Module 3: Structure and Function of DNA and RNA Flashcards
1
Q
What are the basic building blocks for DNA and RNA?
A
Nucleotides
2
Q
Describe an oglionucleotide
A
A nucleic acid containing less than 50 nucleotides
3
Q
Describe a polynucleotide
A
A nucleic acid composed of many nucleotides
4
Q
List the 3 parts to a nucleotide
A
Heterocyclic base
Five-carbon pentose sugar
Phosphate group
5
Q
What is Chargaff’s Rule?
A
In double stranded DNA, there is always an equal percentage of purine and pyramidines
6
Q
T/F
Based on Chargaff’s rule
There will always be the twice the amount of adenosine residues as there is thymidine residues
A
FAlse
There will be always be the same amount of adenosine residues as there is thymidine residues
7
Q
Which is stronger:
CG bond or AT bond. Why?
A
CG bond is stronger because it shares 3 hydrogen bonds compared to the 2 between AT
8
Q
T/F
The two strands of DNA intertwine to form a right-handed double helix
A
True
9
Q
Is the backbone of DNA strand positively charged, neutral, or negatively charged?
A
It is highly negatively charged
10
Q
How are the nitrogenous bases of each nucleotide oriented in the helix?
A
They are oriented towards the center of the helix, allowing them to H-bond with the both bases on the opposite strand
11
Q
In DNA, what is the primary purpose of phosphodiester bonds?
A
To link the nucleotide units together within nucleic acids
12
Q
Describe how a phosphodiester bond is created
A
Occurs when the 5’ phosphate group of one nucleotide links to the 3’ OH group of the next nucleotide
13
Q
What is the major groove on DNA?
A
The wider groove found on the outside of the DNA
14
Q
T/F
The nucleotide sequence is read from the minor groove of DNA
A
False
It is read from the major groove because it is more accessible
15
Q
List the two interactions that stabilize the DNA duplex
A
Hydrophobic stacking
Base pairing
16
Q
Describe how base pairing would provide stabilization to the DNA duplex
A
The bonds between bases are arranged so that they cannot break without simultaneously breaking the other bonds holding them together
17
Q
What are the three main functions of DNA
A
1) Long-term storage for genetic information
2) Acting as a template for DNA replication
3) Coding for proteins
18
Q
T/F
Alterations of DNA are a necessity for its function
A
True
It needs to be altered (eg., strand separation) for replication and transcription to occur
19
Q
What are the four most important internal forces for DNA stability?
A
1) hydrophobic interactions
2) van der Waals interactions
3) Hydrogen bonding between paired bases
4) Ionic interactions
20
Q
T/F
The sugar-phosphate backbone is hydrophobic so it faces internally
A
Flase
It is hydrophilic, it likes the water of the cell
21
Q
In what part of the DNA are van der waal interactions present?
A
In the stacked bases that are interacting through ring structures
22
Q
Describe how ionic interactions work in the DNA helix to promote stability
A
The negative charge of the backbone are neutralized by interactions with cations (such as Na+ and Mg++)
23
Q
What are the four external factors that contribute to DNA stability?
A
1) Temperature
2) Salt
3) Proteins
4) Organic solvents
24
Q
Describe how increasing the temperature of a cell would impact DNA?
A
Heating it too much would cause the DNA to unwind into ssDNA, ultimately destabilzing it
25
Describe what would happen to DNA if there was a salt increase in the cell
The sodium ions would interact with the negatively charged DNA backbone to further the ionic interactions, ultimately making it more stable
26
Describe what would happen if DNA was exposed to excess organic solvents
The organic solvents would disrupt the hydrogen bonds, and solvate the bases, ultimately destabilizing the DNA
27
T/F
There are two types of coding RNAs
False
There is only one; mRNA
28
which non-coding RNA plays a role in genetic regulatioN?
small nuclear RNAs (snRNAs)
29
Which non-coding RNA limits translation by binding to the 3' end of the target mRNAs
MicroRNAs (miRNAs)
30
Which non-coding RNA is involved in the processing of rRNAs?
Small nucleolar RNAs (snoRNAs)
31
Give an example of a catalytic RNA
Ribozymes
32
Describe the one major structural difference between ribose and deoxyribose
Ribose has a OH group at both the 2' and 3' carbon, whereas deoxyribose only has an OH group at the 3' carbon
33
What is the complementary base pair for A in RNA?
U (uracil)
34
T/F
RNA doesn't follow the same rules of Watson-Crick base pairing
True
Sometimes there's A-A or G-U
35
Describe the secondary structure of RNA
Includes regions of unpaired nucleotides that can interact with noncontinuous sequences
This stabilizes the 3D folding of RNA and creates surfaces that can bind other molecules
36
Describe why RNA is more sensitive to alkaline conditions than DNA
Bc RNA has an extra OH group, so it is less stable under alkaline conditions. Bases can interact with the 2' OH, causing hydrolysis of the phosphodiester bond
37
T/F
In tRNA, all of the bases are stacked, even if they are not a part of Watson-Crick base pairing
True
38
(Describe internal loops in RNA secondary structures
Occurs when the dsRNA (ssRNA that has folded back onto itself) separates due to a lack of Watson-Crick base pairing between the two stands
39
Describe a bulge in an RNA secondary structure
A type of internal loop that occurs when there is only one strand that has an unpaired base
40
Describe a hairpin loop in RNA secondary structures
Occurs when an RNA strand folds back onto itself and there is an unpaired loop of bases at the end of a stem region
41
What is the most common type of RNA secondary structure?
Hairpin loops
42
Which RNA secondary structure most represents the structure of DNA?
Helical secondary structures
43
What 4 factors influence RNA structure stability?
of GC vs AU (or GU) base pairs
# of base pairs in a stem region
# of base pairs in a hairpin loop
# of unpaired bases
44
What is the ideal number of base pairs in a hairpin loop?
10>#<5
Anything above or below requires more energy
45
T/F
Unpaired bases decreases the stability of the RNA structure
True
46
What does the molar extinction coefficient measure?
It measures the amount of light absorbed by a 1M solution, with a light path length of 1cm, and is a property specific to the molecule being measured
47
You are comparing two samples with the same amount of nucleotides in them. The difference is that in one sample, the nucleotides are bound, whilst in the other they are free. Which sample do you expect to absorb the most light?
You would expect the sample with free nucleotides to absorb more UV light, as stacking them causes a decreasing in light absorption
48
What does Beer's Law state?
The absorbance of light at a certain wavelength is directly proportional to the concentration of the solution
49
You are measuring the absorbance of UV light via spectroscopy, and increase the concentration, but decrease the path length by the same amount. What do you expect to see?
No change in light absorption
Increasing the concentration would increase UV light absorbance, but decreasing the path length would cause less absorption
50
What is optical density?
The amount of UV light able to pass through a solution at 260nm, and a summation of the optical properties of the bases in the molecule
51
T/F
OD and absorbance are equivalent and can be used interchangeably
False
They are not equivalent, but are used interchangeably
52
Is OD greater in DNA or in protein at 260nm?
The OD of DNA is greater than that of proteins.
53
T/F
The relative order of UV absorption is:
dsDNA < ssDNA < free nucleotides
True
54
What is the hypochromic effect?
Refers to a large decrease in light absorption at 260nm occuring as single strands of DNA anneal to form double-helical strands
55
What is hyperchromicity?
The large increase in light absorption at 260nm tat occurs as double-helical DNA unwinds to form ssDNA
56
At what temperature does DNA denature?
Temps above 80 degrees C
57
T/F
Separation of DNA strands due to extreme conditions occurs after denaturation
True
It is after the dsDNA begins to denature or melt that the strands separate and become two strands of ssDNA
58
What is Tm?
The melting point at which half of the DNA in a sample has denature
59
T/F
Renaturation is a one-step process
Tru-ish
It is a one step process when the DNA is only partially separated
60
What is reannealing?
When two partially separated DNA strands spontaneously rewind to form an intact duplex
61
Describe how two completely separated DNA strands are renatured
In the first, slow step, the two strands "find" each other by random collisions and form a short segment of double helix
In the second, faster step, the remaining unpaired bases come together and the two strands "zipper" themselves together to form the double helix
62
How would a pH of 4 impact DNA hybridization?
It would decrease it by liberating all of the bases from the helix
63
How would a pH of 10 impact DNA hybridization?
It would convert dsDNA into ssDNA
64
How would an increased DNA concentration in a sample impact DNA hybridization?
It would increase it, because more frequent collisions would occur
65
What does stringency refer to regarding DNA hybridization?
The degree of complementarity required between two strands in order for them to hybridize
66
If a DNA segment has high stringency, what does this mean?
Hybridization will only occur when the two strands are highly compatible
67
If a DNA segment has low stringency, what does this mean?
Hybridization will occur even in the presence of some base mismatches
68
What factors increase stringency?
High temperatures
Low salt concentration
Presence of organic solvents
69
What factors decrease stringency?
Low temperatures
High salt concentration
Absence of organic solvents
70
What 4 components are in a PCR reaction mixture?
A DNA sample containing the segment to be amplified
A pair of synthetic oglionucleotide primers
Deoxynucleoside triphosphates (dNTPs)
DNA polymerase
71
List the 5 main steps to a PCR procedure
Denaturation
Annealing
Elongation
Amplification
Repeat
72
List the parameters for designing a good PCR primer set
1) 18-25 nucleotides in length
2) 40-60% GC content
3) Annealing temperature of 50-60 C
4) 1 or 2 GC residues at the 3' end of the DNA strand
5) Minimal secondary structure base repeating
6) Complementary to sequence chosen for amplification
73
A piece of DNA has a Tm of 50 degrees C, what is its annealing temperature?
Ta = Tm -5 C
So the annealing temperature would be 45 degrees C
74
Why is gel electrophoresis used
To determine the size of the PCR product
75
Why is RT-PCR used?
To amplify and sequence gene segments without introns
Can also quantify mRNA levels as a measure of gene expression using qPCR
76
How does quantitative PCR (qPCR) differ from regular PCR?
The amount of DNA product generated by the reaction is quantified after every reaction cycle
77
What is SYBR green? What is it used for
Is a dye added into the reaction mixture in qPCR that fluoresces when it is bound to dsDNA
78
After doing multiple rounds of qPCR, you notice that the green fluorescence has stopped increasing. What would cause this?
The reaction mixture has reached its plateau phase, as the reaction components have become exhausted
79
You have two segments of DNA being amplified by PCR in two different reaction mixtures. One segment is present in greater amounts than the other in the other reaction mixture. What do you expect to see?
The sample with the higher concentration of DNA segment will reach its plateau phase faster
80
What is the cycle threshold (Ct)
The cycle number at which the threshold is first surpassed
81
When you melt your PCR amplification product, what would you expect to see on a melting curve?
A sharp decrease of fluorescence due to a decrease in ds DNA
82
Your melting curve analysis showed a small decrease in fluorescence, followed by a plateau, and then another sharp decrease. What does this mean?
There is more than just the one desired PCR product in the reaction mixture
83
T/F
A DNA segment with low stringency will lead to a decrease in PCR efficiency
True
84
List the 6 steps of Sanger sequencing
1) DNA denaturation
2) Primer
3) Free Nucleotides (dNTPs)
4) Modified Nucleotides (ddNTPs)
5) Chain termination
6) Gel Electrophoresis
85
How is Dye-Terminator Sanger Sequencing different from regular Sanger Sequencing?
Dye-terminator SS all of the ddNTPs are added to the same reaction mixture, and the products of different sizes are separated by size using capillary electrophoresis, which are then excited by a laser and the fluorescent emission (red, yellow, green, blue) is detected, one nucleotide at a time
86
What is molecular cloning?
The isolation and generation of recombinant DNA molecules that are placed in organisms for replication and study
87
Why is molecular cloning useful?
Useful for being able to express a protein-of-interest in cells to be able to study its function
88
What is a cloning vector?
A DNA molecule known to replicate autonomously in a host
89
T/F
Plasmids are useful for cloning fragments which are less than 15000 base pairs in length
True
90
What is a plasmid?
Is a circular DNA molecule found in bacteria that replicates separately from the bacterial chromosome
91
The sequence in a bacteria where replication is initiated is called what?
The origin of replication (Ori)
92
What are restriction sequences?
Sequences that act as targets for restriction endonucleases
Provides sites for where the plasmid can be cut to insert foreign DNA
93
How is the small size of the plasmid beneficial?
It allows its entry into cells and the biochemical manipulation of DNA
94
What are bacterial Artificial Chromosomes (BACs)
Circular vectors with an origin of replication, antibiotic resistance, restriction sites, and often contain a reporter gene
95
What are BACs used for?
Cloning larger segments of DNA that standard plasmid cloning vectors would not be able to handle
96
List 3 limitations of Sanger Sequencing
1) SS is slow and expensive
2) Read lengths are up to 1000-1500 bases
3) Sequences for a large segment must be broken down, analyzed one at a time, then compiled together
97
Describe Next-Generation Sequencing (NGS)
AKA High throughput sequencing
Allows for rapid sequencing of large DNA segments
The large segments are broken down into smaller segments, and then sequenced simultaneously, then are aligned to generate a consensus sequence for the entire DNA segment
98
What sequencing method would be best to use for a long DNA segment?
Next-generation sequencing (NGS)
99
What is another name for Reversible Terminator Sequencing (RTS)
Sequencing by synthesis (SBS)
100
Give a definition for a genome
The complete set of genetic material encoded in a cell or virus
101
T/F
Genetic information that comes from the DNA from an organelle contributes to the organism's genome
False
Any genetic content from organelles (ex., mitochondria, chloroplasts) does not contribute to the organism's overall genome
102
Describe the genome of bacteria
Genome consisting of a single, circular DNA molecule
103
How many chromosomes does the human genome contain?
46
104
T/F
Humans have 22 homologous pairs of autosomes and 2 sex chromosomes
True
105
Describe the structural parts to a chromosome
Consists of a shorter p-arm, and a longer q-arm, separated by a centromeric region
106
When stained with giemsa, the dark bands on the chromosome represent what?
Heterochromatin
These areas stain heavilty
107
What is heterochromatin
The condensed portion of the chromosome that are not transcriptionally active
108
When stained with giemsa, the light bands on the chromosome represent what?
euchromatin
109
What is euchromatin?
Genes that are being actively expressed
110
What are single nucleotide polymorphisms (SNPs)?
They represent a genomic base pair change that helps distinguish one specific from another
111
What are the two kinds of large genomic rearrangements?
Inversions
Fusions
112
Describe inversions
A mutation that results from the inversion of a large segment of DNA in a chromosome
113
Describe fusions
The rearrangement of chromosomal DNA by deletion, duplication, insertion, or transposition to form a 'hybrid gene'
114
What human chromosome is considered a hybrid gene?
Chromosome 2
115
Describe homologs
Any two genes with a demonstrable sequence similarity, whether or not they are closely related by function
116
T/F
Genes that are homologs imply an evolutionary relationship
True
117
Describe orthologs
Two genes in different specific possess a clear sequence AND functional relationship to one another
- These genes are derived from an ancestral gene from the last common ancestor the two species had
118
Describe paralogs
Genes that are similarly related to each other but within a single species
Often arise from gene duplication, followed by specialization of the gene over the course of evolution
119
List the 8 components that are necessary within a genome
1) Coding sequences for RNA and protein molecules
2) Signals for chromatin condensation/remodelling
3) Signals for initiation of replication and chromosomal integrity
4) Control signals for on/off, levels of expression
5) Start and stop sites for transcription
6) Processing signals for primary transcripts
7) Control signals for dynamic access at right time and place
8) Identifiers for coding sequences that must be coordinately or sequentially expressed
120
What is intergenic DNA?
The stretch of DNA sequences located between genes
121
What is unique DNA?
Lengths of DNA with no repetitive sequences
122
What are microsatelites?
A repetitive DNA sequence
123
What are regulatory regions?
Any region that influences the DNA that is transcribed into mRNA and then protein
124
What are genome-wide repeats?
Large repetitive DNA sequences within the genome
125
What are Introns UTRs
Sequences of nucleotides in a gene that are transcribed but removed before the gene is translated
126
What are gene fragments?
Pieces of genes, often ancestral, containing only exons; composed of cDNA
127
What are pseudogenes?
Sections of chromosomes that are an imperfect copy of a functional gene
128
What creates a single gene?
A promotor sequence, exons, and introns
129
What is splicing?
The removal of introns from a primary RNA transcript
130
List the levels of organization within a chromosome from the smallest level to the largest level
Nucleotides
DNA double helix
Histones
Nucleosomes
Chromatin
Mitotic chromosome
131
What are histones?
Proteins that help condense eukaryotic cell DNA
132
What are nucleosomes?
The structural unit for packaging chromatin in eukaryotes
133
T/F
Histones are acidic and positively charged proteins that assemble into octamers
False
They are basic, positively charged proteins that assemble into octamers
134
What does each octamer of a histone contain?
Two copies of the four different histone subunits
135
In the first level of chromosome packaging, DNA is wrapped how many times around the histone octamer?
Twice
136
How does the positive charge interact with the DNA?
It interacts with the negatively charged backbone through electrostatic interactions, which then forms a nucleosome
137
Histones are rich in what amino acids?
Arginine and lysine
138
T/F
Histones H2A and H2B are nearly identical in all eukaryotes
False
Histones H3 and H4 are nearly identical in all eukaryotes
139
When does a solenoidal supercoil form?
Occurs when the histone octamer binds DNA
140
Describe the composition of the histone-fold motif
Composed of a globular domain that consists of 3 a-helices linked by two short loops
141
Describe the composition of a nucleosome
Composed of a head-to-tail dimer of histone fold motifs
142
T/F
The nucleosome interacts with the DNA via the minor groove
True
143
T/F
The presence of multiple A=T base pairs in the minor groove help to facilitate contact with histones
True
GC base pairs can prevent the compression of the minor groove, so they are less helpful
144
Global modifications to DNA are required for what processes?
Mitosis and replication
145
Local modifications of DNA are required for what processes?
Transcription
146
T/F
N-terminal tails of histones are required for condensation into the 30nm filament, but H1 is not
True
Although H1 promotes condensation into the 30nm filament, it is not required, whereas the N-terminal tails are
147
List 4 essential cellular processes that rely on modification of chromosomes
1) Regulation of gene expression
2) DNA replication
3) DNA editing and repair
4) Recombination events
5) The preservation of epigenetic tags
148
What class of enzymes are responsible for opening DNA binding sites to allow binding of transcription factors?
Chromatin remodelling complexes
149
What are the three main functions of chromatin remodelling complexes?
1) Repositioning, or sliding the nucleosome to a different location along the DNA stance
2) Ejecting the nucleosome from the DNA
3) Replacing the nucleosome with one that contains a histone variant
150
T/F
Chromatin remodelling complexes result in the physical movement of the nucleosome
True
151
Describe histone modifying enzymes
Enzymes that covalently modify the N-terminus tails of the histone proteins
They are heritable
152
Describe cis-acting histone modifying enzymes
These affect the chromatin structure solely through modifying the molecule directly
These modifications may result in opening or closing the chromatin by tightening/loosening the arrangement of nucleosomes along the DNA
153
Describe trans-acting histone modifying enzymes
These induce other intermediary molecules
Attract other proteins like transcription factors or chromatin remodelling factors, which will then produce the chromatin change
154
What are the three H2A variants
H2AX
H2AZ
MacroH2A
155
Where do the H2A variants differ from each other?
In the C-terminal tail region
156
What is the H2AX variant associated with?
DNA repair and genetic recombination
157
What is the H2AZ variant associated with?
Nucleosomes located at actively transcribed genes
Thought to stabilize the open-state of chromatin
158
Describe the macroH2A variant
Is abnormally large and contains a unique C-terminal domain
Involved with X chromosome inactivation
159
What are the two variants of H3
H3.3 and CENPA
160
How are the H3 variants different?
The primary differences is the susceptibility of residues in the N-terminal tail to modifications like methylation and phosphorylation
161
Describe the H3.3 variant
Thought to stabilize the open state of chromatin, allowing access of the transcriptional machinery to DNA in actively transcribed regions
162
What is the CENPA variant associated with?
The repeated DNA sequences in centromeres
Also contains a large extension that connects to the kinetochore
163
What are the four main histone tail modifications
1) Acetylation of lysines
2) Methylation of lysines and arginines
3) Phosphorylation of serines
4) Ubiquitination of lysines
164
T/F
Methylation and acetylation of histone tails are reversible
true
165
What are HDACs
Histone deacetylases
Remove the acetyl group from histones
166
What are HATs
Histone acetyltransferases
Add acetyl groups to histones
167
What are HMTs
Histone methyltransferases
Add methyl groups of histones
168
Describe the Jumonji Family
Histone demethylases (KDMs) that remove methyl groups from histones
169
Histone modifications occur most at what position and at what residue?
At N-terminal tails
At the lysine (k) residues
170
Describe the acetylation of lysine
Performed by HATs
The lysine tail is acetylated, neutralizing the positive charge
Associated with enhances accessibility to DNA and subsequent transcriptional activation
HDACs will remove acetyl groups from Lys
171
Describe the methylation of lysine
Can be methylated to monomethyl-, dimethyl- or trimethyllysine
172
Describe the phosphorylation modification
Found on histone tails of H3 and H4
Can only occur on Ser, Thr, or Tyr residues
Results in adding a negative charge onto the histone tail
173
Describe the methylation of arginine residues
Can be double methylated or singly methylated
174
What is chromatin immunoprecipitation (ChIP) used for?
To identify the position of nucleosomes within a genome
175
What is the difference between bromodomains and chromodomains
Bromodomains recognize acetylated Lys residues, whereas chromodomains recognize methylated Lys residues
176
T/F
Chromodomains help promote the closed site of chromatin while bromodomains promote the open site of chromatin
True
177
Define epigenetics
the study of heritable changes in gene function that do not involve changes in the DNA sequence
178
How can heritable changes be transfered?
Either from parent cells to daughter cells during cell division
Or intergenerationally, from parents to their offspring
