Chapters 8 and 9 Flashcards
1
Q
- What are the 3 components of nucleotides?
- What are the 2 components of nucleosides?
- Which bases are Purines and which bases are Pyrimidines?
A
- Nitrogenous base + pentose + phosphate
- Nitrogenous base + pentose
- Purines: Adenine and Guanine, Pyrimidines: Cytosine, Thymine, and Uracil. Complementary bases pairing helps w/ stability, folding, and structure.
2
Q
- What separates DNA form RNA in terms of their base structure?
- Is the ring puckered or planar? What the most important positions in the rings?
- What does this puckered or planar structure help with?
A
- The two pentoses are closed ring forms (furanoses)
- 2’-deoxy-D-ribose is DNA
- D-ribose is RNA
- The ring is puckered. Significant in complex structures. The most important positions in the rings are the the 2’, 3’ and 5’ positions.
- Helps with flexibility and stacking.

3
Q
- What is different about minor bases?
- What nucleotides have additional rings?
A
- Most are methylated forms of the major bases.
- cAMP and cGMP
4
Q
- What type of bonds are nucleotides joined together by?
- What kind of linkages does the backbone have?
- What are oligonucleotides?
A
- Phosphodiester bonds 5’ phosphate + 3’ hydroxyl
- Creates a backbone
- pentose-phosphate-pentose-phosphate: Ester-linkages at the 3’ and 5’ positions.
- Up to 50 bases. 3’-5’ allows for the double turn of DNA.

5
Q
- What part of the molecule is hydrophilic? What group forms the h-bonds?
- What charge does phosphate have at 7.0 pH?
- At what maxima is UV light absorption for nucleotides?
A
- The backbone is hydrophilic, bases are hydrophobic. Pentose hydroxyls form hydrogen bonds with water.
- Negative, because phosphates are completely ionized at pH 7.0.
- 260 nm

6
Q
- Are bases hyrdophobic or hydrophilic?
- What is the reason for h-bonding between the bases of two different strands?
- Why do bases stack?
A
- Bases are hydrophobic, relatively insoluble in water.
- The reason for h-bonding and the bases being hydrophobic is to stabilize binding between two or more strands. (A:T 2 H-bonds) (G:C 3 H-bonds)
- Bases stack to minimize contact with water.

7
Q
- What method did Rosalind Franklin play in helping with the identification of the structure of DNA?
- What was the major observation?
- What were the other observations?
- What are some important features of DNA?
- Structure leads to function: what two grooves are present on the DNA helix?
A
- x-ray diffraction of DNA crystals.
- DNA is a helix with about two periodic sequences.
- Phosphate is on the outside of the helix and DNA has 1-3 strands.
- DNA is a double right-handed helix. Base-pairing is complementary. Backbones are on the outside (phosphate and sugar). Bases face inward. Strands are H-bonded together. Crick deduced that strands are anti-parallel.
- Major and Minor grooves

8
Q
- What kind of mutations does Ethidium bromide cause?
- How can we make this molecule safer?
- What molecule can we use instead and why doesn’t it cross the membrane?
A
- Frameshift mutations
- Ensure it cannot contact DNA, binds to the DNA and causes a frameshift.
- We could use gel red, it does not cross the membrane because it is big and insoluble.

9
Q
- What bonds does DNA rotate about?
- What does thermal fluctuations and the presence/absence of water produce?
- What does this DNA flexibility result in?
A
- Rotation about sugar-phosphate and glycosyl bonds.
- Bending, stretching and melting (broken H-bonds)
- Results in different DNA structures. Natural changes in bonds result in different structures.
10
Q
- What is A-form DNA favored in?
- What is B-form DNA favored in?
- Describe Z-form: Where is it found and what does it play a role in?
A
- Favored in anhydrous solutions. Favored by DNA-RNA hybrids and RNA alone.
- Most stable in biological systems for randome sequence DNA. Called “Watson and Crick” DNA.
- Certain base sequences and high salt favor this form. A left-handed helix, a more elongated than A or B, almost no minor grooves, flat major groove.
- Short stretches found in eukaryotes and prokaryotes.
- Plays a role in gene regulation.

11
Q
- What do four or more Adenosine residues lead to?
- What is a palindromic sequence?
- What are inverted repeats? What type of structure can they lead to?
- What are mirror repeats?
A
- A tight bend in the helix.
- The same sequence forward and backward.
- Occur over 2 strands of DNA.
- Self Complementary: Hairpins and cruciforms. Proteins recognize these loops and turn genes on.
- Occur often on the same strand.

12
Q
- What is Triplex DNA?
- What is Tetraplex DNA?
- What do they play a role in?
A
- B-form of DNA makes additional H-bonds in Major Groove.
- Special H-bonds: Hoogsteen Positions
- Non-Watson-Crick pairing: Hoogsteen Pairing
- 4-strand DNA where there is a high proportion of Guanosine residues, very stable
- DNA replication, recombination, and transcription.

13
Q
- What are the three RNAs?
- What is siRNA (iRNA or RNAi)? What does it do, and how can it be used?
A
- rRNA, mRNA (carries genetic information, DNA to ribosome), tRNA (translates information coded in mRNA into protein.
- Silencing RNA
- Long dsRNA “diced” into siRNA
- siRNA unwinds and becomes ssRNA.
- Binds to mRNA to inhibit translation via endogenous endoribonuclease activity.
- Can be used as a tool to manipulate gene expression.
14
Q
- What is always the product of transcription?
- What kind of helix does it form?
- After transcription, what can complementation produce?
- What form of helix does this product produce?
- What does the final structure of RNA form? What interactions play a role in stabilizing RNA?
A
- A single stranded RNA molecule.
- Right-handed helix.
- Double stranded RNA.
- Complementary strands tend to produce the A-form helix, B-form is not observed.
- Its final structure is complex with many forms. Weak interactions play a role in stabilizing the RNA.

15
Q
- What is DNA at room temperature in aqueous solution?
- What temperature does DNA melt at?
- What kinds of bonds are broken?
- What is disrupted?
- What happens when DNA is unwound and strands separate?
- Does each species have a unique DNA denaturation temperature?
- What would drive the denaturation temperature up?
A
- Viscous
- 80°C and pH extremes
- H-bonds are broken
- Base stacking is disrupted.
- Can be partial or complete, only change is in conformation. Function is not lost.
- Yes, each species has a characteristic denaturation temperature.
- The higher the GC content, the higher the denaturation temperature, due to H-bonding.

16
Q
- Sometimes during Renaturation (Annealing) it happens in one, rapid step. What could this be due to?
- What are the two steps of renaturation if strands are completely separated?
A
- If some small parts of the two DNA strands are still attached, annealing occurs in one step.
- 1st step is slow, strands randomly collide. 2nd step is fast, strands zipper.

17
Q
- What can Denaturation-Renaturation reveal?
- Give an example
- What does the relative number of Hybrid Duplexes reveal?
A
- Evolutionary History
- Melt human and mouse DNA, some sequence anneal together over a few hours.
- Sequence Homology
18
Q
- What is a Southern Blot used for?
- What is the process?
- What is PCR used for?
- What is the process?
A
- Used to show a DNA sequence is present. Others indicate a gene is transcribed and translated.
- Process
- Separate DNA segments by size in a gel.
- Transfer separated segments to membrane.
- Identify segment(s).
- A specific sequence of DNA can be replicated many times using a simple set of reactions
- Heat to denature
- Bind oligonucleotide primers to the specific sequence.
- Add heat-stable DNA poly
- Duplicate the DNA sequence between the primers.
- Repeat the steps above 20-30 times.

19
Q
- What is DNA microarrays used for?
- What is the process?
A
- Identifies gene sequences in a whole genome or plasmid.
- Quantitative
- May identify several sequences on the same array at the same time.
3.

20
Q
- What is Deamination?
- What does deamination of Cytosine result in? How often does it occur? How is it repaired?
- What does deamination of A and G result in? What is it caused by? How often does it occur? How can it be prevented?
A
- The loss of an amine group.
- Results in Uracil
- 1 in 107 per cell per 24 hours.
- Repaired by excision of mutated DNA.
- A to hypoxanthine and G to Xanthine
- Caused by nitrous acid and bisulfite.
- 1 in 105 per cell per 24 hours.
- Prevent bacterial growth in the food industry.

21
Q
- What is Depurination?
- What is the rate of loss?
A
- Hydrolysis of the N-β-glycosidic linkage between the sugar and base.
- Rate of loss is 105 purines per cell per 24 hours.

22
Q
- What does Ultraviolet light result in?
- What does Ionizing radiation and X-rays result in?
- What does oxidative damage drom hydrogen peroxide, superoxide radicals and hydroxyl radicals result in?
A
- Thymidine dimers on the same DNA strand.
- Open ring structures and caused covalent bond breaks in DNA backbone.
- Many alterations in DNA.

23
Q
- What are some other functions of nucleotides?
- How do they operate as cofactors? What happens if you remove adenosine?
- What is a Nucleotide-binding fold? What is it specific for?
- What else may they help with?
- What is The Rossman Fold? What does that area bind? What alternates in this structure?
A
- Chemical energy sources (Phosphate covalently linked to the 5’ hydroxyl of ribonucleotide.
- Adenosine is present in all of the important ones.
- Adenosine does no participate in the chemical reaction.
- Removal of adenosine reduces cofactor function.
- A domain present in many enzymes. Specific for adenosine nucleotides over others.
- May assist in substrate/active site induced fit.
- A binding motif or super-secondary structure. Area of a protein that binds nucleotides with specificity (As a Motif, they are a pattern of secondary structure found in many proteins. Alternating beta strands (6) and alpha helices (4).

24
Q
- Can nucleotides act as electron carriers?
- What do the Nitrogens in Riboflavin do?
- Nucleotides as Regulatory molecules?
A
- Yes!
- N picks up an electron and a proton, and another does the same, for a total of 2 e- and 2 protons.
- Can act as signal transducers and second messengers.

25
Q
- What is the overall goal of the Sangar method?
- What do we prepare the 4 tubes with?
- Why can’t ddNTPs covalently bind another NTP at 5’ position?
- How do we separate labeled fragments?
- What is the result?
A
- To obtain 4 sets of complements of original DNA. Each set is labeled at a different position of 5’ end.
- DNA as a template, All 4 dNTPs, DNA Poly, DNA primer. After add a small amount of ddNTPs
- There is no 3’ hydroxyl, as a result DNA polymerization stops at positions with that complementary base. Fragment sizes correspond to relative position of 1 base in sequence.
- Separate with by electrophoresis.
- As as result, a pattern forms in each lane for each of 4 bases.

26
Q
- What does restriction endonucleases types 1 and 3 do?
- What does restriction endonucleases type 2 do?
- What does DNA ligase do?
A
- Random or cut at 25 bp from recog. sequence. ATP dependent
- Cleave phosphodiester bond at very specific sequences. Produce sticky or blunt ends. Use recombinant DNA tech.
- re-form the phosphodiester bonds.

27
Q
How do we get the DNA into the cloning vector? How does it make many copies?
A
