Exam I - Lecture (1-5) Flashcards
1
Q
Transcription is reversible?
A
yes
2
Q
translation is reversible?
A
no
3
Q
Genetic material must ___ very large amounts of ___
A
store; information
4
Q
Genetic material must have the capacity to be ___ accurately to be ____ “unchanged” to the next generation
A
Replicated; transmitted
5
Q
Genetic material must ___ _____
A
Encode; phenotype
6
Q
Transformation principle = DNA
A
Avery MacLeod and Mccarty
7
Q
Transformation
A
F. Griffith
8
Q
Bacteriophage genetic material = DNA
A
Hersey and Chase
9
Q
Griffiths experiment used what organism?
A
Streptococcus pneumoniae
10
Q
S Strain - Smooth
A
virulent → mouse dies
11
Q
R strain
A
non virulent → mouse lives
12
Q
Transformation
A
some cellular component is taken up by live R bacteria (Streptococcus pneumonia) from dead S bacteria, making them virulent.
13
Q
Who postulated that genes = DNA
A
Avery
14
Q
Mechanism of genetic transformation
A
CapS (capsule gene) fragment released when the S cell is being heat killed. The CapS fragment enters the chromosome of the CapR cell → recombination and cell division leads to a virulent S cell.
15
Q
Bacteriophage components
A
50% protein and 50% DNA
16
Q
Phage attaches to E. Coli and injects its chromosome ..
A
- Phage chromosome replicates
- Expression of phage genes produces phage structural components
- Progeny phage particles assemble
- Bacterial wall lyses, releasing progeny phages
17
Q
1952 Hersey and Chase experiment
A
Concluded that DNA - not protein - is the genetic material in bacteriophages
18
Q
How a centrifuge works
A
Larger, denser molecules move toward the bottom of the tube more quickly.
19
Q
How did the Hershey and Chase experiment work?
A
32P DNA → infect bacteria → Break bacterial cell walls away from any viral material remaining outside them → centrifuge → little 32P in supernatant → bacteria lyse → 32P labeled DNA in progeny phage
20
Q
what does a nucleotide consist of
A
5-carbon sugar + phosphate + nitrogenous base (4 options)
21
Q
Nucleotides (monomer) are connected by ___ bonds to form a nucleic acid (polymer)
A
phosphodiester bonds
22
Q
What atom is is in the 2’ carbon of ribose (RNA)
A
OH
23
Q
What atom is is in the 2’ carbon of deoxyribose (DNA)
A
H
24
Q
The nitrogenous base is attached to carbon ___ of the sugar
A
carbon 1
25
The phosphate is attached to carbon ___ of the sugar
carbon 5
26
DNA sugar
deoxyribose
27
RNA sugar
Ribose
28
Which group of a nucleotide is an acid?
The phosphate group → it releases H + ions
29
Why are DNA and RNA negatively charged molecules?
Because the phosphate releases H+ ions
30
Ribose has ___ in its 2' position
**OH**
RNA is not as stable as DNA because OH is reactive
31
deoxyribose has ___ in its 2' position
**H**
Makes DNA have very high stability
32
ribose and deoxyribose are ___ sugars
pentose
33
Purine
Has two rings
Adenine and Guanine
34
Pyrimidine
1 ring
Cytosine, Uracil, Thymine
(uracil and thymine differ in a methyl group thats found in thymine)
35
Nucleoside
Pentose sugar + nitrogenous base
36
Linking nucleotides by phospohodiester bonds or linkages (covalent)
5' phosphate group of one nucleotide is linked to the 3' hydroxyl group of the next nucleotide
37
Ester linkage (phosphodiester bond) involves
loss of water
38
Nucleic acids have polarity
3' end ends with hydroxyl (-OH)
5' end ends with phosphate group
39
Chargaff's rules
A = T, C = G
40
Rosalind Franklin - Diffraction data
discovered the helical DNA structure - but Watson and Crick received the prestige
41
width of helix
2.0 nm
42
distance between bases
0.34 nm
43
length of one complete turn of helix
3.4 nm
44
what bond is between the nitrogenous bases
hydrogen (weak) → but strong enough to stabilize the molecule
45
DNA is
* complementary
* Constant width
* antiparallel
46
Ideal DNA structure
B Form
* Spiral staircase with bases being treads
* flat base pairs perpendicular to the backbone (sugar+phosphate)
47
The **B** Form
* Hydrated in vivo conditions
* Right handed double helix
* On the outside → major and minor grooves (transcription factors)
* 10bp/turn (theoretically)
* In the cell, it is closer to 10.5 bp/turn
48
Most of our DNA is ___ because our cells are mostly composed of \_\_
B; water
49
**A-** Form of Double Helix
* Shorter
* 11 bp/turn
* bases are tilted
* Right handed
* DNA when **dehydrated**
* In vivo, dsRNA and RNA/DNA hybrids resemble A-form
50
**Z form -** of DNA double helix
* Longer and thinner
* 12 bp/turn
* backbone a zigzag
* **Left handed**
* Formation favored by high conc. of +charged ions and long GC or AT stretches (in vitro)
* **Never reported in vivo**
51
DNA triple helix
H DNA
Hoogsteen Base pairs - **H-DNA -** sideways pairing forms
## Footnote
**C=G=C or T=A=T**
**_One purine interacting with 2 pyrimidines_**
52
Why is it very unlikely to have a triple helix DNA strand in the cell?
because it's pH is very low. Our body's pH is 7.2
53
DNA tetraplex or quadruplex
Guanosine tetraplex
**only DNA sequences with high portions of G (end of chromosomes → telomeres)**
54
Polypeptides made of AA are attached to each other via ___ \_\_\_
Peptide bonds
55
Building blocks of proteins
amino acids
56
Amino acid structure
R group → distinguishes one aa from another
Amino group
Carboxyl group
Hydrogren
All attached to an Alpha carbon
57
non-polar, aliphatic R groups
Glycine, Alanine, Proline, Valine, Leucine, Isoleucine, Methionine
58
Polar, uncharged R groups
Serine, Threonine, Cysteine, Asparagine (N) , Glutamine (Q)
59
Polar, Negatively charged R groups
Aspartate (D), Glutamate (E)
60
Polar, Positive Charged R groups
Histidine, Lysine (K), Arginine (R)
61
Non-polar, aromatic, R grou[s
Phenylalanine (F) , Tyrosine (Y) , Tryptophan (W)
62
Peptides are \_\_\_linked together by \_\_
amino acids; peptide bonds
63
O=C-NH
Peptide bond
64
R groups face the __ direction of each other
opposite
## Footnote
**trans-conformation → most common**
65
secondary structure
regular repeats resulting from **hydrogen bonds** involving the backbone
66
Types of secondary protein structure
**Alpha helix →** coil (10-15 aa long). Right handed spiral (1.2nm)
**Beta pleated sheet →** a fold (strands 3-10 aa long)
67
what sequences of aa can form a-helix
* No consecutive bulky or long R groups
* No consecutive like-charged R groups → would cause repulsion
* Few with polar R groups (Ser, Thr, Asn, Asp) → these destabilize the helix
* Infrequent glycine (very flexible) or proline (inflexible)
68
What type of beta strand is most stable?
Antiparallel. Alternating C-N-C-N terminus
69
Beta sheets consist of at least 2 beta strands
Parallel / Antiparallel
70
Super-secondary structural elements
Helix-Helix Turn
Coiled-coil
Four-helix-bundle
71
Helix - turn - Helix (HTH)
in proteins, HTH is a major structural motif capable of binding DNA
AA sequence forms a turn
72
How does HTH bind DNA?
C-terminal binds to a major groove. N-terminal helps to position the complex
73
Types of super-secondary **beta** structural elements
Beta hairpin
Antiparallel Beta sheet
Beta barrel → can form forks in the cell membrane
74
**Mixed** Super-secondary structural elements
* Beta-alpha-beta
* A/B barrel → beta sheets on the inside and alpha helices on outside → higher stability
75
Tertiary structure of protein
is the overall folded 3D shape of a single polypeptide chain
30 structure is determined by 10 and 20 structures combined with interactions between R groups and the environment
76
What proteins are on the inside of a protein
Non-polar AA
## Footnote
**Hydrophobic exclusion**
77
Interactions important to keep 3D shape + functions
Hydrogen bond
**Disulfide Bridge**
Ionic Bond
Van der Waals → hydrophobic interactions
78
Hemoglobin
40 structure
contains 2 alpha + 2 beta subunits
79
Quartenary protein interactions
Hydrogen bonds
ionic bonds
Hydrophobic and Van der Waals interactions
80
oligomers
proteins composed of multiple peptide chains
1. Homooligomers
2. Heterooligomers
81
Homooligomers
identical subunits
82
heterooligomers
nonidentical subunits
83
Intrinsically unstructured proteins
⅓ of all proteins in higher eukaryotes may lack fixed 30 structure entirely or at least contain unstructured sections or regions → diversity in binding
84
example of homooligomer (homodimer)
E.Coli - Beta clamp
85
example of heterooligomer
hemoglobin
86
Protein function
* Defense
* communication
* enzymes
* transport
* storage
* structure
87
karyotype
arranges chromosomes by size
88
homo sapiens has __ chromosomes
46
89
Bacteria's chromosomes are \_\_
circular
90
In cells, ___ molecules are much longer than the cell diameter (or nucleus) - require \_\_\_
DNA ; compacting
91
prokaryotes
no nuclear membrane separating genetic material from other cellular compartments
92
DNA supercoiling: First level of compaction
in bacteria
“a coiled coil”
Relaxed circular DNA
* add two turns → over-rotate → positive supercoil
* remove two turns → under-rotate → negative supercoil
93
DNA topoisomerases
catalyzes DNA under-winding and relaxation
94
DNA gyrase
Type II Topoisomerase → introduces **negative** supercoils to bacterial chromosomes
95
typical bacterial chromosome contains
50 giant loops of supercoiled DNA (- charge) arranged around a protein scaffold (+ charge)
96
Chromatin in eukaryotes
DNA and proteins (50/50)
97
basic unit of chromatin
nucleosome
98
Nucleosomes: beads on a string
Histone core of nucleosome + linker DNA of nucleosome
99
Histone proteins
isolate proteins bound to DNA and run on a gel
100
H1
half the amount as the other histone proteins
101
what histones are approximately equimolar
H2A, H2B, H3, H4
SDS page gel electrophoresis
101
what histones are approximately equimolar
H2A, H2B, H3, H4
SDS page gel electrophoresis
101
what histones are approximately equimolar
H2A, H2B, H3, H4
SDS page gel electrophoresis
102
Histone proteins are
highly conserved, positively charged proteins
103
Histone proteins are
highly conserved, positively charged proteins
104
about 25 % of each histone
lysine (K) and Arginine (R)
105
Histone protein structure
all _have N terminal “tails”_ that are important for the _regulation of chromatin structure_
N terminus - unstructured domain
106
histone fold domain
3 alpha helices
107
histone fold domain
3 alpha helices
108
which histones have longer C termini
H2A + H2B
109
the histone fold motif
supersecondary structure
Loop1, Loop 2, alpha helix 1, alpha helix 2, alpha helix 3
110
histone octamers ____ \_\_\_\_ into repeating units
organize; DNA
No DNA present → no histone octamer
111
H2A and H2B form a
dimer
112
H3 and H4 form a
tetramer
113
what does one histone octamer consist of?
2x each core histone
* H2A, H2B, H3, H4
* left handed supercoil of **146 bp** winds 1.67 times around the histone octamer
114
what is responsible for binding protein interactions between core histones as well as binging DNA to form nucleosomes?
Histone folds
Hydrogen bonds
115
Regulation of chromosome structure
* Chromatin remodeling complexes
* Histone Modifications by enzymes
116
Chromatin Remodeling Complex
Nucleosomes can be arranged by this ATP driven complex.
Consists of 2-18 proteins
**These complexes change in some way the association between the histone cores with the DNA wrapped around.**
"Promoter regions has to be accessible for transcription factors to begin transcription
117
Chromatin Remodeling Complex in humans
H. Sapiens
hBRM - ATPase
10 - Number of subunits
Bromodomain - Domain
Activation - Effect on transcription
118
H3.3
maintaining transcriptionally active open state
119
Open chromatin (transcriptionally active)
exposed promoter - not too condense (chromatin is available)
120
CENPA
maintaining kinetochore attachement
121
Kinetochore attachment sites to the spindle fibers
separates the sister chromatids
122
H2AX
attracting DNA repair enzymes
123
double strand break in histone/chromatin
H2AX → phosphorylation attracts repair proteins (when there is a break - in the DNA)
124
modifications of ___ alter chromatin structure
histone tails
125
histone modifying enzymes attach \_\_\_
chemical groups to aa of nucleosome subunits
126
Modifications: closed → open
**cis:** if the outcome is a direct result of the modification
**Trans:** if the modification attracts another protein that performs the histone modifying function
127
histone tail modifications
will either affect the clamping together nucleosomes closer or pushing them further away, altering DNA accessibility
128
Acetylation of Lysine in residues
decreases the positivity of histone→ **enhances DNA accessibility (looser DNA → enhances transcription of the DNA)**
129
HAT
histone acetyl transferase
more transcription
130
HD = HDAC
histone deacetylase
Less transcription
131
tails compromise ___ of mass of histones
25-30%
132
Histone tails are rich in
Lys (K)
Ser (S)
Arg (R)
133
Lysine (K) in tails
acetylation
methylation
ubiqutination
134
Serine (S) in tails
phosphorylation
135
Arg (R) in tails
Methylation
136
Linker histone H1 (large protein)
binds the nucleosome and “packs” adjacent nucleosomes
137
chromatin is more compacted when ___ binds the nucleosome
H1
138
H1 helps nucleosomes condense into a higher level of packaging
30 nm filament
139
How is the 30nm. fiber maintained?
By interactions between the tails of the adjacent nucleosomes as well as compaction by H1
140
*euchromatin*
undergoes condensation and de-condensation during cell cycle - may become transcriptionally active
141
*heterochromatin*
stays more compacted
142
mitotic chromosomes is ___ shorter than its naked DNA
50,000 X
143
basic unit of heredity
gene
144
A gene is a …
basic unit of heredity and a sequence of nucleotides in DNA and RNA that encodes the synthesis of a gene product, either RNA or protein
145
a gene segment of a nucleic acid
carries the code for a protein or a ncRNA (functional non-coding RNA)
146
prokaryote vs eukaryote
Both have cell membrane
Prokaryote: circular DNA/chromosome, no nucleus
Eukaryote: linear DNA, nuclear membrane
147
what separates living cells from gene creatures
plasma membrane
148
cloning
making identical copies
149
General steps of DNA cloning
**Recombinant DNA Technology or Genetic Engineering**
1. Isolate a fragment of chromosome (DNA segment) to be cloned by digestion with restriction enzymes (endonuclease)
2. select a small molecule of DNA, capable of self replication (cloning vectors) and digest with restriction enzymes
3. Join two DNA fragments covalently (DNA ligase) - recombinant DNA
4. Move recombinant DNA from the test tube to a host cell (most common E.Coli); provide the machinery for DNA replication
1. Identify/select host cells containing recombinant DNA (selectable markers; permit the growth of a cell or kill the cell under a defined set of conditions).
150
endonucleases → cut the nucleic acids in the middle of the molecule “molecular scissors”
restriction enzymes
151
Type II restriction endonuclease
Cleaves DNA at specific base sequences
152
DNA ligase
Joins two DNA molecules together
153
Nucleases (Exo and Endo) used for degradation of nucleic acids
Exo-nuclease → degrades from either 3' or 5' end.
154
recognition sequence (type II restriction endonucleases)
usually 4 to 8bp long and palindromic
155
plasmids
circular DNA molecule that replicates separately from the host chromosome
156
frequency with a particular recognition sequence occurs in the DNA
the longer the recognition sequence the lower the probability!
4bp → 4^4 = 256bp
6bp → 4^6 = 4,096bp
157
plasmids(up to 5kb)
bacterial artificial chromosomes
158
Origin of replication
(ori)
159
what is used to clone DNA
pBR322
160
gel electrophoresis
1. DNA samples containing fragments of different sizes are placed in wells in an agarose gel
2. An electrical current is passed through the gel
3. All DNA fragments move froward the positive pole; smaller fragments move faster than large fragments. After electrophoresis, fragments of different sizes have migrated different distances
4. A dye specific for nucleic acids is added to the gel.
5. DNA fragments appear orange under UV light
161
PCR components
1. template DNA
2. oligonucleotide primers
3. DNA polymerase
4. dNTPs
5. Buffer to maintain pH and provide Mg2+
162
PCR components
1. template DNA
2. oligonucleotide primers
3. DNA polymerase
4. dNTPs
5. Buffer to maintain pH and provide Mg2+
163
primers
synthetic oligonucleotides
164
Primers (short nucleotide strand/oligonucleotide) synthesize the ______ direction
5' → 3'
165
Taq DNA polymerase
stable at very high temperatures
166
stages of PCR (repeat 25-30 times)
denaturation, primer annealing and elongation
167
denaturation
first, the denaturation of double stranded DNA into single stranded DNA occurs at high temp. (94-96C)
168
annealing
the reaction temperature is lowered and DNA primers bind to single-stranded DNA with complementary sequences. The annealing temperature varies for each primer set and is dictated by length and sequence
169
Extention/elongation PCR
following annealing, the temperature is raised to 72 for **elongation** by DNA polymerase. DNA polymerase uses the primers, template and dNTPs to make new DNA strands
