Midterm 1 Flashcards
Ace this midterm! (147 cards)
1
Q
Central Dogma
A
The flow of genetic information goes from DNA to RNA to Proteins
2
Q
Beadle and Tatum
A
They worked with bread mold and showed that each gene controlled a single protein (one gene: one enzyme hypothesis)
3
Q
Griffith Experiment
A
The lethal strain S has a smooth capsule that allows it to evade the host’s immune system. The nonlethal strain was called the R strain. Griffith showed that the genetic material from the heat killed S strain turned the R strain into lethal cells.
4
Q
Griffith’s conclusion
A
The S strain was able to transform the R strain into a virulent strain.
5
Q
Avery, Macleod, and McCarty
A
They took Griffith’s experiment and did it in vivo. Then, they treated cells with DNase, RNase, and Proteinase to see which would affect the transforming ability. Since DNase prevented transformation, they concluded that DNA is responsible.
6
Q
Hershey Chase Experiment
A
Used S and P radioisotopes to label the protein capsid and DNA of bacteriophage. They saw that most of the P was in the pellet where the cells are while the S was in the supernatant. This concluded that bacteriophages inject their DNA into the host cell and only this genetic info directs the creation of more progeny. DNA = genetic info!
7
Q
Building blocks of DNA
A
Nucleotides!
Purines: Adenine and Guanine
Pyrimidines: Cytosine and Thymine
8
Q
Polarity of DNA
A
Polarity comes from phosphodiester linkages, and has a net negative charge.
9
Q
Chargaff’s rules
A
G and C are in same amount and A and T are in same amount
10
Q
Rosalind Franklin and Maurice Wilkins
A
Performed X-ray diffraction studies on DNA and their data showed that DNA was in helical form with “ladder-like” rungs connecting parts of it
11
Q
Watson and Crick
A
They proposed 3D structure of DNA to be double helix
12
Q
Structural characteristics of DNA
A
Has sugar phosphate backbone, bases project inwards, one turn is about 10.5 bp, has major and minor grooves.
13
Q
Mica experiment
A
DNA has about 10.5 bp per turn in solution
14
Q
Gel Electrophoresis
A
Separates DNA molecules according to their weight. DNA travels to positive side because it is negatively charged
15
Q
Ethidium Bromide
A
Intercalates between bases and can be seen under UV light
16
Q
B form of DNA
A
Represents an ideal form of DNA with about 10 bp per turn. But DNA is not perfectly regular like this
17
Q
Z form DNA
A
DNA is more elongated and slim, about 12 bp per turn. It is also left handed
18
Q
Why is DNA so stable?
A
Large number of weak h-bonds, and also stacking interactions
19
Q
Why is major groove rich in chemical information?
A
Proteins can tell by the order of hydrogen bonds/acceptors which base pairs are there. AADH = GC and ADAM = AT
20
Q
Denaturation
A
Can be done with high heat or changing pH (the OH- concentration). Separates the double helix into single strands
21
Q
Conditions for denaturation
A
high temp, lower salt concentration, high pH because they break the h-bonds
22
Q
GC content and denaturation
A
The more GC present in DNA, the more stable it is and therefore the more heat is required for denaturation.
23
Q
Wavelength that DNA absorbs
A
260nm
24
Q
DNA sequence homology
A
similarity between the sequences of two DNA molecules
25
DNA hybridization
the pairing between complementary ssDNA or RNA. Only occurs when the strands have homology. (Used in southern blots)
26
Southern vs. Northern vs. Western blots
```
Southern = DNA size
Northern = RNA probed with DNA probe
Western = proteins probed with antibodies
```
27
DNA supercoiling
relaxed circular DNA has about 10.5 bp per turn but supercoiled has more. It is caused by some sort of structural strain on the DNA, like underwinding
28
Linking number
The number of times that each strand winds around the other. L = Twist + Writhe, when there are no supercoils, L=T
29
Topoisomerases (general)
Enzymes that increase or decrease the linking number by underwinding the DNA. They break one strand, allowing the DNA to unwind and then religate
30
Type 1 and 2 topoisomerases
Type 1 relax supercoiled DNA without ATP
| Type 2 need ATP to relax the DNA or to introduce supercoils
31
Topoisomerase inhibitors
They are used in chemotherapy to stop fast cell division, and stop topoisomerases from doing their jobs
32
Pulse-Chase
In the pulse, we expose the cells to labeled precursors which the cell will use when making macromolecules like DNA, RNA or proteins. In the chase, we wash out the label and let cell grow. Then, we use x-ray imaging to see where the labels end up
33
What labels are used in Pulse-chase experiments?
```
Protein = 35S-methionine
DNA = 3H-thymine
RNA = 3H-uracil
```
34
Conclusion of Pulse-chase experiments
RNA is synthesized in the nucleus and then migrates to the cytoplasm where it partakes in protein synthesis. Confirms central dogma
35
RNA characteristics
- Has ribose instead of deoxyribose
- Uracil instead of thymine
- Single stranded and has secondary structure
36
Why is RNA less stable in alkali?
Since the ribose sugar has a hydroxyl group, it can be deprotonated and this O- can attack the phosphodiester bond and degrade the RNA
37
Types of RNA
mRNA, tRNA, rRNA, miRNA, siRNA, and ribozymes
38
Secondary structures for RNA
Stem-loop, bulge, loop, RNA tends to fold on itself where there are complementary sequences
39
What form of helix does RNA take?
A form rather than B form like DNA
40
Does RNA follow Watson-Crick base pairing? Why or why not?
No, you can find GU and GA very commonly in RNA. Triple base-pairing is also possible for stabilization
41
Uses for Mg2+ and K+ in RNA
Since they are positively charged, they shield the negative charge of the backbone and provide stability. Also helps RNA pack more tightly
42
RNase P
Its an endoribonuclease that cleaves off a leader segment from 5' end of precursor tRNA and changes it into functional mature tRNA
43
Hammerhead Ribozyme?
Another ribonuclease that is self-cleaving RNA
44
Primary structure of proteins
Chain of amino acids called a polypeptide
45
Protein homology
Either an identical amino acid, or an amino acid with similar properties. If an amino acid is similar enough, it shouldn't change the protein function or structure that much
46
Secondary structure of proteins
Beta sheets, alpha helices, random coils, and turns
47
Protein tertiary structure
Stable 3D structure
48
Protein quaternary structure
The number of polypeptide subunits together
| Ex) hemoglobin has 4 subunits
49
What interactions are occurring in protein secondary structure?
Secondary structures are stabilized by H-bonds between the peptide bonds in the backbone
50
Homodimer
Both polypeptides are identical
51
Heterodimer
Non-identical polypeptides (2) could be more if it wasn't a dimer
52
Oligomer
Composed of multiple polypeptide chains
53
Protomer
Individual polypeptide chains
54
Structural domain of a protein
- A part of a single polypeptide chain that has folded onto itself.
- Domains can have independent functions, or all be used for one function
- Protein function is usually based on the combination of different domains
55
Dimerization region
The region where two different polypeptides interact
56
Antibodies
Their quaternary structure creates an extremely specific antibody-antigen interaction
-contain disulfide linkages for added stability
57
Motif
A combination of secondary structures found in many proteins
| EX) Beta barrel, coiled coil, and helix loop helix motif
58
4 Different types of protein folding
1) Spontaneous
2) Chaperone assisted folding
3) denaturing
4) renaturing
59
How does major groove information?
Through its pattern of h-bond donor and acceptor groups. Bases can be read by this characteristic pattern
60
Do protein H-bonds interfere with base-pair bonding?
Nope!
61
Protein to protein interactions
- hydrophobic
| - may be affected by post-translational modifications like methylation or acetylation
62
Enzymes
Very specific catalyst that provides an environment for a reaction to occur rapidly
(Lower the activation energy of the reaction)
63
Protein levels modified by...
- Transcriptional regulation
- Post-transcriptional (RNA)
- Translational
- Post-translational (modifications)
- Allosteric regulation
64
Proteolytic modification
Removes certain peptide segments in order to produce usable insulin. It's activated in response to specific conditions
65
Def of Phosphorylation
Adds phosphate using ATP
66
Def of Adenylylation
Adds adenine using ATP
67
Def of Acetylation
Adds acetyl group from Acetyl-CoA
68
Def of Methylation
Adds methyl group from S methionine
69
Kd
Represents the dissociation constant and is the concentration of the ligand at which half of all the ligand binding sites on the protein are occupied
70
What does a high Kd mean?
It means the protein has a low affinity for the ligand
71
Ways to purify protein
Separation, detection using immunological techniques, and sequence or structural analysis
72
Protein separation
1) Lyse cells
2) Centrifuge
3) Chromatography
4) Gel electrophoresis
73
What does detergent do to cells?
Makes holes in the plasma membrane
74
Ion exchange chromatography
Separates molecules according to their charge. Positively charged particles will come through column first and negatively charged particles are bound to the beads (can be eluted later). The opposite will happen if you use negatively charge beads
75
Gel filtration chromatography
Large proteins come out first because the smaller proteins go through the beads and can get stuck.
76
Affinity chromatography
Use a competing molecule, salt or change in pH to remove the protein of interest from the column
77
Antibody affinity chromatography
Separated molecules according to their affinity for a specific ligand like an antibody. The beads have the antibody so the protein will stick to the beads. The antibody-binding proteins can be eluted by lowering the pH
78
Immunoprecipitation
Antibody is again coupled to the bead and this makes the protein heavier for centrifugation
79
Epitope
A sequence of 7-10 amino acids recognized by an antibody
80
What wavelength do we measure for proteins?
280nm
81
How can we measure beta-galactosidase activity?
Instead of giving the cell lactose, we give it ONPG. Bgal will also recognize this fake sugar and will cleave it to create galactose and ONP which turns yellow
82
What does SDS do?
It linearizes the protein and coats it with a negative charge
83
Beta-mercaptoethanol
Reduces disulfide bonds
84
What does SDS-PAGE separate by?
It separates proteins according to their molecular weights. Low MW will travel farther down and high MW will travel through the gel more slowly.
85
2D gel electrophoresis
The first gel separates the proteins by their IEP. While the second separates by size.
86
IEP
Stands for the isoelectric point. It is the pH at which the protein no longer has any charge.
87
Western blot
Transfer the proteins from SDS PAGE gel to a nitrocellulose membrane. Then add an antibody that will bind tightly to the antigen. Next, add a secondary antibody that contains a marker that will bind to the primary antibody.
88
ELISA
Wells are coated with an antigen. Next add plasma from the subject and if they have the antibody, it will bind to the antigen. Then add a secondary antibody to bind to that first one. Finally, if that secondary antibody binds, it should chnge the color of the solution
89
Edman degradation
A type of protein sequencing that takes off one amino acid at a time and can identify which amino acid it was through column chromatogrpahy.
90
What is mass spectrometry used for?
It's a fast an accurate way to measure the molecular weight of a protein. It can also determine the sequence of proteins
91
Proteomics
The main goal is to identify the full set of proteins produced by a cell under a certain set of conditions
92
G
Glycine
93
A
Alanine
94
V
Valine
95
I
Isoleucine
96
W
Tryptophan
97
F
Phenylalanine
98
P
Proline
99
M
Methionine
100
L
Leucine
101
D
Aspartate
102
E
Glutamate
103
K
Lysine
104
R
Arginine
105
H
Histidine
106
S
Serine
107
T
Threonine
108
Y
Tyrosine
109
N
Asparagine
110
Q
Glutamine
111
X-ray crystallography
An x-ray beam is shot through a crystallized protein and from the diffraction pattern, we can determine the protein's structure.
112
EMSA
Stands for the Electrophoretic mobility shift assay. The protein is mixed with radio-labeled DNA and if the protein sticks to a certain binding site, it will produce a different band in western blot than the rest of the DNA
113
DNA footprinting
Another way of finding out where the protein is binding to the DNA. Put a radio-labelon end of DNA and allow protein to bind. Then cute DNA at different sites and see where the DNA wasn't cut. This is usually the location of the protein.
114
ChIP
Chromatin Immunoprecipitation.
1) Proteins are attached to DNA.
2) Antibody against the protein of interest is added
3) Precipitate the sample with the primary antibody attached
4) Take off protein and antibody and amplify the sequence of DNA using PCR
115
Restriction enzymes
Bacterial enzymes that recognize specific, symmetric (palindromes) sequences in DNA and cleave at these sites
116
Homologous genes
Genes with similar sequences that indicate a common ancestor
117
What year was the human genome finally sequenced?
2003
118
DNA in bacteria
They have circular chromosomes and extrachromosomal elements like plasmids
119
What causes lower chromosomal gene density?
As an organism's complexity increases, it's gene density decreases because the DNA is filled with repeating sequences and introns rather than the actual gene
120
What happens to introns?
They are removed form the RNA after transcription during RNA splicing
121
Intergenic vs. Intragenic
Intergenic is within the gene itself while intragenic is between different genes
122
Pseudogenes
They are integrated into the genome after reverse transcription but can't be expressed because they lack the right regulatory sequences to direct their expression
123
Transposable elements
Sequences that can move form one place in the genome to another. (Transposition)
124
Paralogs
Homologous genes within a species
125
Orthologs
Homologous genes between two species
126
Synteny
Conserved linear order of the genes. It's a good illustration of common ancestry. We have quite a bit of synteny with the mouse genome
127
Protein families
Proteins related in amino acid sequence and 3D structure
128
Single nucleotide polymorphism
A difference in one nucleotide
129
The four main factors that drive evolution
1) Mutation rate
2) Natural selection
3) Genetic drift
4) Migration
130
Chromatin
DNA mixed with protein
131
Number of nucleotides between nucleosomes
20 to 60 bp
132
How many base pairs per nucleosome?
200 base pairs per nucleosomes
133
Structure of nucleosome
Has 2 copies of each H2A, H2B, H3, and H4. Only one copy of H1. H2A and H2B form a dimer and H3 and H4 come together as a tetramer
134
What charge do nucleosomes carry?
They are positively charged
135
Histone motif
Has three alpha helices and two loops connecting them
136
Where do histone interact with the DNA?
They interact between the histone fold and the phosphide ester backbone of the minor groove
137
Function of histone tails
They stick out of the histone and are sites of post-translational modifications like methylation, acetylation, and phosphorylation. They aren't needed for nucleosome formation though. They also interact with adjacent nucleosomes to further pack the DNA tightly together
138
Function of H1
It binds to the linker DNA at the end of the nucleosome and also the middle of the associated 147 bp. It leads to more compact defined structure.
139
Order of DNA packing
DNA ➡ nucleosomes ➡ 30nm filament ➡ extended form of chromosome ➡ condensed section of chromosome ➡ the mitotic chromosome!
140
Nucleosome remodelers
They are enzymes that loosen the interactions between the DNA and the nucleosomes in order to increase its accessibility
141
A sliding
Slides the DNA off the nucleosome to expose the genes needed
142
B transfer
Transfers nucleosomes to a different DNA so that that strand is now free
143
H2A and H2B exchange
The third form of unwrapping DNA exchanges the subunits in a nucleosome in order to loosen up the DNA
144
Signal of acetylation
It tells cells to start transcription of this gene. It is done by adding an acetyl group from acetylcoA to a lysine or other amino acid
145
Signal from Methylation
It's usually tells the cells that this gene is not needed and signals the nucleosome to bind more tightly to the DNA. It is done by adding a methyl group to arginine
146
Method of DNA replication
Semi-conservative!
147
Meselson and Stahl 1958
They did an experiment to prove that DNA replicates semi-conservatively. They grew the cells in a medium containing 15N and then transferred the cells to media containing 14N. If the DNA is conservative, we should see 2 bands (HL HH) while the semi conservative should only have HL. They observed only one band. In the next doubling, they saw two bands HL and LL. this further proved that this was a semi-conservative process
