Exam II Flashcards
1
Q
What is the difference between forward and reverse genetics?
A
Forward: Find genetic basis of a phenotype/trait
Reverse: Find what phenotypes arise as a result of particular genetic sequences
2
Q
What are two things we need to edit a gene?
A
- DNA binding factor that recognizes only the desired sequence
- Molecular scissors
3
Q
Restriction enzymes recognize [short/long] specific sequences.
A
short
4
Q
What are the two things needed in transposon-based gene editing?
A
- Separate inverted repeats flanking the gene of interest
- Transposase gene
5
Q
Is transposon-based gene editing random or site-specific?
A
Random
6
Q
Describe meganucleases (homing endonucleases).
A
- Contain both DNA recognition and cleavage functionalities
- Has large recognition site
- Expressed in bacteria, phages, fungi, yeast, algae, and some plants
7
Q
What are domains in proteins?
A
Proteins are formed by distinct domains, which are functional units.
Domains can be swapped to engineer chimeric/synthetic proteins.
8
Q
Each zinc finger module can recognize __ base pairs.
A
3
9
Q
There are ___ zinc finger proteins encoded within the human genome, and almost all of them are ______.
A
700, transcription factors
10
Q
Stringing zinc finger proteins enables what?
A
More specificity
11
Q
What are zinc finger nucleases?
A
Zinc fingers fused to Fok1 endonuclease
12
Q
How many fingers does each zinc finger nuclease have?
A
3 (Thus a 9 nucleotide recognition site since each finger recognizes 3 base pairs)
13
Q
All zinc finger nucleases come in triplets. True or false?
A
False. They come in pairs.
14
Q
Fok1 only works as a [monomer/dimer/trimer/tetramer].
A
dimer
15
Q
What are TALEs?
A
Transcription-activator-like effector proteins
16
Q
How many nucleotides do individual TALEs recognize?
A
One
17
Q
What do TALEs bind to?
A
Repeat variable diresidue (RVD) positions in DNA
Each RVD binds to a specific DNA base
18
Q
What are TALENs?
A
Transcription activator-like effector nucleases
TALEs infused with the Fokl endonuclease
19
Q
What is the limitation of TALENs?
A
Difficult to construct and express
20
Q
What does CRISPR stand for?
A
Clustered regularly interspaced palindromic repeats
21
Q
How does the bacterial immune system work?
A
- Foreign DNA acquisition
- CRISPR RNA processing
- RNA-guided targeting of viral element (Viral DNA gets degraded)
22
Q
Watch a video on CRISPR-Cas9.
A
Ok
23
Q
What is PAM?
A
Protospacer adjacent motif
- Located in the target sequence
- Needed to generate double strand break
- Recognized by Cas9
24
Q
Simply, CRISPR/Cas9 is Cas plus _____ and what?
A
sgRNA (crRNA/tracrRNA)
Functional artificial CRISPR/Cas9 nuclease
25
What is the benefit that Cas9/CRISPR has over ZFNs and TALENs?
Cas9/CRISPR is efficient; you can modify multiple genes at once by adding more sgRNAs.
With ZFNs and TALENs, you can only modify one gene at a time.
26
What are the advantages of CRISPR/Cas9?
1. DNA recognition depends on sgRNA instead of protein domains
2. Very easy to design/clone sgRNAs
3. Highly specific
27
Rank these methods for gene editing by least to most feasible.
TALENs, ZFNs, meganucleases, CRISPR/Cas9
1. Meganucleases
2. ZFNs
3. TALENs
4. CRISPR/Cas9
28
What is the structural difference between ribose and deoxyribose?
Ribose has an -OH group at both the 2' and 3' carbon.
Deoxyribose has on -OH group only at the 2' carbon.
29
What is the structural difference between uracil and thymine?
Thymine has an extra methyl group.
30
What do DNA replication and transcription need to incorporate NTPs into the polynucleotide chain?
DNA template strand
31
Where is the energy coming from during the polymerization of DNA and RNA?
The hydrolysis of the phosphodiester bond of the incoming nucleotide.
32
What is the first step in DNA replication or transcription?
Unwinding the DNA
33
What percentage of DNA in the genome is replicated? Transcribed?
All is replicated. Less than 1-2% is transcribed.
34
What is the difference between DNA polymerase and RNA polymerase?
DNA polymerase requires a primer. RNA polymerase doesn't.
35
Transcription is asymmetric. What does this mean?
Only one strand of DNA is ever transcribed.
36
In transcription, which strand is the template strand?
Either of them.
37
In replication, which strand is the template?
Both
38
As the RNA chain is synthesized, does it remain hydrogen-bonded to the template?
No
39
Genes are transcribed at the same level. True or false?
False. They're transcribed at different levels. This can vary depending on the cell type and environmental conditions.
40
In transcription, which strand has the same sequence as the transcribed RNA?
The sense/coding strand (Not the template strand)
41
Does transcription and replication work at the same time?
No
42
For replication and transcription, is the product processed?
Replication: New DNA is not processed
Transcription: mRNA is processed (5' cap, introns spliced out, 3' polyA tail, transport to cytoplasm)
43
What are the three steps of transcription in bacteria?
1. Initiation
2. Elongation
3. Termination
44
Describe transcription initiation in bacteria.
1. RNA polymerase weakly binds to DNA and scans for a promoter
2. Sigma factor binds to promoter sequence to aid in promoter recognition
3. RNA polymerase + sigma factor unwind a bit of DNA
4. Formation of phosphodiester bond between first two NTPs. RNA polymerase couples ~10 nucleotides a few times.
5. RNA polymerase breaks free of promoter sequence and sigma factor.
45
Describe the elongation phase of transcription.
RNA polymerase synthesizes RNA at a high rate
46
True or false? RNA polymerase has a higher error rate than DNA polymerase. Why?
True because RNA polymerase lacks proofreading activity.
47
During elongation, DNA in front of RNA polymerase becomes [positively/negatively] supercoiled while trailing DNA becomes [positively/negatively] supercoiled.
positively, negatively
48
Which enzymes releases supercoiling?
Topoisomerase
49
What is the difference between Topoisomerase 1 and Topoisomerase 2?
Topoisomerase 1 doesn't need ATP and generates nicks.
Topoisomerase 2 needs ATP and generates double strand breaks.
50
What happens during transcription termination in prokaryotes?
RNA polymerase reaches a specific DNA sequence (The Terminator) consisting of:
1. A GC-rich inverted repeat (Forms hairpin loop that causes RNA polymerase to stop)
2. 6-8 consecutive adenines (Causes RNA polymerase to dissociate)
51
How is mRNA processed in eukaryotes? Why?
5' cap and 3' polyA tail
RNA polymerase doesn't terminate as precisely as it does in prokaryotes.
PolyA tail is required for mRNA nuclear export, translation, and stability.
52
How is the polyA tail made?
Poly A polymerase (PAP) binds to the 3' end and adds ~200 adenosines one at a time (No template required)
53
Poly-A binding proteins bind to the poly-A tail. What do these proteins do?
They define the length of the poly-A tail and play a role in mRNA stability, quality control, and translation initiation.
54
What is the role of the 5' cap?
Protects the mRNA from degradation by 5'-3' nucleases
55
What is the role of the polyA tail?
Protects the 3' end from 3'-5' nucleases
56
Are the 5' cap and polyA tail required for translation initiation?
Yes
57
When the polyA tail is shortened below a critical length, what happens to the mRNA?
It's rapidly degraded.
58
What is chromatin?
The complex of DNA and proteins
59
What percentage of proteins are histones?
50%
60
What are the core histones?
H2a, H2b, H3, and H4
61
Are core histones small or large?
Small (100 amino acid residues)
62
Are core histones acidic or basic?
Basic (Many lysine and arginine residues)
63
Core histones form what structure?
Quaternary octamer structure
64
Describe the N-terminal tails of core histones.
They're unstructured/flexible. They're modified to regulate the accessibility of nonhistone proteins to the DNA.
65
Describe the octamer histone core assembly.
H3 + H4 to make a dimer. Two of these associate to form a H3-H4 tetramer.
H2a and H2b form dimers (Not tetramers).
N-terminal tails stick out.
66
During octamer core assembly, where is DNA wrapped?
Around the H3-H4 tetramer
67
What is a nucleosome?
DNA wrapped around an octamer core
68
DNA is wrapped ___ times around the core [clockwise/counterclockwise].
1.75, clockwise
69
What is the histone H1?
The linker histone that binds where DNA enters and exits the octamer, covers linker DNA, and sets an angle for further condensation.
70
What role do N-terminal tails play in compaction?
The N-terminal tail of an H4 subunit can interact with the tails of the H2a and H2b subunits of the next nucleosome to promote 30 nm fiber formation.
71
How do nonhistone proteins contribute to compaction?
Some recognize specific DNA sequences and bind more tightly than octamer cores.
This contributes to nucleosome phasing, which can help with optimal compaction.
72
How do nonhistone proteins bind to DNA?
DNA can unravel from the octamer core for a sufficient amount of time to permit a nonhistone protein to bind.
73
What is the major reason to slide nucleosomes? What carries this process out?
To position them optimally for 30 nm fiber compaction
ATP-dependent chromatin remodeling complex
74
The composition of octamer cores can be changed by what?
Chromatin remodeling complexes
75
What are the reasons for exchanging cores?
1. Introduce variant forms of core histones
2. Remove cores during transcription and replication
76
What are DNase I hypersensitive sites?
Chromatin-sensitive, nucleosome-free regions sensitive to cleavage by DNase I
77
What is the histone variant H2AX used for?
DNA repair and recombination
78
What is the histone code?
Covalent modifications of the N-terminal tails
Acetylation = Active chromatin (DNA being transcribed)
Methylation = Silenced chromatin (DNA not able to be transcribed)
79
RNA polymerase is an apoenzyme. True or false?
False. It's a holoenzyme.
80
What are the five core subunits of RNA polymerase?
Two alpha subunits
Beta subunit
Beta' subunit
Omega subunit
81
What is the function of the alpha subunits in RNA polymerase?
Complex assembly
82
What is the function of the beta subunit in RNA polymerase?
Catalytic subunit
83
What is the function of the beta' subunit in RNA polymerase?
DNA binding
84
What is the function of the omega subunit in RNA polymerase?
Necessary for folding/stability of RNA polymerase complex
85
What does a gene consist of?
1. Start and stop codon
2. Promoter
3. Regulatory sequences
86
What is a consensus sequence?
The most commonly observed bases when aligning multiple sequences
87
In what regions are the consensus sequences located in bacterial promoters?
-10 and -35 region
88
What does promoter strength indicate?
How often transcription is initiated per unit of time
89
What defines a strong vs weak promoter?
Strong: High rate of transcriptional initiation, good fit to consensus sequence
Weak: Low rate of transcriptional initation, poorer fit to consensus sequence
90
What are the two varieties of promoter mutations?
1. Down: Decreased RNA synthesis
2. Up: Increased RNA synthesis
91
How does the sigma factor provide specificity to promoter recognition?
Free sigma factor directly binds to the DNA at the promoter site in the -35 and -10 regions
92
In bacteria, much of gene regulation occurs in response to what?
Nutrient conditions in cell environment
(Bacteria don't waste energy by synthesizing things they don't need)
93
What does a repressor do?
Reduces or blocks RNA polymerase-promoter interactions
94
What is the function of activators?
Increase RNA polymerase activity/interactions with promoter
95
What is the function of effectors?
Bind to repressor/activator and induce a conformational change that increase/decrease its activity
96
What is negative regulation?
In the absence of regulatory factors, gene is transcribed.
Repressor inhibits transcription.
97
What is positive regulation?
In the absence of regulatory factors, gene is not transcribed.
Activator increases ability of RNA polymerase to bind to promoter.
98
What is an operon?
A cluster of genes sharing a promoter and regulatory sequences
99
An operon is transcribed into what?
A polycistronic mRNA (Encoding multiple proteins)
100
Each coding sequence in an operon has its own translational initiation and termination site. True or false?
True
101
What is an operator?
The binding sequence for transcriptional regulators
102
What does the trp operon consist of?
Five genes required for the biosynthesis of tryptophan
103
The trp repressor binds to its operator in what conditions?
In the presence of tryptophan
104
What happens when trp binds to the trp repressor?
The trp repressor changes shape so that it can bind to DNA, blocking expression.
105
What does the lac operon consist of?
Three genes required for the utilization of lactose as an alternative energy source when glucose is absent.
106
What are the three genes in the lac operon? What are their functions?
1. lacZ: Cleaves lactose to yield glucose and galactose
2. lacY: Transports lactose into the cell
3. lacA
107
The lac operon relies on what kind of regulation?
Both positive and negative
108
When glucose is abundant, is transcription of the lac operon repressed or not?
Yes, it's repressed.
109
How does cAMP relate to glucose levels in bacteria?
cAMP synthesis is increased when glucose is depleted, activating the binding of CAP protein at the promoter.
cAMP signals that glucose supplies are low and the cell should start using alternatives.
110
Transcription of the lac operon requires what two things?
Presence of lactose and absence of glucose
111
When lactose is low, what happens?
The repressor has no lactose bound and binds to the operator, blocking RNA polymerase.
112
What happens when lactose is high?
Lactose binds to the repressor, causing an allosteric change and making it not able to bind to the operator.
113
Regulation of gene expression in eukaryotes is much more complex than in prokaryotes. Why?
Eukaryotes have:
1. Multiple cell types
2. More genes per cell
3. Chromatin packaging of DNA (Limiting accessibility)
114
How do the number RNA polymerases in eukaryotes compare to prokaryotes?
Prokaryotic cells have one RNA polymerase form to transcribe all RNA.
Eukaryotic cells have multiple to transcribe different classes of RNA.
115
What is the difference in promoter recognition between prokaryotes and eukaryotes?
Prokaryotic RNA pol: Requires sigma factor (one at a time)
Eukaryotic RNA pol: Needs multiple general transcription factors (GTFs)
116
How many general transcription factors are required for promoter recognition and initiation in eukaryotes?
Six
117
What is the first GTF to bind to the promoter and nucleates formation of the initiation complex?
TFIID
118
What is TFIID composed of?
1. TATA binding protein subunit
2. 12 additional subunits (TAFs)
119
What is the function of TBP?
Binds to TATA box, recognizing the minor groove of DNA and causing a bend in it
120
What is the function of TFIIA?
Binds next and stabilizes TFIID binding to DNA at TATA box
121
What is the function of TFIIB?
Recognizes complex between TFIID and DNA and binds to DNA that has been bent
122
RNA pol II is escorted by ___ to the transcription start site.
TFIIF
123
Summarize GTFs and what they do.
1. TFIID + TBP bind first, binds TATA box, bends DNA
2. TFIIA binds, stabilizes TFIID/DNA interaction
3. TFIIB binds, recognizes bend
4. RNA pol II comes in, bringing TFIIF
5. TFIIE comes in and stabilizes everything
6. TFIIH: The unwinder
124
The carboxy terminal domain (CTD) is only found where?
On RNA pol II (Unique to eukaryotes)
125
What is epigenetics?
Affecting gene expression without changing the nucleotide sequence
126
What are the two epigenetic marks?
1. Covalent DNA modifications (Not nucleotides)
2. Covalent histone modifications at the tails
127
How is DNA methylated?
Addition of methyl group to cytosine residues at the 5' position
128
Does methylation affect the base pairing properties of cytosine?
No
129
What do maintenance methylases do?
They recognize hemi-methylated DNA sites and add an additional methyl group on the opposite strand.
130
What are the two ways that activators can make promoters more accessible?
1. Rearrange nucleosomes to provide access to other proteins
2. Recruit other factors that modify the nucleosome structure/organization
131
What are three examples of histone modifications?
1. Acetylation: Lysine
2. Methylation: Lysine, arginine
3. Phosphorylation: Ser, Thr, Tyr
132
Histone methylation is involved in gene [silencing/expression] while acetylation is involved in gene [silencing/expression].
silencing, expression
133
"Writer" enzymes are usually transcriptional [coactivators/corepressors] while "eraser" enzymes are transcriptional [coactivators/corepressors].
coactivators, corepressors
134
What are the "writer" enzymes?
HATs, KDMs, histone kinases
135
What are the "eraser" enzymes?
HDACs, HMTs, histone kinases
136
Coded histone tails interact with what to recognize modifications?
Code readers
137
What is a chromodomain?
The protein domain that binds methylated lysine
138
What is a bromodomain?
The protein domain that binds acetylated lysine
139
What percentage of histones are recycled in newly replicated DNA?
50%
(Meaning that their modifications end up in new nucleosomes)
140
Is it possible to manipulate the epigenome?
Yes
