have brain cell genes expressed as heterochromatin. It forms under specific circumstances to silence gene expression. This is regulated through: DNA methylation (which will inhibit gene expression), and parent to offspring epigenetics.
Okay, so we’re still talking about chromatin. We now establish the existence of: chromatin elements! There are going to influence gene expression pretty heavily. The first one we’ll start with are locus control regions. This is essentially an ordered sequence of genes that share a singular control region that upstream from gene clusters. And these will control the condensation of genes. The next one we’ll discuss is scaffold-associated regions – we’ve touched on scaffolds before. So, scaffold (or matrix) is what the chromatin loops used to bind to, and the scaffold-associated regions are the AT rich regions on the chromatin that are used in order to bind to the scaffold. AT makes a lot of sense because we should be able to easily condense and uncondense in order to go through gene expression. Next, we’re covering insulators. So far we’ve discussed locus control regions, scaffold associated regions, and now we’re touching on insulators. These are regulatory domains in the DNA that define domains of gene expression. Essentially, they’re used to separate the genes.
Okay, so before we discussed centromeres being constitutive heterochromatin. If we associate centromeres with its respective proteins, we get a structure called the kinetochore.
Telomeres were also mentioned as heterochromatin. These are specialized regions at the end of chromosomes, while centromeres were (fittingly), at the center. Telomeres are going to protect the chromosome from degradation and shortening during replication through looping of the 3’ overhang – a t-loop. A great diagram is attached below:
There’s also the use of nonhistone proteins here. There is the attachment of these chromatin fibers into nonhistone protein complexes, also known as scaffold/matrix. This allows for looped domains to maintain their shape, as they kind of look like worms attached to a floor. Those scaffold attachment regions (SARs) are also known as matrix attachment regions (MARs). I explained before that MARS and SARs are AT rich – I’d like to clarify now that there’s no consensus sequence attached to M/SARs. It’s just AT rich. Topoisomerase II (which we talked about earlier, responsible for chromatin packing), is pretty relevant to this conversation we have about M/SARs. For this reason, we have an attachment site for topoii.
Another nonhistone protein we’ve got going on is one that works constantly on chromosome repair. SMC – structural maintenance of chromosome. These are a family of ATPases that participate in higher order chromatin structure.
DNA replication proteins – do pretty much exactly what it says on the bottle!
TFs, chaperone factors. All of these are used in chromatin organization.
that previous answer was so long and detailed please make it into different questions
that previous answer was so long and detailed please make it into different questions
What is unusual about the amino acid composition of histones? How is the function of histones related to their amino acid composition?
Histones are rich in lysine and arginine. These make them highly basic and positive, able to remove tension while the DNA wraps around the histones.
Okay, we’re going to talk about histone proteins again.
• All highly conserved, esp H4
• H1, the linker protein, is the one that’s the least conserved
• H5 is a protein present in fish/amphibians/reptiles/birds. It is an extreme variant of H1. So really 6 histone proteins, but only 5 present at one time in a cell.
• Centromeres have their own version of H3 – known as CenH3.
