Midterm 1 Flashcards Preview

LS3 > Midterm 1 > Flashcards

Flashcards in Midterm 1 Deck (147):
1

Central Dogma

The flow of genetic information goes from DNA to RNA to Proteins

2

Beadle and Tatum

They worked with bread mold and showed that each gene controlled a single protein (one gene: one enzyme hypothesis)

3

Griffith Experiment

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

Griffith's conclusion

The S strain was able to transform the R strain into a virulent strain.

5

Avery, Macleod, and McCarty

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

Hershey Chase Experiment

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

Building blocks of DNA

Nucleotides!
Purines: Adenine and Guanine
Pyrimidines: Cytosine and Thymine

8

Polarity of DNA

Polarity comes from phosphodiester linkages, and has a net negative charge.

9

Chargaff's rules

G and C are in same amount and A and T are in same amount

10

Rosalind Franklin and Maurice Wilkins

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

Watson and Crick

They proposed 3D structure of DNA to be double helix

12

Structural characteristics of DNA

Has sugar phosphate backbone, bases project inwards, one turn is about 10.5 bp, has major and minor grooves.

13

Mica experiment

DNA has about 10.5 bp per turn in solution

14

Gel Electrophoresis

Separates DNA molecules according to their weight. DNA travels to positive side because it is negatively charged

15

Ethidium Bromide

Intercalates between bases and can be seen under UV light

16

B form of DNA

Represents an ideal form of DNA with about 10 bp per turn. But DNA is not perfectly regular like this

17

Z form DNA

DNA is more elongated and slim, about 12 bp per turn. It is also left handed

18

Why is DNA so stable?

Large number of weak h-bonds, and also stacking interactions

19

Why is major groove rich in chemical information?

Proteins can tell by the order of hydrogen bonds/acceptors which base pairs are there. AADH = GC and ADAM = AT

20

Denaturation

Can be done with high heat or changing pH (the OH- concentration). Separates the double helix into single strands

21

Conditions for denaturation

high temp, lower salt concentration, high pH because they break the h-bonds

22

GC content and denaturation

The more GC present in DNA, the more stable it is and therefore the more heat is required for denaturation.

23

Wavelength that DNA absorbs

260nm

24

DNA sequence homology

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