Module I Flashcards
What do the letters dNTP stand for?
Deoxyribonucleoside triphosphate (these are the precursor building blocks to nucleic acids/nucleotides
Which form of DNA is predominant in vivo?
B-DNA
What is the significance of the major and minor grooves of DNA? How are they formed?
The sugar-phosphate backbone of DNA isn’t spaced equally, resulting in a major and minor groove. The Major groove exposes the base letters, allowing binding proteins to “read” the exposed nucleotides. Specifically, the solvent-exposed N and O atoms in the nucleotides form H-bonds with the binding protein’s amino acid side chains, and the difference in the donor-acceptor patterns tells the binding protein which pair of nucleotides it’s attached to.
The width of the minor groove is dependent on which nucleotides are present in the surrounding DNA. For example, a short section of adenine (an A-tract) forms a narrow minor groove. The Minor groove is thought to be harder to read because the H-bond patterns are the same regardless of which way the base pair is flipped, meaning that the only difference in pattern would be between AT and GC pairs. However, there are some DNA-binding proteins that can recognize the minor groove, such as the TATA-binding protein (TBP), which binds at the TATA box and plays a role in initiating gene transcription in eukaryotes.
Why does G+C count affect the melting temperature of DNA?
Because G+C base pairs are held together with 3 H-bonds instead of the 2 found in A+T base pairs, it takes more energy to cleave the base pairs apart, and therefore a greater temperature during melting.
What are some other methods to denature DNA that aren’t heat?
Lowering salt concentration (this removes the cations that shield the negative charges on two strands from each other)
High pH (disrupts H-bonds)
Organic solvents (disrupts H-bonds)
Why does cooling time matter for renaturation?
If denatured single strand DNa is cooled rapidly, it will remain single stranded. However, if it is cooled slowly, the complementary sequences will find each other and form a new double helix.
What happens when we change the values of one of the variables in the Cot curve equation?
Cot Curve Equation: C/C。= 1/(1+tKC。)
This whole equation is really just a ratio. Increasing one variable makes all other variables increase.
Why could Cot ½ of one species be greater than that of another?
The larger the genome size, the longer it will take for any sequence to encounter its complementary sequence. So unless the two species in question have identical genome sizes, the Cot ½ of each species will be different.
Why do some species have Cot curves that follow the ideal model, while others vary?
In the graph of a Cot curve, the fraction of DNA that reanneals faster represents repeated DNA sequences. Because they are found more often in the genome, they have a greater probability of finding a matching sequence and reannealing faster. The remaining fraction of the genome reanneals more slowly because it is made of unique sequences and has a lower probability of finding its one matching sequence.
What is the significance of supercoiling in vivo?
Negative Supercoiling
Puts Potential Energy into DNA, making it easier to pull the helix apart during replication
Plays a role in replication, transcription, and recombination
Transitions between B-DNA and Z-DNA may be triggered by negative supercoiling
Positive Supercoiling
Found in thermophilic archaea, as it is more energetically stable and resistant to degradation at higher temperatures
Under what conditions does B-DNA form? What is its applications?
High humidity (95%)
Low salt concentrations
Predominant helical form in vivo
Under what conditions does A-DNA form? What is its applications?
Low humidity (75%)
High salt concentrations
Found in RNA and in vitro
Under what conditions does Z-DNA form? What is its applications?
High MgCL2, NaCl, or ethanol
Found only in the presence of methylated cytosine
Some evidence for in vivo, but is predominantly the product of bored lab technicians.
How does DNA denaturation depend on G+C content and salt concentration?
High G+C content → Increased melting temperature
Low salt concentration → Increased denaturation speed
How does melting affect relative absorbance at 260 nm?
Once the strands have been separated/melted, the UV absorbance at 260nm increased by 40%, a phenomenon called hyperchromicity.
What are the different types of unusual DNA secondary structures?
Slipped structure, cruciform structure, triple DNA helix, G-Quadruplex
How are slipped structures formed, where are they expressed, and how are they used?
Forms at tandem repeats in which misalignment leads to single stranded loops
Found upstream of regulatory sequences
Possible recognition sites for binding proteins
How are cruciform structures formed, where are they expressed, and how are they used?
Forms when AT rich regions unwind and form a stem-loop cruciform.
For a sequence to form a cruciform, it must be a palindrome, or inverted along its 5’ →3’ directionality
Found in vitro in plasmids and bacteriophages
May potentially be a regulatory element of replication and gene expression, but could also potentially lead to genetic instability and cancer
How are triple helix structures formed, where are they expressed, and how are they used?
Forms when a third strand intertwines
Found in mirror repeat symmetrical sequences
The third strand is either intramolecular (from the same helix) or extramolecular (from a separated DNA molecule)
Uses Hoogsteen base pairs between AT and GC to form
Requires a lower pH (between 4-5) to form
Found in Friedreich’s ataxia
How are G-Quadruplex structures formed, where are they expressed, and how are they used?
Four stranded structure formed in stretches of tandem guanines
Uses Hoogsteen bonds to form the planar G-Tetrad
Stabilized with a central monovalent cation.
Multiple G-tetrads then stack and interconnect with loops, forming the full G-Quadruplex
Found in telomeres and promoter regions
What’s the difference between a classic Watson-Crick G+C nucleotide pair and a GU Wobble?
A GU wobble uses 2 H-bonds, and the classic G+C base pair requires 3 H-bonds.
How does RNA differ from DNA in primary structure? What about secondary structure?
Primary → RNA uses uracil in place of DNA’s thymine
Secondary → RNA forms a single stranded helix, DNA forms a double stranded helix
One tertiary structure found in RNA is a tetraloop, in which four nucleotides form a loop. What kind of interactions stabilize this structure?
Hydrophobic and Van der Waals interactions, and base stacking
Why was the term ribozyme created? How is it different from an enzyme?
The classic definition of an enzyme doesn’t always apply to RNA-enzyme molecules. Enzymes never permanently change their shape and are used over and over ad nauseum. But RNA-enzymes often self-cleave, meaning that they are used only once. So a new term was created to describe these misfit molecules: ribozymes.
The Hammerhead Ribozyme is a ribozyme that self-cleaves at a phosphodiester linkage with the 2’ hydroxyl group acting as the attacking nucleophile. Why does this happen only in the Hammerhead Ribozyme? Phrased another way, why don’t all RNA molecules spontaneously self-cleave?
RNA molecules don’t spontaneously self-cleave because of the way the 2’ OHs are folded into the RNA’s 3D tertiary structure. The molecules simply aren’t close enough together to trigger this self-cleaving reaction. It’s only when the hammerhead ribozyme brings these two sites closer together that they can react and cleave apart.
What are noncanonical pairs, and why are they useful for the RNA molecule?
Noncanonical pairs are any nucleotide pairs outside the classic Watson-Crick AT GC binary. They are useful for the RNA molecule because they widen the major groove and make it more accessible to ligands.
How does the RNaseP RNA differ in E. coli and in humans?
E. coli → True enzyme, no chemical transformation
Humans → Protein-controlled ribozyme/catalytic RNP enzyme
The “RNA World Hypothesis” states that life first existed with RNA, and that only later did DNA and proteins evolve from RNA. What is the evidence for this hypothesis?
Proteins can’t replicate themselves
RNA has all the structural prerequisites for replication
RNA genomes are widespread among viruses
RNA can do everything proteins can do and more
What is coaxial stacking? List two examples where coaxial stacking can be seen in RNA.
Coaxial stacking occurs when bases of two separate stems stack, align, and form what ~appears~ to be a continuous helix, but they aren’t actually bonded to each other. They are pseudo-continuous. This can be found in tRNA. The stacked arms form its L shape.
Why does RNA need hyper specific binding proteins to create its secondary and tertiary structures?
Because RNA is negatively charged, formation of tertiary structure requires charge neutralization via either basic proteins, or monovalent or bivalent metal ions.
Also, only a few variations of secondary and tertiary structure in RNA leads to any sort of function. Therefore, the more binding proteins and chaperones the RNA has at its disposal to ensure it gets to a functional structure, the better.
Why do RNA genomes typically have a very high mutation rate?
There is a fitness trade off between replication rate and replication fidelity. For organisms with RNA genomes, their high mutation rate indicates that it was more evolutionarily advantageous to multiply their genomes quickly than it was to multiply their genomes accurately.
What structural features allow proteins to fulfill their functions?
The functional groups found on amino acids
What are some advantages of intrinsic disorder within proteins? What are some disadvantages?
Advantages → conformational flexibility lets the protein fulfill more than one function
Disadvantages → these proteins are more prone to misfolding and aggregation
Why is phosphorylation the most important part of post-transcriptional regulation? What are some of the things it can do?
Phosphorylation can:
Cause change in a protein’s shape
Mask or unmask a catalytic/active site or functional domain
Provide a recognition point or binding motif
Promote dissociation in multiprotein complexes
How do histone structures compare between archaea and eukaryotes?
Archaean genomes are more compact with fewer intergenic regions. Some have histones, but they are only found in a single histone fold domain, and they lack N and C terminal tail extensions. Additionally, DNA wraps only once around archaeal histones.
Why is mitochondrial inheritance almost always predominantly maternal?
The mitochondria of sperm cells is not located in the head of the cell. Because only the head of the cell enters the egg in fertilization, the paternal mitochondrial genome is not inherited in the offspring.
What are some atypical mitochondrial inheritance patterns and where have these patterns been documented?
mtDNA leakage from sperm
Some fungi, slime molds, and plants have biparental inheritance
Some land plants and algae have strictly parental inheritance
How does heteroplasmy impact mitochondrial diseases?
When not all of the copies of mtDNA in a mitochondria are identical, any harmful mutations within them are spread unevenly across the different copies of the mitochondrial genomes. If a greater percentage of those genome copies have the harmful mutations, the severity of the symptoms for the disease it causes will increase.
Compare and contrast the eukaryotic genome with the bacterial genome.
Bacteria have circular DNA that is condensed into a nucleoid. They also carry some plasmids, but those are replicated and transcribed separately from the genome.
How does DNA wrap around histones? (What kind of supercoils does the DNA form?)
DNA wraps around histones twice and restrains it into negative (lefthand) supercoils.
Major interactions between DNA and core histones are probably electrostatic. What evidence supports this hypothesis?
Histones can be removed from DNA by high salt concentrations.
Why do chromatin regions naturally repel each other, despite the presence of core histones, and how is this problem solved by cellular machinery?
The negative charges of DNA’s phosphate backbone are only partially shielded by the positive charges of core histones, making chromatin naturally repellent of itself. This issue is solved by using cations, linker histones, and miscellaneous positively charged proteins to shield the backbone’s negative charges
How does the DNA condense further after being wrapped into nucleosomes?
The condensin complex and ATP hydrolyzing enzymes further condense the chromatin.
What decides the location of the centromere on each chromosome?
The formation of a specialized chromatin structure, a nucleosome with a centromere-specific histone H3 variant called CENP-A or CenH3, decides centromere location.
DNA binding proteins gain access to the chromatin at three different levels of regulation. Name each of these levels, then provide at least one example of their regulation.
- DNA Sequence
- Wrapping of the DNA around the histone core octamer inhibits access to this part of the DNA sequence
- Wrapping of the DNA around linker histones allows access to the DNA sequence
- Segments within a chromosome can switch between different transcriptional states - Chromatin
- Changes in how the DNA is wrapped around the histones results in uncoiling and decondensation
- Nucleosome positions are determined partially by differing affinities for DNA sequences
- Can be displaced and recruited by competing or cooperating with other protein factors - Positioning of the chromosomes in the nucleus
- Nucleosome can be actively moved and displaced by remodeling complexes
In what type of DNA does gene silencing occur?
Heterochromatin
What is HP1⍺ and what characteristic of its structure allows it to fulfill its function?
HP1⍺ (heterochromatin protein 1⍺) spreads along long regions of the genome. Its intrinsically disordered regions promote phase separation which allow it to silence genes.
What is the C-Value paradox and what are some of its current accepted resolutions?
The C-Value paradox states that the amount of DNA in a haploid genomes doesn’t seem to correspond with the complexity of the organism. Some accepted resolutions are that most genomic DNA consists of various classes of repeats that originate from the replication of “jumping genes”, and that the majority of the human genomes is just intergenic regions (regions that aren’t under selective pressure, where mutations are maintained and transmitted, and that consist of unique and medium-to-highly repetitive sequences).
What does mtDNA typically code for?
Proteins responsible for oxidative phosphorylation and the mitochondrial electron transport chain.
What types of symptoms do mitochondrial diseases have and why? In carriers of these diseases, at what point are the symptoms expressed?
Symptoms include degeneration of the brain and muscles, muscle paralysis, and blindness, really any tissue that heavily relies on the mitochondria’s oxidative phosphorylation. In carriers of these diseases, symptoms are usually expressed once over 60% of the mtDNA is mutated.
Some examples of mitochondrial diseases include Leber’s Hereditary Optic Neuropathy and Kearn-Sayre Syndrome. How are these diseases inherited?
Maternally
What is heteroplasmy as it relates to mitochondrial and chloroplastic DNA, and how does it affect the functioning of these organelles?
Heterplasmy is when not all of the mitochondria or chloroplasts within a cell have the same genome. Some of the mitochondrial or chloroplastic genomes have mutations. The functioning of these organelles depends on the severity and magnitude of the mutations. Cell functioning is dependent on the total number of mutated mitochondria or chloroplasts and their genome’s severity of mutations.
Compare and contrast archeal genome organization with eukaryotic genome organization.
Archaeal genomes are more compact than eukaryotic genomes, with fewer intergenic regions. Some have histones, but these histones have only a single histone fold domain, lack N and C terminals tail extensions, and the DNA wraps only once around them. Additionally, where eukaryotic histones can only heterodimerize, archaeal histones can heterodimerize AND homodimerize.
Why can’t DNA polymerase initiate DNA synthesis de novo?
Because dNTPs are present usually only in low concentrations, DNA polymerases wouldn’t have evolved to bind to 2 dNTPs at the same time. Therefore, DNA polymerase can’t form the initial phosphodiester bond, and needs other cellular machinery to make that first bond in DNA synthesis.
What characteristics must primers have?
Nucleotide primers must be able to bind to the 3’ OH- group, so primers must have a free 3’ OH-. Primers must also be made of small RNA segments.
How does DNA polymerase replicate both the leading and lagging strands of DNA despite being able to add primers only in the 5’→3’ direction?
DNA replication is semi-discontinuous. The leading strand is synthesized start-to-end, and the lagging strand is synthesized in short, discontinuous fragments, and later glued together to form one long strand.
What are some differences in genome replication between bacteria and eukaryotes?
Bacteria
- One single, well defined replication origin
- Recruits helicase to the origin immediately after initiation proteins accumulate there
Eukaryotes
- Multiple replication origins
- Separate origin selection from initiation with a pre-replication complex, in order to prevent overreplicaiton of the genome
Origin sequences tend to be rich in which sequences? Why?
Origin sequences tend to be rich in AT sequences because they are bound with 2 hydrogen bonds, unlike GC sequences which are bound with 3 H-bonds. The smaller number of bonds requires less energy to break, making AT rich sequences energetically ideal to start replication.
Where are mammalian replication origins located?
In active or open chromatin, however much of the genome is capable of supporting low levels of replication initiation events. Mammals lack an easily identifiable consensus origin sequence.
What is the rate of genome replication dependent on?
The number of origins used and the rate at which they fire.
Topoisomerase I doesn’t create free ends when cleaving the phosphodiester bond between two adjacent nucleotides. How does it do this?
It binds to a circular DNA molecule with one negative supercoil and unwinds the double helix. Instead of creating fee ends, it becomes covalently attached to one of the two broken ends of the DNA. Which end depends on the specific enzyme. While winding the broken ends, the enzyme then passes the unbroken strand through the break and ligates the cut ends, thereby increasing the linking number of the DNA by one. The oxygen of the tyrosine hydroxyl group in the active site of the topoisomerase attacks a DNA phosphorus, forming a covalent phosphotyrosine link between the DNA and the enzyme, and breaking a DNA phosphodiester linkage at the same time. Rejoining of the DNA strand occurs by the reverse process. The oxygen of the free DNA 3’-OH group attacks the phosphorus of the phosphotyrosine link, breaking the covalent bond between the protein and the DNA, and reforming the phosphodiester linkage between adjacent nucleotides in the DNA chain.
What happens when gyrase is inhibited?
There is a decrease in replication initiation at the origin. This is because negative supercoiling makes it easier to separate the two strands of the double helix at the origin.
How do Type II topoisomerases work?
Once cut, the ends of DNA are separated and a second DNA double helix is passed through the temporary gap, also called a DNA linked “protein gate.” The DNA is then relegated. This reaction allows the Type II topoisomerases to increase or decrease the linking number of a DNA by two units and promotes chromosome disentanglement.
Example: Wrapping of the DNa around gyrase places one part of the DNA double helix, the T segment, over another part of the helix, the G segment. Upon ATP binding, one subunit of gyrase (GyrB) forms a dimer that captures the T segment, while at the same time the G segment is transiently cleaved. Hydrolysis of one ATP then allows GyrB to rotate, and the other subunit (GyrA) opens. The T segment drops into this opening and moves through the cleaved G segment. Religating of that G segment puts negative supercoils into the DNA. Finally, the T segment is released from the gyrase and hydrolysis of a second ATP resets gyrase back into its original shape.
