BIOL 226 FINAL Flashcards
1
Q
What does snRNA do?
A
Involved in the splicing process when mRNA is formed
2
Q
What does rRNA do?
A
combines with proteins to form ribosomes
Once in the cytoplasm, the mRNA binds to a ribosome, which is a large complex comprised of several ribosomal RNA (rRNA) and many proteins. The ribosome is the site of protein assembly. The bases of the nucleotides in the mRNA are read three at a time by the ribosome. Each of these base triplets is called a codon and specifies one amino acid.
3
Q
What does tRNA do?
A
Brings amino acids to sites of mRNA during protein synthesis
a cloverleaf‑shaped molecule containing an anticodon complementary to an mRNA codon and is attached to an amino acid specific to the anticodon. The interaction between the codons in the mRNA and the complementary tRNA anticodons dictates the order that amino acids are delivered to the ribosome. Enzymes within the ribosome join the amino acids from the tRNAs to form the peptide chain. When at least three amino acids are linked, the chain is considered a polypeptide.
4
Q
What does siRNA do?
A
eliminates the expression of an undesirable gene
5
Q
What does mRNA do?
A
Coding - carries genetic information to ribosomes for protein synthesis
a linear sequence of ribonucleotides transcribed from DNA that carries instructions for protein production
6
Q
What does miRNA do?
A
inhibits the translation of mRNA into proteins
7
Q
What is common about the promoter in both pro and eukaryotes?
A
In both prokaryotes and eukaryotes, the promoter is located in the 5′ direction, upstream from the transcription start site.
8
Q
What does the promoter often contain in eukaryotic transcription?
A
In eukaryotes, the promoter often contains a TATA box, which is where the TATA‑binding protein binds. The binding of the TATA‑binding protein is the first step in eukaryotic transcription. When the TATA‑binding protein binds, it is part of a larger transcription factor complex, which then attracts other general transcription factors and the RNA polymerase, together forming the preinitiation complex.
9
Q
What does the promoter contain in prokaryotic transcription?
A
In prokaryotes, the promoter contains a −35 and −10 region upstream of the transcription start site, which is recognized by the RNA polymerase holoenzyme. The RNA polymerase holoenzyme is composed of five units that make up the core enzyme plus a sigma factor subunit. After the RNA polymerase holoenzyme binds to both the −35 and −10 regions, transcription is initiated at the start site.
10
Q
What occurs in the first step of transcription - initiation?
A
initiation, during which an enzyme called RNA polymerase binds to the DNA template at a specialized sequence called a promoter.
Before RNA polymerase can bind to the promoter, transcription factors must first bind and prime the promoter region.
Once bound, RNA polymerase unwinds local regions of the DNA double helix which enables the genetic code to be copied.
11
Q
What occurs in the second step of transcription - elongation?
A
Once the DNA is unwound and the nucleotides are exposed, the elongation step begins. RNA polymerase reads the DNA template strand and adds nucleotides to the 3’ end of the growing chain.
12
Q
What occurs in the last step of transcription - termination?
A
RNA polymerase continues to transcribe the DNA until it encounters a sequence that signals the end of the gene.
RNA polymerase detaches from the DNA and the string of RNA bases is released as a single‑stranded transcript.
The newly formed RNA transcript, also known as messenger RNA, is ready to be translated into a protein.
13
Q
Which strand is the template strand in transcription?
A
3’
14
Q
What is transcription?
A
The process by which DNA is copied to RNA
15
Q
What is translation?
A
The process of using RNA to produce proteins
16
Q
What are the 8 brief steps of eukaryotic transcription?
A
RNA polymerase II binds to DNA promoter element
DNA is unzipped by RNA polymerase
Ribonucleotides align with complementary DNA nucleotides
RNA polymerase links triphosphate ribonucleotides together
Guanine cap added to 5’ end of mRNA
RNA polymerase released from DNA
Splicing and polyadenylation (polyA tail) events modify mRNA
Processed mRNA leaves the nucleus
17
Q
What is a transcription factor?
A
A protein that binds to a specific DNA sequence to regulate transcription
Can bind either upstream or downstream,
Can activate - recruits RNA polymerase
Can inhibit - blocks RNA polymerase from binding
18
Q
What is a polyA tail?
A
Sequence of adenine nucleotides added onto the end of premRNA, added onto 3’ end
Facilitates export of mature mRNA from nucleus and protects from degradation
Stabilizes the trailing end of mRNA
19
Q
What is an intron?
A
noncoding segment of DNA that lies between coding regions, is removed from premRNA
20
Q
What is an exon?
A
Coding portion of DNA that is present in mature mRNA
exons are eventually expressed by being translated into AA sequences
21
Q
What is alternative splicing and why is it important in eukaryotic organisms?
A
Alternative splicing is done by the spliceosome where it splices in slightly different places
Produces related but distinct proteins called isoforms
It increases the variety of proteins that can be produced without altering the genome size
22
Q
What is the purpose of the 5’ cap?
A
Assists mRNA in binding to the small ribosomal subunit
Assists in RNA splicing
23
Q
What happens in the three phases of translation?
A
During initiation, the small subunit of the ribosome and the initiator tRNA bind to the mRNA. This complex, along with other proteins known as initiation factors, scans the beginning of the mRNA for the first start codon, AUG. The 5′ cap of the mRNA is important for ribosome binding and helps to ensure that ribosomes load before the first start codon. Once the start codon is recognized, the large ribosomal subunit binds to the small ribosomal subunit and translation can& begin.
During elongation, additional amino acids are added to the growing polypeptide chain. For each codon after the start codon, a tRNA complementary to the codon binds to the ribosome and mRNA. This tRNA carries with it the amino acid specified by the codon. The ribosome catalyzes the addition of the amino acid from the new tRNA to the amino acid carried by the initiator tRNA, forming a peptide bond. Using energy, the ribosome and elongation factors remove the initiator tRNA, move the ribosome forward, and accept a new tRNA complementary to the next codon. This process continues to repeat until the ribosome reaches a stop codon.
During termination, the ribosome reaches a stop codon and dissociates from the mRNA. Unlike other codons, the stop codon is not recognized by a tRNA, but instead is recognized by a protein, known as a release factor. Release factor proteins are shaped like a tRNA. The release factor protein and other factors catalyze the release of the polypeptide chain and the dissociation of the ribosome from the mRNA.
24
Q
What are the sizes for each subunit of ribosomes in pro and eukaryotes?
A
prokaryotes - 70s, small subunit 30s, large subunit 50s
Eukaryotes - 80s, small subunit 40s, large subunit 60s
25
Which type of organism has multiple ribosome binding sites?
Prokaryotes can have multiple binding sites because a single mRNA can encode many proteins, but in eukaryotes mRNA only encodes a single gene and therefore only has a single binding site.
26
Which DNA bases are purines and which are pyrimidines?
Purines - A, G (larger)
Pyrimidines - C, T, U (smaller)
27
What is the difference between a transition and transverse missense mutation?
A transition mutation causes a purine - purine or pyrimidine - pyrimidine substitution
a transversion mutation causes a purine - pyrimidine substitution
28
What are two differences between DSBR and SDSA? (Pathway after homologous recombination)
In DSBR the non crossover product contains DNA from the homologous chromosome and only forms after a Holliday junction resolution
In SDSA the non crossover product does not contain DNA from the homologous chromosome, and there are only non crossover products
29
What is a mutagen and what can they do?
A mutagen is a chemical or physical force that can increase the mutation rate above the spontaneous mutation rate
Replace a base pair in the DNA strand
Alter a base pair leading to mismatch
Damage a base pair so that it cannot pair with anything else
30
How does homologous recombination repair double stranded breaks in DNA (simplified/)
It uses complementary sequence on a homologous chromosome as a template to extend DNA past breakpoint
31
Which repair pathways detect DNA damage during transcription?
nonhomologous end joining (NHEJ)
base excision repair (BER)
nucleotide excision repair (NER)
direct repair (kinda)
These DONT (during replication instead)
translesion synthesis
mismatch repair
homologous recombination
32
How is cDNA generated from a eukaryotic mRNA?
Reverse transcriptase generates a single-stranded cDNA, and then DNA polymerase synthesizes the complementary strand.
33
What are three ways scientists can confirm that a cell has incorporated recombinant DNA?
Screen for the expression of a reporter gene (GFP)
Grow cells in the presence of an antibiotic
PCR if needed
34
To inactivate a gene by RNAi what do you need?
the sequence of the target gene
35
What is an operon?
A gene cluster or group of genes that act like a single gene for regulation and transcription
36
What is the function of the repressor in the lac operon?
A repressor is a type of protein that inactivates the expression of the lac operon genes by binding to the DNA of the lac operon - protein transcribed in the regulatory gene
37
What happens in the lac operon when lactose is scarce in the environment?
The repressor is activated in the absence of lactose
The repressor binds to the operator (normally would bind to lactose)
RNA polymerase cant move
Lactose enzyme genes are not transcribed
The cell can no longer digest lactose
38
What is the central dogma of biochemistry?
Transcription of DNA to RNA,
Translation of RNA to Amino Acid chains
The folding of amino acids to proteins
39
What are four differences between DNA and RNA?
Nucleotides used - U vs T
Sugar used - Ribose vs Deoxyribose
Function - various vs data storage
State - single vs double stranded
40
What are the purines?
Adenine and Guanine
41
What are the pyrimidines?
Cytosine, Thymine and Uracil
42
Four basic steps in the overview of transcription
DNA is unwound
RNA is synthesized 5-3 following DNA sequence by RNA polymerase
DNA rewinds
mRNA is released
43
What is RNA in reference to the coding and template strand?
RNA is complementary to the template strand, because it is synthesized off the complementary strand
RNA is identical to the coding strand
44
Can either DNA strand be the template strand?
Yes
45
What are other names for the template and coding strand?
Template - antisense
Coding - sense
46
Where is RNA synthesized, where is it transported to?
Synthesized in the nucleus, transported to the cytoplasm
47
Is mRNA synthesis and degradation fast?
mRNA is rapidly synthesized and rapidly degraded, know bc of phage infection experiments with ecoli
48
RNA is translated from?
The template strand
49
3 stages of transcription in prokaryotes?
Initiation
elongation
termination
50
What are the functions of the 5 subunits in PROKARYOTIC RNA polymerase? (only need to rlly know two)
Sigma - initiation of transcription, recognizes promoter
Omega - chaperone activity, ensures correct folding
Alpha - assembly of core
Beta - ribonucleotide triphosphate binding site
Beta prime - DNA template binding region
51
What are promoters? What is the site where transcription starts? What are the two non coding regions of mRNA transcript?
Short orderly sequences of cis elements that are recognized by sigma RNA polymerase subunit
Only present on template strand to ensure the proper sense mRNA is made
+1 site is where transcription starts, between promoter and ATG start codon
Untranslated regions - UTR, 5UTR before AUG and 3UTR after stop codon
52
Generally, what are cis and trans elements?
Cis elements - same side - DNA shid
Trans elements - other side - Proteins n shid
53
Why might promoter regions have different sequences?
Polymerase binding is not perfect so variation does not affect function
Different genes are translated at different rates/ times so different promoter regions can call for that
54
What are the two kinds of termination in RNA transcription?
Factor dependent termination - requires a trans element (p-dependent) (rho)
Intrinsic termination - requires cis elements in the end of transcript (p-independent)
55
Explain factor dependent termination
RNA polymerase pauses at termination site
Rho binds RUT sequence
Rho destabilizes the transcriptional complex
mRNA transcript dissociates from RNA polymerase
56
Explain intrinsic termination
Poly A pauses RNA polymerase
GC rich inverted repeat forms a hairpin structure in mRNA
Causes dissociation and termination
57
What is the function of the sigma subunit in RNA polymerase?
Helps with initiation
58
5 differences between prokaryotic and eukaryotic transcription
Prokaryotic vs Eukaryotic
- One RNA polymerase vs. three RNA polymerases.
- Sigma factors vs. multi-subunit general transcription factors.
- Co-transcriptional translation vs. spatially separated transcription and translation.
- Simple transcript vs. processed transcript (cap, introns/exons, poly A tail).
- DNA (almost naked) vs. chromatin.
59
Four special challenges in eukaryotic transcription
Harder to locate the promoter because genome is bigger and genes are more spaced out
Transcription and translation do not happen at the same time due to eukaryotes having a nucleus
Eukaryotic DNA is wrapped up in histone complexes that need to be removed for transcription to occur
Transcription is more complex - polymerase has more subunits, transcription factors are required to recruit the polymerase
60
How many RNA polymerases are there in eukaryotes?
5, 2 are plant exclusive
61
What is a homozygote?
Each copy of the gene has the same allele
62
what does the 5' cap do?
Protects mRNA from nucleases
Recognition for signal translation`
**is a guanine
Added during transcription to prevent degradation
63
What are R loops formed from?
Introns that need to be spliced
64
What is the branch point adenine for?
Alternative splicing
65
What are the two main mechanisms for splicing in eukaryotes?
Self splicing - primary transcript with enzymatic activity - no protein involvement, no energy required
RNA Protein complex mediated splicing - enzymes and snRNAs needed to recognize and mediate intron excision (spliceosome) - requires ATP
66
What is a cofactor?
Compound or chemical used to catalyze a reaction - not a protein
67
4 true things about splicing
Only present in eukaryotes
Can be done with or without proteins
is a huge source of disease due to mistakes
occurs during transcription
68
What are isoforms?
Related but distinct proteins produced from alternative splicing
69
Are all RNA translated?
NO - some are introns that get cut out
70
What type of RNA is used in translation?
tRNA
71
What do the three RNA polymerases do in translation?
Pol1 - mRNA coding
Pol2 - decoding all RNA but 5sRNA
Pol3 - decoding tRNA and 5sRNA
72
What are the two ends of a tRNA
The anticodon pairs with the mRNA
The amino acid is covalently attached to the 3' end
73
What attaches an amino acid to its tRNA?
Aminoacyl-tRNA synthetase (ATS)
74
Where does codon specificity reside?
The anticodon, not the attached amino acid
75
How does rRNA fold?
by intramolecular base pairing
Structure is key for function
76
Explain the Shine-Dalgarno sequence
Process of initiation of translation in prokaryotes
sequence binds to complementary sequence in rRNA
-docks the small ribosomal subunit
77
3 steps of initiation of translation in prokaryotes
Small subunit 30s binds shine-Dalgarno sequence
tRNA binds to Psite
Large subunit 50s binds to 30s
78
4 steps of initiation of translation in eukaryotes
Small subunit 40s binds MET tRNA in Psite
Small subunit binds to 5'cap
Small subunit walks along mRNA to start codon
Large subunit 60s binds to 40s
79
How is translation terminated?
Release factor binds Asite with stop codon - stop codons do not have a corresponding tRNA
80
A mutation in an aminoacyl-tRNA synthetase causes the amino acid alanine to bind to the tRNA for tyrosine - What does this cause?
The protein will always have an alanine instead of a tyrosine
81
A ribosome is mutated so theres no functional A site, what is the first effect
Elongation cannot occur
82
Decoding mRNA happens at the ...
Aminoacyl (A) site
83
How many possible 3 base triplet combinations are there?
64 - but we only make 20 different amino acids - redundancy
84
What is the start codon usually?
MET - AUG/ATG
85
What happens when a single base pair is added into a coding sequence?
Frameshift mutation
86
Which coding sequence error is the least serious?
A silent mutation, because the functionality is the same
87
6 properties of the genetic code
-The genetic code is composed of nucleotide triplets
* The genetic code is non-overlapping
-coding sequences are never shared between genes (on the same or different frames)
* The genetic code is comma-free
-a mature transcript carries the whole, non stop, coding sequence of a gene
* The genetic code is degenerate
-there are more than one codon for a given amino acid
- wobbly third position
* The genetic code contains start and stop codons
* The genetic code is nearly universal
88
What is an amber mutation?
Premature stop codon introduced, also known as a nonsense mutation
89
What is a suppression mutation?
A mutation in the TRNA genes that change anticodon specificity
Basically if an amber mutation codes for a stop codon, but the trna mutation ignores the early stop codon and translation continues as normal - translated as tyr instead
90
What are prions?
self producing pathogenic proteins
91
Why do we think life might have started as RNA?
Can store info - not as well as DNA
Can transduce DNA messages to protein
Can read information and catalyze reactions
Can synthesize peptides
92
Somatic vs germinal mutations
Germinal mutations occur before the germline, in the germ cells, these mutations will be transmitted to progeny
Somatic mutations only impact the individual
93
Spontaneous versus induced mutations
spontaneous
- mostly from replication errors
- polymerase induced mistakes - slippage
- followed by defects in DNA repair mechanism
- occurs via the spontaneous mutation rate MU which is usually very low
Induced
- via known chemical and physical agents (mutagens)
- much higher frequency
94
What is a point mutation?
A single base pair change - SIngle nucleotide polymorphism (SNP)
-Can occur anywhere
- can be caused by DNA polymerase slippage causing indels
Base substitutions
- Transition - purine purine or pyrimidine pyrimidine
- transversion - purine pyrimidine
Base deletions - removal of one
Base insertions - addition of one
95
What is a silent mutation
encodes the same amino acid
no effect on phenotype
96
What is a missense mutation
Codes for a different amino acid
can be conservative where the amino acid is similar enough to retain protein function or chemically different and non conservative
97
What can point mutations affect (4)
Gene expression
mRNA stability
splicing events
how much protein is made
98
List four examples of DNA damage
abasic sites
base analogs
intercalation
backbone breaks
99
Mutation cause - Nucleotide mispairing
Altering the chemical geometry of nucleotides can change favorable hydrogen bonding and cause nucleotide to bond to the wrong one
100
Mutation cause - cellular environment (3)
Depurination - loss of a purine
Deamination - loss of an amine
Oxidative damage - idfk
101
What is a mutagen?
A chemical or physical force that can increase the mutation rate above baseline mu
102
What are the three things mutagens can do?
Replace a base in the DNA strand
Chemically alter a base pair leading to mismatch
Damage a base pair so that it cannot pair with anything else
103
6 types of mutagens
Chemical agents
- Alkylating agents
- ROS
- Intercalating agents - distorts double helix
- DNA adducts
- Base analogs
Physical agents
- UV/ radiation - forms pyrimidine dimers
104
What is the difference between a carcinogen and a mutagen?
All carcinogens are mutagens but not all mutagens are carcinogens
carcinogens cause cancer rip lol
105
What is a revertant?
Where an organism with a non viable mutation has an additional mutation that 'fixes' the effects of the original mutation
106
What is the mutagenic potential of mutagens>
The frequency of mutant animals that can survive after exposure to a mutagen reflects the number of times the reversion mutation (mutation 2) occurred in the same position in the same gene because of exposure to a mutagen
107
What is the Ames test?
A test used to help determine the mutagenicity ratio
done in Salmonella his complexes
Salmonella can only grow when HIS is on the growing medium, test to see if reversion happens, if it does salmonella will be able to grow on the plate without his added
Mutageneitc compounds used to increase mutation chance
every salmonella observed on plates indicate different events of reversion mutations
108
How is the mutagenicity ratio determined?
MR= total number of revertants / number of spontaneous revertants
test plate / control
No mutagenic effect - MR= 1
Mutagen - MR > 1
Kills cells - MR < 1
109
What are 6 major DNA repair systems?
Base excision repair BER
Nucleotide excision repair NER
Mismatch repair MMR
Translesion synthesis TLS
Homologous recombination repair HR
Non homologous end joining NHEJ
110
Four steps to DNA repair
Surveillance - must detect error post replication
Excision - enzyme removes or alters BPs involved
Polymerization - uses undamaged template to re polymerase bases
Ligation - reconnects any sugar sugar bonds
111
What is and what are the steps to base excision repair?
Removes damaged bases
relies on non affected strand
DNA glycosylases removes base
Damaged strand nicked by nuclease and removed by DNA polymerase
DNA polymerase adds correct base
Ligase seals strand
112
What is nucleotide excision repair?
For large damage affecting multiple base pairs, bulky adducts and pyrimidine dimers
Steps of NER:
1. damage detection
* global genome surveillance
* stalled RNA polymerase (transcription coupled
2. strand separation (helicases)
3. incision (nickase)
4. excision (~20bps) (nuclease)
5. polymerization (DNA replication factors and polymerases)
6. Ligation (DNA ligase
113
What is mismatch repair
Conserved from bacteria to eukaryotes.
* Active during DNA replication.
* Loss of MMR leads to an increase in mutation frequency due to
replication errors of ~100 fold.
1. Detection
* Mismatches during replication cause distortions in the DNA strand (loops) that are recognized
* Must detect new strand vs template strand
2. Incision – nick to allow excision
3. Excision – helicase unwinds and region around mismatch removed by nucleases
4. Synthesis – DNA polymerase fills the gap
5. Ligation - Ligase closes the incisions
114
Two types of double stranded break repair
Homologous Recombination (HR)
* Uses homologous chromosome as template.
* Active post-DNA replication
* More error-proof
* Used in meiosis to produce recombinant and non- recombinant chromosomes.
* Used in CRISPR gene editing to introduce precise changes or whole new gene sections
Non-Homologous End Joining (NHEJ)
* DNA strands join ends independently of complementarity.
* Active in dividing and non-dividing cells (does not require a template strand)
* More prone to introducing errors
* Used in CRISPR gene editing to introduce random indels
115
Non-Homologous End Joining (NHEJ)
Steps
1. Detection (Surveillance)
-- Proteins bind each end of the break to suppress further damage.
2. Strand Resection
o Recruitment of kinase and nuclease proteins
o remove 5’ and 3’ overhangs
o makes blunt ends
3. Polymerization
o DNA polymerase fills ends for ligation
4. Ligation
o Ligase ligates the two ends.
116
Homologous Recombination (HR)
Requires an undamaged template
o Sister chromatid
o Homologous chromosome
Steps
1. Detection
2. Strand resection
* Creates 3’ overhangs (sticky ends)
* required for strand exchange
3. Strand Exchange/invasion:
o 3’ overhang invades template strands
o Recombinase mediated
o forms a D-loop (displacement loop)
4. Polymerization
o Using the homolog/sister as template, the invading strand is
extended by polymerases.
These intermediates can then be processed by either
1. Synthesis Dependent Strand Annealing Pathway (SDSA)
2. Double Strand Break Repair Pathway (DSBR)
117
Homologous Recombination - Synthesis Dependent Strand Annealing Pathway (SDSA)
(reconstitution of original strand – no crossovers)
DNA helicases break invading off the homologous
template after polymerization.
* Original strands re-anneal
* Gaps are filled by DNA polymerase
* Strands re-connected by DNA ligase.
* There is no chance of strand exchange in this
scenario.
118
Homologous Recombination - Double Strand Break Repair Pathway
(results in crossed over recombinants)
1. Second strand invades, associates with template.
* This forms a two crossover intermediate structure referred as a double Holliday Junctions (Robin Holliday).
2. Resolution of intermediates
o endonucleases cleave HJs to release recombined or non-recombined products.
3. Polymerization and ligation
o Polymerases fill gaps between the strands and DNA ligases connect the cleaved strands.
119
What is the function of ligation?
To reattach the sugar phosphate backbone
120
What are the two things needed for gene cloning and what does it produce?
Introducing a desired DNA (insert) into a viable host molecule (vector)
Creates a recombinant molecule that can be propagated
121
What is a vector?
What we clone the desired gene into - can be viral or plasmid (bacterial DNA)
Vector is then introduced into host cell to replicate vector
122
What are plasmid vectors?
Circular, double-stranded DNA molecules present in bacteria.
* Range from 1 kb to over 200 kb.
* Foreign/recombinant DNA insert up to ~10kb.
* Replicate autonomously.
* Many carry antibiotic-resistance genes, which can be used as selectable markers.
123
3 essential assets of a functional plasmid for cloning
1 ORI - origin of replication
- allows plasmid replication independently of bacterial DNA
2 AMPR - selective marker for ampicillin resistance
3 POLYLINKER - specific location for inserting DNA
- has several viable restriction sites
124
How do you fuse DNA into a recombinant molecule?
restriction endonucleases/enzymes make site-specific cuts in DNA
* The nucleotide cut sequences are called restriction sites
- blunt end cutting
- sticky end cutting - easy to restick together
125
A primer is...
A short single stranded piece of DNA meant to prime in vitro reactions
126
What are selection strategies in cloning?
Select for AMPR - they will die if they don't have it
Select for presence of an insert - color based reactions to determine - Bgal turns blue in Xgal - can tell difference
127
How is phage selection done?
By plaque formation - want clear plaque = full of viruses
128
What is genetic engineering?
use of recombinant DNA tech to alter a phenotype
129
What is a transgene?
genetically engineered DNA to be introduced into a genome
130
How can you deliver transgenes into plant cells?
the Ti vector via agrobacterium, will contain elements that promote insertion of cloned sequences into host genome
131
How does CRISPR work?
Guide RNA (~20nt)
* designed to match specific site of interest
* Adjacent to PAM (NGG) sequence
2. Cas9 protein cuts DNA at a specific site
* Binds using guide RNA
* Causes double stranded break
3. DNA double stranded break is repaired by the cell
* NHEJ – errors likely
* HR – can insert sequence of interest
* By adding homologous DNA
132
4 things CRISPR can do
CRISPR can inactivate a gene of interest
* relies on NHEJ
* using 2 cut sites increases the likelihood of gene loss
CRISPR can add genes of interest
* relies on HR
* using 2 cut sites increases the likelihood wanted repair
* Added DNA contains homologous flanking regions and
gene of interest
* Replace gene (Ins1GFP)
* Add gene (Ins1 Ins1+GFP)
CRISPR can modify a gene/locus of interest
* relies on HR
* using 2 cut sites increases the likelihood wanted repair
* Added DNA contains homologous flanking regions and
modified gene
* Ins1 Ins1 with specific break to look at function
CRISPR can add modify gene regulation
* Cas9 protein modified as a gene regulator
* Binds to promoter region
* Can turn off/on specific genes as a transcription
factor (Topic 9)
* Causes permanently active Ins1
133
What do genome and cDNA libraries allow for?
Studying the genome and transcriptome (RNA) respectively
134
What is Sanger sequencing?
For short sequences with many copies – from PCR
* Works almost the same as PCR, copying DNA
* Uses: DNA polymerase Primers, dNTPs
* Also uses: dideoxynucleotides (ddNTPs) with fluorescent tags
* Polymerase adds ddNTPs to DNA chain which terminates polymerization
* Happens rarely and random as DNA is synthesized
135
What is the basic strategy for sequencing whole genomes (steps)
Most chromosomes are too large to be sequenced at once (for
now)- Instead, genomes are broken down, sequenced and re- assembled using software.
1. Break the genome into a pool of small, overlapping fragments.
* Uses multiple genome copies
2. Sequence fragments to create ”reads”.
3. Use computer algorithms to identify overlapping sequences
o Assemble reads together into larger continuous sequences (‘contigs’)
o The draft genome is then updated as gaps are filled
o better sequencing needed to correct errors or work out complex regions (usually because of repetitive sequences)
Shotgun sequencing
Illumina sequencing
136
Why does sequencing often require multiple copies of the same fragment?
To tell real signal from noise
To produce a signal bright enough to detect
To reduce the impact of synthesis mistakes
137
A contig is...
The assembled pieces of NGS
138
What are homologs, orthologs and paralogs?
Homologs - share a common ancestor and display conservation
Orthologs - Homologs that are syntenic - located in the same locus in closely related species
Paralogs - homologous genes that evolved as duplicates - same species
- genes often duplicate as genomes evolve
139
What happened with the cis elements in the androgen receptor
Why men dont have penile spines lmfao
140
What dictates phenotypes?
Gene expression
141
What are constitutive genes?
Genes that are continuously expressed in most cells
142
What are activators, what kind of regulation are they in?
Positive regulation
an activator is required to bind RNA polymerase and start transcription
Activators bind to activator binding sites
143
What are repressors, what kind of regulation are they in?
Negative regulation
Repressors prevent RNA polymerase from binding and starting transcription
Repressors bind to operators
144
What are activators and repressors?
Kinds of transcription factors?
145
How are transcription factors regulated?
on allosteric sites by inducers and co repressors
- named for how they affect transcription NOT transcription factors
Activator + inducer = ON transcription
Activator + co repressor = OFF no transcription
Repressor + inducer = ON transcription
Repressor + co repressor = OFF no transcription
146
What are inductible systems?
OFF by default
Negative control - Add inducer to repressor to remove it, enabling transcription
Positive control - Add inducer to allow activator to bind, enabling transcription
147
What is a repressible system?
ON by default
Negative control - Add a co repressor to a repressor to allow it to repress, blocking transcription
Positive control - Add a co repressor to an activator to remove it, blocking transcription
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What is an operon?
In prokaryotes, a group of genes that function as a single gene for transcription and regulation
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What is and what type of operon is the LAC operon?
Inductible - OFF by default
Converts lactose to glucose and lactose - lac operon is normally off, unless there's lactose (the inducer)
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What can the LAC operon only be transcribed in the presence of?
Lactose, cAMP-CAP
cAMP-CAP is inducer, will not be present in high glucose levels
You have to both remove the repressor and add CAMP to do it
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4 components of LACO
I - Inhibitor/ repressor - in trans
O - operator - in cis
P - promoter - in cis
ZYA - structural genes
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What happens when ZYA are mutated?
Cannot metabolize lactose, doesn't work
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What happens when I or O is mutated?
Disrupts ability of cells to inhibit LACO, meaning its ALWAYS on
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What happens when P is mutated?
Disrupts ability of RNA polymerase to bind meaning it is ALWAYS off
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Whats the deal with heterozygotic mutations in LAC operon?
Basically one functional I can go to either copy, only needs one functional copy to work
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Whats the deal with I^s?
Is dominant over I+, super repressor, means inducer cant bind, ALWAYS OFF no matter waht
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How is gene transcription controlled in DNA?
Both intracellular signaling and intercellular communication are important for transcriptional
regulation in eukaryotes
* Gene expression can be induced by environmental factors such as heat and light, and by
signaling molecules such as hormones and growth factors.
* Positive and negative gene regulatory proteins called transcription factors bind to specific
regions of DNA (response elements) and stimulate or inhibit transcription.
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A response element ... (enhancer)
is a cis factor that binds a transcription factor
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Overview of transcriptional regulation in prokaryotes and eukaryotes
In Bacteria RNA polymerase can bind all promoters (s factor); its function is increased by activators and decreased by repressors.
In Eukaryotes RNA polymerase typically cannot bind promoters on its own and several steps of protein association to DNA must happen before it can bind promoters with enough affinity to trigger transcription.
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What are two other types of transcription factors and response elements?
Enhancers (Cis)
* normally far from the gene
* often tissue specific.
* bind special transcription factors
* normally specific to a subset of genes
* bind to RNA polymerase or other TFs
Promoter-proximal elements (Cis)
* precede the promoter
* bind general transcription factors
* generic transcription factors bind promoter regions
* facilitates RNA polymerase binding
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What is the mediator complex?
is a co activator complex that promotes DNA activation but does not bind DNA
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A mutation causes the gene for a wing transcription factor to be within range for setae, what will happen?
Nothing, transcription factors act in trans, doesnt matter where DNA is, however if the mutation is in the ENHANCER (which act in cis,) it could have an effect
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What do enhancers do?
form enhanceosomes that help recruit transcriptional machinery
regulate promoter region via chromatin loops
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What is the GAL pathway?
Eukaryotic genes are not functionally organized in operons.
* Synchronous transcription of genes in the same pathway often relies on the
use of similar transcription factors.
* In the presence of galactose, the expression of the genes required for breaking
galactose to glucose are induced 1000x.
* This requires the independent transcriptional activation of all Gal genes at the
same time.
* a single TF, Gal4 binds an enhancer (UAS) present in the 5’ of all Gal4 genes
Gal4 has 2 functionally independent domains: DNA binding and Activation
Many transcriptional activator proteins are
modular: domains can be separated
functionally.
* Gal4 is an example for modular activator.
The DNA binding domain brings the Activation
Domain to the promoter, so it can help in
activating RNA polymerase
The domains are independent of each other
* the DNA binding domains does not
require the activation domain to bind DNA
* the activation domain can still bind
proteins associating with RNA pol, without
the DNA binding domain
What turns the Gal gene off?
* Off by default – needs galactose
* glucose
* Gal80 inhibits Gal4 activity in the absence of
galactose
* galactose-sensing inhibitory protein
* binds the activation domain of Gal4,
preventing in from activating genes in the
absence of galactose
* ‘co-repressor’
* When galactose is present, Gal80 disassociates
from Gal4, allowing it to activate genes
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What does RNAi RNA interference do?
Regulate gene expression
Reduces expression of target genes by removing mRNA to prevent translation
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3 things RNAi does
RNAi (Dicer) detects dsRNA
dsRNA is bound by Dicer
* Dicer is a nuclease that cleaves dsRNA into smaller (21- 22bp) RNAs
* called small interfering RNAs (siRNA) if derived from long dsRNAs
* or mature micro RNAs (miRNA)s if produced from miRNAs
RNAi (RISC) detects (siRNA / miRNA) (RNA-induced silencing complex)
Dicer products (siRNA/miRNA) have overhangs important for recognition by RISC
* RISC process siRNAs/miRNAs
* only the complementary RNA strand (anti-sense/guide RNA) remains
* Sense strand/mRNA removed
RNAi (RISC) detects and destroys complementary RNA
RISC mediates the association (via the guide RNA sequence) and cleavage of the mRNA substrate, essentially reducing gene expression for the respective gene
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What is DNA wound around and what does this form
DNA is wound around histones to form chromatin
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How does chromatin influence gene transcription?
MACROLEVEL:
* Chromatin compacts DNA uniformly along chromosomes
* Regions with high chromatin level tend to be silenced transcriptionally
* Regions with loose chromatin are more exposed to the transcriptional machinery
GENE LEVEL:
* Nucleosome can be rearranged, and the DNA exposed at specific loci.
* This depends on reversible chemical modifications on the histone sequence that change the affinity of histones to other proteins and DNA and reconfigure the local chromatin arrangements, exposing regulatory sites on the 5’ of promoters or hiding them.
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What can spreading heterochromatin cause?
silences genes, covers it, causes position effect variegation
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Position effect variegation is responsible for ...
Variable expressivity
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What are insulators?
Insulator – cis-element that blocks the action of an
enhancer (sometimes called a ‘silencer’)
* Insulator-binding proteins form complexes that can fold DNA and restrict long range DNA interactions
* These regions block the effect of enhancers by changing their optimal distance to the targeted promoter.
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What is chromatin remodelling?
Chromatin remodeling - the process of changing histone position
* Chromatin remodeling helps transcription by removing histones blocking the promoter, enhancers, etc..
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Can histones be modified?
yes, very much so, can have writiers, readers, erasers, remodelers that do stupid shit
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Heterochromatin ...
grows and shrinks
prevents transcription
can be modified via protein remodelers
made up of nucleosomes
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What does DNA methylation do?
Methylation is heritable
~20% in humans (Villicaña and Bell 2021)
Methylation can indicate tissue type
turn off brain proteins in your muscles
Play a role in disease
methylation of the promoter PM20D1 affects Alzheimers
DNA methylation (Vertebrates)
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What can genetic drift do?
Can remove or fix alleles in a population
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What is the gene pool?
Sum of all alleles present in the breeding members of the population at any given time
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Assumptions in hardy Weinberg ideal population
1. Infinite in size (no inbreeding)
2. Have random mating (no sexual selection)
3. All genotypes are equally fit (no differential death)
4. No migration (no external influence)
5. No mutations (no new alleles)
In an ideal population, allele frequencies will not change
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What is gene flow?
The transfer of alleles between populations
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What is genetic drift?
Allele frequencies will change between generations due to chance
Affect is stronger in small populations
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What is the inbreeding coefficent?
F = (1/2)^n where n is number of individuals in the inbreeding loop not counting the one individual
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3 forms of selection
Directional selection
* Allele frequencies move in one direction until the allele is fixed or lost
* Positive selection: increasing the frequency of a favorable mutation (towards fixation). - Ex. When a positive allele confers a new adaptation
* Purifying selection: decreasing the frequency of deleterious mutation (towards
elimination).
Balancing selection
* Heterozygotes (A/a) are better adapted than either homozygote
* More common with multiple alleles or non-mendelian inheritance
- Can lead to lots of genetic diversity
Disruptive selection
* Heterozygotes (A/a) less fit than either homozygote
* More common with multiple alleles or non-mendelian inheritance
* Can lead to speciation – splitting phenotypes
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What is a selective sweep?
The fixation of a selected haplotype in a population
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Simplified LAC operon pathway
Glucose present = no cAMP = operon is inactive
Glucose absent = cAMP = operon turns on BUT ONLY IF LSCTOSE IS THERE
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Whats important to know about snRNAs?
snRNAs can associate with proteins to form SNRPs which are a part of spliceosomes that splice mRNA
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What does each of the repair pathways fix?
BER - non bulky damage, alkylation, oxidation, deamination, depurination
NER - Pyrimidine dimers and bulky adducts
MMR - replication errors like base mismatch and loops
NHEJ (quick fix) - double stranded break
HR - (better fix) - double stranded break
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4 steps to fixing DNA damage?
Surveillance
Excision - nuclease
Polymerization - DNA polymerase
Ligation - ligase
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APE sites
A - aminoacyl tRNA binds
P - forms peptide chain
E - exit, where tRNA goes after dropping AA
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What do i actually need to know about the GAL pathway?
Its the eukaryotic equivalent to the operon
multiple genes involved in making galactose
off by default
GAL4 is transcription factor - inducer
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Lytic vs Lysogenic phage cycles
Lytic - replicates in cell, kills cell, fast and easy to study
Lysogenic cycle - integrates into genome, does not kill cell
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What do enhancers do?
- cis
- enhance gene regulation
- specific
- binds TF
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A neutral allele appears, what wont affect its frequency?
Selection pressure
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What is linkage disequilibrium?
D = P(ab) - Pa(Pb)
if it does not equal 0 then linkage disequilibrium could be in play
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Fitness (W)
w > 1, growing population
w= 1, stable population
w < 1, declining population
