1
Q
What did Fredrick Griffith demonstrate?
A
Demonstrated that cells could
be transformed
2
Q
What is transformation?
A
Transformation - uptake of
genetic material from an external source resulting in new traits.
3
Q
When did Griffith demonstrate transformation?
A
1928
4
Q
What did Avery, MacLeod and McCarty do?
A
The first demonstration
that DNA was the genetic material.
- Perpetuated that the transforming principle is DNA
5
Q
When did Avery, MacLeod and McCarty demonstrate DNA being the genetic material
A
1944
6
Q
What did Hershey and Chase do?
A
Alfred Hersey and Martha
Chase: Demonstrated that DNA not protein is transmitted or passed on to progeny.
7
Q
When did Hershey and Chase make their discovery?
A
1952
8
Q
What was bacteria infected with to label proteins?
A
Bacteria infected with
phage that has 35S
labeled coat protein.
9
Q
What was bacteria infected with to label DNA?
A
Bacteria infected
with phage that has
32P labeled DNA
10
Q
What was found in Hershey and Chase’s results?
A
Labeled protein NOT
detected in progeny, while labeled DNA WAS detected in the progeny (the cell debris as opposed to the supernatant).
11
Q
What was the conclusion of Hershey and chase?
A
DNA - not protein - is the genetic material in bacteriophages.
12
Q
What were the three steps in the Hershey chase experiment?
A
- infect
- blend
- centrifuge
13
Q
What did Kossel discover?
A
Albrecht Kossel: Nucleic acid contains four nitrogenous bases: Adenine (A), Cytosine
(C), Guanine (G), and Thymine (T).
14
Q
When did Kossel discover the nucleotides?
A
This happened in the 1800s
15
Q
What did Levene discover?
A
Aaron Levene: DNA is made of
repeating units called nucleotides.
16
Q
When did Levene make his discovery?
A
He made his discovery in 1910
17
Q
What did Chargaff discover?
A
Erwin Chargaff: Analyzed the
nucleotide composition of
DNA - A=T; G=C.
- regularity in base ratios of DNA
18
Q
When did Chargaff make his discovery?
A
1948
19
Q
What did Franklin and Wilkins (Watson and Crick) discover and when?
A
Devise the secondary structure for DNA in 1953
20
Q
What three items make up nucleotides and how are they connected?
A
Phosphate group: attached to sugar 5’ carbon
Deoxyribose sugar: 2 attached
Base: AGCT attached to 1’ C.
21
Q
What is the structure of purines?
A
Purines have two rings
22
Q
What are the two purines?
A
A and G are the purines
(Pure As Gold)
23
Q
What is the structure of pyrimindines?
A
They have one ring
24
Q
What are the two pyramidines?
A
C and T
(Cut The Py)
25
Difference between nucleotide and nucleoside?
Sugar and base = nucleoside
sugar base and phosphate = nucleotide
26
Difference between RNA and DNA?
- OH group at 2'
- RIBOSE sugar
- Pyrimidine base uracil instead of thymine
27
What were the two important observations that Chargaff made?
1) Base composition is species specific.
2) The relative amount of adenine (A) is the same as the amount of thymine (T), similarly with guanine (G) and cytosine (C)
28
What is Chargaff's rule?
Chargaff’s Rule
purines (A + G ) /
pyrimidines (T + C)
≈ 1.0
29
What did Franklin and Wilkins discover?
Rosalind Franklin & Maurice Wilkins, using x-ray
diffraction, determined that DNA was a helix of
constant diameter.
30
What information was helpful for determining DNA structure?
Nucleotide structure & Erwin Chargaff’s rule
provided information on base pairing and
positioning of the nucleotides within the helix.
31
What did Watson and Crick think originally?
James Watson and Francis Crick used model
building techniques developed by Linus Pauling.
Watson and Crick initially favored a model with
bases on outside. Pauling also favored a base-out
model, but with 3 strands.
32
What did Franklin point out and what did this change?
Franklin pointed out that phosphates on the inside would make molecule unstable, hence phosphates were on the outside. Watson and Crick using Franklin’s data proposed the structure for DNA.
33
What makes up the backbone of each DNA strand?
The backbone of each DNA strand is a repeating deoxyribose sugar-phosphate polymer
34
Where do the flat bases stack?
The planar (flat) bases stack on top of each other, perpendicular to the helix axis
35
Anti-parallel arrangement?
The strands of DNA are anti-parallel, spiraling around the
helix axis in opposite directions
36
What is the sequence of bass determined by?
The sequences of bases in the
two strands are determined by hydrogen bonding between
adenine and thymine or guanine and cytosine
37
What form does the structure form?
The B-DNA structure is a right-
handed helix with 10 base pairs per rotation of the helix
38
DNA Grooves?
Two grooves: major and minor
groove.
39
What does structure depend on?
Structure does not depend on any particular sequence, structure is more conserved than sequence.
40
What is melting?
“Melting” is the separation of two DNA strands.
41
Is separation irreversible?
Separation is reversible (renature)
42
What does denaturation and renaturation allow?
Process allows artificial hybrid molecules to form (strands from a different sources).
43
What are the ways to denature?
Ways to “melt” or denature DNA: increase temperature; reduce salt concentration; increase pH; solvents
44
What is the melting temperature?
Melting temperature (Tm)
by definition is the
temperature at which one
half of the DNA duplex will
dissociate to become
single stranded and
indicates the duplex
stability..
45
How do we monitor DNA melting/denaturation?
DNA melting or denaturation can be monitored by measuring absorbance.
46
What is the hyper chromic shift?
As the DNA duplex separates - absorbance increases (hyperchromic shift).
47
What does Tm indicate?
Tm is an indication of the stability of the hybridized DNA molecule.
48
Higher Tm = ?
The higher the Tm the more stable the DNA helix.
49
What increases Tm?
The higher the GC content (% G+C), the higher melting
temperature.
50
Why is GC content equal higher Tm?
Because G-C bonds are triple bonds whereas A-T bonds are double bonds - it takes more energy to break triple bonds!!
51
What destabilizes the double helix?
The repulsion between the
negatively charged phosphate
backbones destabilizes the double helix.
52
What shields the negative charges?
The negative charges are shielded by salt ions (e.g. Na+) in the solution, this stabilizes the helix and increase the melting temperature.
53
Classifying based on Tm?
Classify organism e.g. bacteria, because the GC content in
the DNA is species specific
54
Detecting mutation using Tm?
Rare gene mutations can be detected because mutated DNA sequences melt at different temperatures than ‘normal’ ranges
55
DNA melting and Tm role?
Process of DNA melting plays an important role in molecular
biology techniques, e.g. polymerase chain reaction and southern
blotting.
56
What does Tm equal?
Tm = 81.5 + 16.6 log[M] + 0.41(%GC) - 675/L
M = molar concentration of ions in solvent
%GC = percentage of G’s + C’s
L = length of DNA measured in base pairs (bp)
57
What does Tm depend on?
Tm depends on ionic strength of buffer, GC content, and length of DNA.
58
What model of replication is correct?
SEMICONSERVATIVE!!!!!!!
- one template strand is maintained with the replicated strand!!!!!
59
Where was E. coli grown?
Grew E. coli on 15N medium
for many generations.
60
What were the cells swapped to?
Switched some cells to 14N
medium.
61
How did we determine the composition of DNA?
Used equilibrium density
gradient centrifugation to
determine isotope composition of DNA
62
After just heavy isotope?
ALL DNA was heavy
63
One cycle after lighter isotopes?
ALL WAS MIXED
64
Two cycles of light?
1/2 LIGHT, 1/2 MIXED
65
How is the parental DNA separated?
Separation of the two DNA
strands of the parental molecule.
66
What do the strands of parent DNA serve as?
Each parental strand serves as a template that determines the order of nucleotides along the newly synthesized strand.
67
What do the daughter DNA molecules consist of?
Each “daughter” DNA molecule consists of one parental strand and one newly synthesized strand.
68
What are the four requirements for DNA synthesis?
1) Template of single-stranded DNA (ssDNA)
2) All 4 deoxyribonucleoside 5’ triphosphates (dNTPs)
3) DNA polymerase and other
enzymes and proteins
4) Free 3’-OH group
69
What direction is DNA synthesized to the template?
New strand is synthesized antiparallel to the template strand, bases added to the 3' end.
70
What does catalysis of phosphate group allow?
This creates the energy needed to bind the DNA molecules!!!
71
What does DNA polymerase use?
Uses deoxyribonucleoside 5’ triphosphates (dNTPs)
72
What does DNA polymerase catalyze?
Catalyze phosphodiester bonds
73
What is the absolute requirement for DNA polymerase?
Has an absolute requirement for a preexisting 3’ OH.
74
What can't DNA polymerase do?
Cannot make a DNA chain de novo. In other words it can
only extend a chain.
75
In what direction does elongation occur?
Always elongates chain in
the 5’ to 3’ direction.
76
In what direction is the template strand read?
Template strand is always
read in the 3’ to 5’ direction.
77
Where does replication begin?
Replication begins at a specific nucleotide sequence - origin of replication.
78
Where does synthesis take place?
Synthesis takes place within a replication bubble.
79
Are the strand synthesized at different times?
No. Both DNA strands are synthesized simultaneously at the replication fork.
80
What is a replicon?
Replicon is any DNA molecule or a region of DNA that replicates from a single origin of replication.
81
Synthesis is always what direction?
Synthesis is ALWAYS 5’ to 3’. How are both antiparallel DNA
strands copied simultaneously at the replication fork?
82
Replication is...?
Replication is
semidiscontinuous
83
Leading strand?
Leading strand: Synthesis is
continuous and in direction of
the fork
84
Lagging strand?
Lagging strand: Synthesis is
discontinuous and occurs in
opposite direction of the fork.
- Okazaki fragments formed!!
85
Types of replication in circular genomes?
Circular genomes
1) Theta replication (bacteria, e.g. E. Coli)
2) Rolling circle replication (viruses)
86
Type of replication for linear genomes?
Linear genomes
3) Linear replication (eukaryotes)
87
How does theta replication work? Replicon, replication type and forks?
Single replicon (for bacteria = entire chromosome).
* Bidirectional replication – two replication forks within a replication bubble.
* Replication is semidiscontinuous at both replication forks.
88
How does rolling circle replication work?
No replication bubble.
* Uncoupling of the replication of the two strands of the DNA molecule.
* Replication is continuous
89
What do we have multiple of in linear replication?
Multiple……
….replicons.
…origins of replication.
… replication bubbles.
90
Bidirectional linear replication? Continuity?
Bidirectional replication – two replication forks within a replication bubble.
* Replication is semi discontinuous at both
replication forks.
91
Four stages of replication?
1. Initiation
2. Unwinding
3. Elongation
4. Termination
92
What do initiator proteins do?
Initiator proteins bind to the
origin of replication (oriC).
93
How do proteins bind during initiation?
A short section of DNA is
unwound and proteins bind to
the ssDNA.
94
Role of SSBPs during initiation?
Single-strand-binding proteins
keep DNA strands separated.
95
Role of helicase in initiation?
Helicase binds to lagging strand template; breaks hydrogen
bonds.
96
What occurs in unwinding due to helices and gyrase?
DNA helicase separates the two DNA strands by breaking the hydrogen bonds .
- DNA gyrase (topoisomerase) travels ahead of the replication fork and alleviates supercoiling caused by unwinding.
97
What occurs during chain elongation?
Short stretch of RNA nucleotides (RNA primer) is
synthesized by Primase.
* RNA primer provides a free 3’OH for the DNA polymerase to use.
* The RNA primer is later removed and replaced with DNA nucleotides.
98
Why does chain elongation involve an RNA primer?
Because the production of RNA does not require a 3’ end
99
E. coli polymerases? amount and type of activity?
Five DNA polymerase in E. coli (Pol I to Pol V)
* All five have 5’ to 3’ polymerase activity.
100
Exonuclease activity in E. coli?
Some polymerase have exonuclease activity (to remove a newly incorporated nucleotide that does not match the template strand)
101
Pol III?
Pol III is the principle replication enzyme.
102
Pol I?
Pol I removes and replaces RNA primers with DNA.
103
Role of DNA Pol I exonuclease?
DNA Pol I 5' -> 3' exonuclease
activity removes RNA primers
starting at the 5’ ends.
104
Role of DNA Pol II polymerase?
DNA Pol I 5’ -> 3’ polymerase
activity fills in the gap with DNA nucleotides.
105
Role of DNA ligase?
DNA ligase seals the nick in
the sugar phosphate
backbone.
106
What is DNA replication?
DNA replication is the process by which DNA makes a
copy of itself during cell division.
107
a-DNA polymerase?
Polymerase activity, no exonuclease activity.
- initiation of nuclear DNA synthesis and DNA repair; primase activity
108
delta-DNA polymerase?
Polymerase and exonuclease activity
- lagging strand synthesis, DNA repair and translation DNA syntheiss
109
epsilon-DNA polymerase?
Polymerase and exonuclease activity
- leading strand synthesis
110
How are origins activated and what are these called?
Origins of replication are
activated in clusters (20-80
at a time). Each cluster is
known as a replication unit.
111
How do we control initiation?
Controlled initiation:
1) An origin must be selected or “licensed” by replication licensing
factors.
2) Origin is then activated and replication begins.
3) Once activated/replicated an origin is deactivated.
112
Rate of E. coli fork speed, duration and origins?
Genome: 4.6 million
Fork speed: 1000 bases/sec
Duration: 40min
Origins: 1
113
Rate of human fork speed, duration and origins?
Genome: >3 billion
Fork Speed: 50 bases/sec
Duration: 8 hours
Origins: >10,000
114
Why do we have multiple origins?
In eukaryotes S phase would last about a month if
only one origin.
* Multiple origins ensure efficient genome replication
in limited time.
115
What else needs to be made more of?
Eukaryotic DNA is packaged into chromatin.
* Need to disassemble, produce more histones and
reassemble nucleosomes
116
What is the issue with telomeres?
Telomeres:
* are the ends of linear
chromosomes.
* are made up of G-rich short
repeated sequence.
* stabilize chromosomes.
Each round of replication
leaves up to 200 bp DNA
unreplicated at the 3' end.
117
Telomerase role?
Telomerase
- Specialized reverse
transcriptase.
- Extends the end of the parental DNA by RNA-templated DNA synthesis.
- Responsible for the replication of the chromosome ends
118
What do we get when something is transcribed and translated?
Transcription + Translation gives us the expression of a gene!
119
What is the central dogma of biology?
DNA gets transcribed to RNA which gets translated into protein.
120
What is an extra process in the central dogma?
We also have reverse transcription, a backwards process. This is mainly in viruses.
121
What does gene expression refer to?
The process of transcription and translation together refers to gene expression
122
What is DNA replication?
Information is transferred from one DNA molecule to
another
123
What is the process of transcription?
Information transferred from DNA to an RNA molecule
124
What is the process of translation?
Information is transferred from RNA to a protein through a code that specify the amino acid sequence
125
What is different in prokaryotes compared to eukaryotes?
Prokaryotic cells have no nucleus, and thus no separation. This means that translation and transcription occur at the same time.
126
How dos the process of expression work in eukaryotes?
Transcription occurs in the nucleus, is processed into mRNA and then exported to the cytoplasm where translation occurs.
127
What are the three types of RNA in both eukaryotes?
Messenger RNA (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
128
What are the types of RNA in only eukaryotes?
Pre-messenger RNA (pre-mRNA)
Small nuclear RNA (snRNA)
Small nucleolar RNA (snoRNA)
MicroRNA (miRNA)
Small interfering RNA (siRNA)
Piwi-interacting RNA (piRNA)
129
What RNA is only produced in prokaryotes?
CRISPR RNA (crRNA)
130
What is the typical structure of RNA?
RNA is usually single-stranded
but it can fold into complex
secondary structures called
hairpin-loops and stem-loops
131
What is the main difference between DNA and RNA?
RNA has a hydroxyl group on the 2' carbon atom of its sugar component, whereas DNA has a hydrogen atom. This makes RNA more reactive than DNA.
132
What is the other difference between RNA and DNA?
Instead of thymine, RNA has uracil
133
Transcription is a ______ process?
Transcription is a selective process, the selective synthesis of RNA.
134
Is all DNA transcribed?
Not all of the DNA in a given cell is transcribed.
135
How does RNA synthesis occur?
Synthesis is complementary and antiparallel to the DNA template strand.
136
What is the nontemplate strand?
The non-template strand is identical to the RNA molecule that we are making using the template strand.
137
Describe the initiation of transcription?
Initiation does not require a primer: chain synthesis begins de novo.
138
Where are ribonucleotides added?
Ribonucleotides are added to the 3’ -OH group of the growing RNA chain.
139
Where does DNA unwind and what happens when synthesis is done?
DNA unwinds at the front of the transcription bubble and then the molecule rewinds!
140
What are the three requirements for transcription?
1. DNA template
2. RNA nucleotides (rNTPs)
3. RNA polymerase and other proteins
141
What is RNA synthesized from?
RNA is synthesized from one of the two DNA strands
142
What can be used as the template strand?
Either DNA strand can be used as the template strand/
143
How is the template read?
Template is always read in the 3’ -> 5’ direction.
144
In what direction is RNA synthesized?
RNA is synthesized in the 5’ -> 3’ direction.
145
What is the transcription unit?
Region of DNA that codes for an RNA molecule and the sequences necessary for transcription
146
What are the three critical regions of the transcriptional unit?
PROMOTER (upstream of start site, adjacent to gene)
RNA CODING REGION (downstream of start site)
TERMINATION SITE (downstream of start site)
147
Which region is transcribed?
Only the RNA coding region is transcribed.
148
What is the promotor?
The PROMOTER is a DNA sequence that is recognized and bound by the
transcription apparatus (RNA polymerase plus other proteins).
149
What does the promoter indictate?
The promoter
indicates the direction of transcription.
150
What does the binding of RNA polymerase do?
Binding of the RNA polymerase to the
promoter orients the enzyme towards the start site
151
What is initiation?
Initiation is the assembly of transcription apparatus on the
promoter and begins synthesis of RNA
152
What is elongation?
DNA is threaded through RNA
polymerase, unwinds the DNA, adds new nucleotides to the
3’ end of the growing RNA strand
153
What is termination?
Termination is the recognition of the end of transcription
154
What do bacteria have?
Many bacteria have multiple
types of sigma factors, which
help in the recognition of
multiple classes of promoters.
155
What happens without sigma factors?
Without sigma, core enzyme
initiates transcription randomly
156
What is holoenzyme?
Holoenzyme is the complete
enzyme complex composed of
the core RNA polymerase and
the sigma factor.
157
What are consensus sequences?
Promoters contain short stretch of DNA that are conserved among promoters of different genes. These are called consensus sequences.
- they are the most common DNA sequence when we compare sequences.
158
What are the most common consensus sequences?
Most common encountered sequences (or elements) are at -10 (Pribnow box) and -35 nucleotides upstream of the start site.
159
What does the binding to the consensus sequence do?
Binding of transcription apparatus to these sequence orients the RNA polymerase towards the start site.
160
Are the -35 and -10 elements ubiquitous?
-35 and -10 elements are not
identical in all promoters.
161
How do the elements vary?
Each is a variation on a theme, i.e. consensus sequence.
162
What does the variation affect?
Variation affects the strength of the promoter. (strength = frequency of transcription)
163
What is recA?
recA is a strong promotor (matches consensus sequence)
164
What are down mutations?
base substitutions that make the sequence less similar to the consensus sequences reduce the rate of transcription
165
What are up mutations?
sequence becomes more similar to the consensus sequences
166
When does transcription end?
Transcription ends after a terminator sequence is transcribed.
167
What are the two major types of terminators in bacteria?
Rho-dependent (requires Rho protein)
Rho-independent (also called intrinsic terminator)
168
What are the three steps of termination that is rho-dependent?
1) Rho binds to RNA upstream of the terminator.
2) RNA polymerase pauses when it reaches the terminator sequence
and Rho catches up.
3) Rho unwinds DNA-RNA
hybrid using helicase
activity.
169
What are the several steps categorizing rho-independent termination?
- Polymerase pauses at Us
- Hairpin formation destabilize DNA-RNA hybrid
- A:U base pairing is relatively weak.
- RNA transcript dissociates from RNA polymerase, and DNA reanneals
170
What composes the RNA polymerase II promotor?
Promoter = Core Promoter + Regulatory Promoter
- and an enhancer sequence
171
Can the core promotor work alone?
Yes, the core promotor is able to initiate a minimal level of transcription
172
Where is the core promotor?
Extend upstream/downstream of transcription start site.
173
What is the core promotor needed for?
Minimal sequence required for accurate transcription initiation.
174
What does the core promotor include?
Includes a number of consensus sequences (common elements: TFIIB, TATA, Initiator and DCE) for transcription factor binding.
175
Where is the regulatory promotor located?
Located upstream of the core promoter, exact location can be variable.
176
What proteins bind to the regulatory promotor?
Transcriptional activator
proteins bind to consensus
sequences and affect the
rate of transcription.
177
What composes the basal transcription apparatus?
The TATA binding protein and general transcription factors as well as RNA polymerase
178
What sequence does transcription end at?
Transcription does not end at aspecific sequence.
179
What does transcription require?
Termination requires cleavage of the mRNA at a specific site.
180
What is the role of the exonuclease?
A 5’->3’ exonuclease degrades
the remaining mRNA terminating transcription.
181
What is an example of the 5'->3' exonuclease?
Rat1!!
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