Final Review (after midterm) Flashcards
1
Q
Central dogma
A
DNA (transcription)- RNA (translation)- amino acid chain (folding)- protein
Explains the flow of genetic information from DNA to phenotype
2
Q
RNA
A
RNA is typically single stranded
Ribonucleic acid
RNA’s shape can be as important as its sequence
Has OH group in position 2 of the ribose
3
Q
DNA
A
Is double stranded.
Has H group in position 2 of the ribose
4
Q
RNA Nucleosides
A
Pyrimidines
Purines
5
Q
Messenger RNAs
A
mRNAS
coding
Carries gentic information from DNA to the ribosomes
Short lived mobile blueprint molecules for protein synthesis
6
Q
Small nuclear RNAs
A
(snRNAs)
Non-coding
Structural componenets of spliceosomes
7
Q
Transfer RNAs
A
(tRNAs)
Non-coding
Adaptors between amino acids and mRNA codons
8
Q
Ribosomal RNAs
A
(rRNAs)
Non-coding
Structural and catalytic components of ribosomes
9
Q
Micro RNAs
A
(miRNAs)
Non-coding
Short single-stranded RNAs that block expression of complementary mRNAs
10
Q
Overview of Transcription
A
- DNA is unwound
- RNA is synthesized following DNA sequence by RNA polymerase (5’-3’)
- DNA rewinds
- mRNA is released
11
Q
RNA synthesis
A
The precursors are ribonucleotide triphosphates.
Only one strand of DNA is used as a template
RNA chains can be initiated de novo (no primer required)
Uracyl instead of thymine
Catalyzed by RNA polymerases
12
Q
How fast is RNA synthesized?
A
50+nt/s Prokaryotes
20nt/s Eukaryotes
13
Q
How long does mRNA last?
A
Seconds to minutes: prokaryotes
Minutes to days: eukaryotes
14
Q
Cis elements
A
same side->DNA
15
Q
Trans elements
A
Across->proteins
16
Q
Initiation of Transcription (1):
A
RNA polymerase binds to promoter.
Promoter recognized by the RNA polymerase sigma subunit.
ONLY present on the template strand, ensures only sense mRNA is made.
17
Q
Promoters
A
Short specific DNA sequence (cis element)
18
Q
Initiation of Transcription (2)
A
RNA polymerase unwinds the two DNA strands to expose a single stranded template.
Formation of phosphodiester bonds between the first few ribonucleotides in the nascent RNA chain
Sigma is released
19
Q
Elongation
A
RNA chain grows from 5’ to 3’
RNA polymerase continues to unwind DNA: includes helicase activity
DNA re-winding: reforming hydrogen bonds between the two DNA strands: no energy cost
20
Q
Termination
A
RNA polymerase decouples from DNA template, RNA strand is released.
21
Q
Intrinsic termination
A
Required cis elements at the end of transcript (p-independent)
GC rich region creates hairpin
Pulls mRNA off
22
Q
Factor-dependent termination
A
Requires a trans-element rho (p-dependent)
Binds to Rho-utilization site
Disassembles RNA polymerase
23
Q
RNA polymerase II
A
Transcribes mRNA and some functional (non-coding) RNAs
Assisted by transcription factors-protein complexes that help it recognize and initiate transcription at the promoter
Most promoters contain a TATA box
24
Q
“TATA”
A
less promoters use other elements to direct RNA polymerase II
25
Special challenges in eukaryotic transcription
Harder to locate promoter
Transcription and translation are decoupled
Eukaryotic DNA is wrapped up around proteins
Eukaryotic transcription is more complex
26
Transcription initiation in eukaryotes
Ordered addition of transcription factors pre-initiation complex.
Recruits RNA polymerase II
RNA polymerase starts synthesis
Phosphorylation of RNA polymerase C-terminal domain recruits mRNA processing proteins in order: CTD- domain at -C end of a protein, capping splicing and poly-adenylation
27
Capping
Co-transcriptional processing of RNA during elongation:
unusual 5'-5' phosphodiester bond, methylated guanine
protects mRNA from nucleases. recognition signal for translation
28
SPLICING
Co-transcriptional processing of RNA during elongation:
Most eukaryotic genes contain noncoding sequences called introns that interrupt the coding sequences, or exons.
Introns are excised from the RNA transcripts prior to their transport to the cytoplasm
29
Introns
Intragenic regions
Only eukaryotes have them: certain viruses carry sequences from host eukaryotic genomes with introns.
Noncoding sequences located between coding sequences.
Removed from the pre-mRNA and are not present in the processed/mature mRNA.
Are variable in size and may be very large
30
EXOns
Expressed regions
Are composed of the sequences that remain in the mature mRNA after splicing.
Comprise the coding region.
31
two main mechanisms of spicing evolved in eukaryotes
self splicing
RNA/protein complex mediated splicing
32
Self splicing
Primary transcript with enzymatic activity (ribozyme). No protein involvement.
No energy required
33
RNA/protein complex mediated splicing
Enzymes/snRNAs needed to recognize and mediate intron excision (spliceosome).
Reconfiguration of the splicing machinery requires ATP.
34
In some protozoa
Introns splice themselves
A guanosine is used as a co-factor
35
Co-factor
A compound/chemical used to catalyze a reaction
Not a protein
36
Example of self splicing:
Autocatalytic Splicing of rRNA in tetrahymena
37
Spliceosome-dependent splicing
RNA/protein structure
Excises introns from nuclear pre-mRNA
five snRNAs: U1, U2, U4, U5 and U6 (small nuclear RNAs)
Some snRNAs associate with proteins to form SNRP
38
Splicing and disease
60% disease-causing mutations in humans affect splicing (not coding sequences)
Abnormal splicing common in cancer cells
39
Alternative splicing produces related but distininct proteins
isoforms
40
POLY ADENYLATION
Co-transcriptional processing of ENA during elongation
41
The 3' poly (A) tail-poly adenylation
Polymerase stalls-end of transcription signal at the 3' end (GT rich)-DSE
Endonuclease activity cleaves transcript downstream of an AU rich region- AAUAAA
Poly A polymerase recognizes processed transcripts as templates to add poly A tail
42
Purpose of the 3' poly (A) tail
Enhances mRNA stability in the cytoplasm
Mediates mRNA transport across the nuclear envelope
43
RNA editing and modification
RNA can be changed after transcription the functions are not all clear affects RNA structure, function and stability.
44
Transfer RNAs (tRNAs)
Small (90bp)
Adaptors between mRNA and amino acids.
Two ends: anticodone (pairs with the mRNA), amino acid (covalently attached to the 3' end)
Each anticodon has its own tRNA with a specific amino acid.
Contains chemically modified nucleosides to avoid mispairing with the codon
45
The structure of transfer RNA
tRNA folds to formspecific 3D structures, common among tRNAs.
The 3D structure of the tRNA is important for its function: serves as substrate for amino acid linkage, enters and moves across ribosomal compartments.
46
Aminoacyl-tRNA synthetase (ATS)
Attaches an amino acid to its specific tRNA
21 different ATS exist, one for each amino acid that specifically interacts its corresponding tRNAs
47
The specificity of a tRNA depends on
matching the correct residue (aa) to the corresponding anticodon.
48
The aa specificity depends
primarily on the activity of aminoacyl tRNA synthases.
Connects the right amino acid to the right tRNA
49
Ribosomes
decoding hubs
50
E.coli
Seven rRNA genes distributed among three sites on the chromosome.
51
rRNA folds up by
intramolecular base pairing
52
Initiation in prokaryotes
The Shine-Dalgarno sequence.
1. Small subunit (30S) binds Shine-Dalgarno sequence: with the help of initiation factors.
2. tRNA binds to P site: special formyl-Methionine (fMet) only used for initiation, with the help of initiation factors.
3. Large subunit (50S) binds to 305: with the help of initiation factors.
52
Mechanism of translation
Stages: polypeptide chain initiation, chain elongation (peptide bonds), chain termination
53
Initiation in eukaryotes
1. Small subunit (40S) binds to Met-tRNA in P-site: with the help of initiation factors.
2. Small subunit (40S) binds to mRNA 5'-cap: with the help of initiation factors.
3. Small subunit 'walks' along mRNA to start codon (AUG): lands at P site, with the help of initiation factors
4. Large subunit (60S) binds to 40S: with the help of initiation factors.
54
Termination of translation
1. Stop codons bind release factors, not tRNAs: stop codons are the only codons in the genetic code without a corresponding tRNA in nature.
2. Release factor binds A site with stop codon.
3.Translation machinery disassembles: the absence of tRNA terminates translation.
55
Stop codons
UAG
UAA
UGA
56
Properties of the genetic code
Composed of nucleotide triplets.
Is non-overlapping: coding sequences are never shared between genes.
Is comma free: a mature transcript carries the whole, no stop, coding sequence of a gene.
Is degenerate: there are more than one codon for a given amino acid.
Contains start and stop codons.
Nearly universl
57
The genetic code in non-overlapping
Genes have one single coding frame, such that every nucleotide only participates of a single codon, never two or three.
58
The genetic code is comma-free
There are no pauses in the coding transcript
Once the mRNA is processed, the entire information from a gene must flow from the first ATG to the first STOP codon without interruptions.
59
Purines
Adenosine and Guanosine
60
Pyrimidines
Cytosine
Uridine and thymine
61
Inosine (I)
is a purine RNA dericative formed by deamination of adenine-> RNA modification
62
Wobble rules
identifies base pair interactions between mRNA (3' end of codon) and tRNA (5' end of anticodon) that do not follow normal pairing rules (A-T and C-G)
63
PRION
Protein and infection
'Self replicable' proteins in the analogous sense that DNA or RNA are self replicable nucleic acids
64
Prion diseases
Ex: Creutzfeld-Jacob (transmissible spongiform encephalopathy)
Rare, degenrative fatal brain disease, Chracterized by protein aggregates in the brain, triggered by presence of a misfolded prion protein
65
Germinal mutations
Only mutations in the germ cells will be transmitted to the progeny.
66
Somatic mutations
Will impact the individual where the mutation occurs but are evolutionary irrelevant.
67
Mutations
Create phenotypic variability but also threatens the cell.
Are random and hertiable changes in the sequence of DNA that can no longer be repaired.
Often have deleterious consewuences, but they can also be innocuous or beneficial to the cell/organism
Key for the process of evolution
68
The reigning "paradigm" of mutations
Mutations can occur in any cell at any time, and their occurence is 'random'
69
DNA types of damage
Abasic sites (loss of nucleotide, not backbone)
Base mismatches
Modified bases
Inter and intra strand crosslinks
Double stranded breaks (DSBs)
70
DNA damage
occurs due to chemical or physical stress on the double helix
71
Spontaneous mutations
Mostly from replication errors.
Polymerase-induced mistakes cuasing mutations (slippage)
Followed by defects in the DNA repair mechanism
72
Spontaneous mutation rate
m is very low and varies according to: gene size, domains on the chromosome, genome chracteristics (species-specific), cell age
Low frequency
Eukaryotes: once every 100,000,000 replications
73
Induced mutations
via known chemical or physical agents (mutagens)
Base analogs, hydroxylating agents, alkylating agents, deaminating agents, intercalating agaents, UV radiation, ionizing radiation.
Higher frequency
Ethylmethanesulfonate (once every 100 replications)
Commonly used to induce mutations and study gene function
74
Point Mutations
Single base pair change=single nucleotide polymorphism (SNP)
Can occur anywhere in the genome: coding regions, intergenic regions, non-coding regions of genes (promoters, UTRs, Introns)
75
Types of point mutations
Base substitutions
Base deletion
Base insertion
76
Base substitutions
(4) Transition (purine replaced by a purine)[A=G];[G=A] or (pyrimidine replaced by a pyrimidine) [C=T]:[T=C]
(8) transversion: (purine replaced by a pyrimidine)[A=C];[A=T]:[G=C]:[G=T]
(pyrimidine replaced by a purine) {C=A]:[C=G]:[T=A]:[T=G]
77
Base deletion
the removal of one base pair
78
Base insertion
the addition of one base pair-> indels (insertions and deletions)
79
Silent mutations
encodes the same amino acid.
Usually the third position of a codon: degenerate nucleotide
No effect on the phenotype
Synonymous
80
Missense Mutations
Non-synonymous
Codes for a different amino acid:
chemically similar- conservative: may retain protein function
Chemically different- Non-conservative: more likely to affect protein function
81
Nonsense Mutations
Non-synonymous
Changes to a STOP codon (UAA, UAG, UGA)
Generates truncated protein
Likely severe
82
Frameshift mutation
Removal or addition of base pairs disrupts the triplet reading frame.
The result is the translation of an abnormal series of aa downstream from the indel and the increased chances of a premature STOP codon producing a truncated protein.
Indels (insetions/deletions) that are multiple of three reconstitute the frame downstream of the last mutation site
83
Mutation cause I
Error in replication
DNA polymerase slippage causes indels during replication: usually of repeated regions
84
Mutation cause II
Nucleotide mispairing
Energetically favorable hydrogen bonding ensures DNA strand complementarity
85
Ionization
Gain or loss of an electron
86
Mutation cause III
Cellular environment
Depurination
Deamination
Oxidative Damage
87
Depurination
Loss of a purine base (G or A)
Efficiently repaired by endogenous DNA repair mechanisms
88
Deamination
removal of an amine (-NH2) group from C, A and G
Can alter base pairing
89
Oxidative Damage
Reactive Oxygen Species (ROS) [H2O2, O2-, OH-]: byproducts of aerobic metabolism in mitochondria, induced by ionizing radiation
Damages DNA: creates mismatched base pairing, by directly breaking DNA double strands
90
Mutagen
A chemical or physical force that can increase the mutation rate above background
91
Mutagens can:
a) replace a base pair in the DNA strand.
b)chemically alter a base pair leading to a mismatch
c) damage a base pair such that it cannot base pair with any other nucleotide
92
Type of mutagens
Chemical agents: alkylating agents, ROS, intercalating agents, DNA adducts, Base analogs
Physical agents: UV/ionizing radiation
93
Alkylating Agents
Addition of an alkyl group (usually CH3 or C2H5) to a nucleotide.
disrupts correct base pairing
94
Bulky adducts
Attach nitrogenous bases: removal of the adduct causes apurinic site-mutation
95
Base Analogs
Chemically similar to A,C,T,G
Can be incorrectly incorporated by DNA polymerase
Can be mutagenic if analog is likely to mispair
96
Intercalating agents
Inserts between base pairs
Distorts double helix
Increases DNA polymerase slippage
97
UV
Covalently links neighboring pyrimidines: forms pyrimidine dimers, can't be recognized by DNA polymerase, stops DNA repliaction
Often results in transition mutations
98
Ionizing Radiation
Indirect: causes reactive ozygen species (ROS)
ROS: creates mismatched base pairing, directly breaking DNA double strands( abasic sites, single and double strand DNA breaks)
99
Carcinogens
Compounds that can lead to tumor development.
Many induce mutations that de-regulate a cell's ability to stop proliferating
100
Mutagenicity Ration (MR)=
total number of revertants/number of spontaneous revertants
If a compound has not mutagenic effect, MR=1
If a compound shows signs of being mutagenic, MR>1, if a compound kills the cells, MR<1
101
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)
102
The modus operandi of DNA repair systems
Surveillance(error detection post replication)
Excision(enzyme removes or alters the bp(s) involved)
Polymerization(uses undamaged template/homolog to re-polymerase the removed bases)
Strand ligation (reconnects any sugar-sugar bonds in the repaired strands)
103
Direct repair
Some DNA damage can be directly reverted using specific enzymes that identify altered specific nucleotides
104
Base Excision Repair
Removes and replaces damaged bases: caused by alkylation, oxidation and deamination.
Relies on complementarity on the non-affected strand to correct the mistake
105
Base Excision Repair steps
1. Detection
2. Excision
3. Polymerization
4. Ligation
106
Nucleotide Excision Repair
For large damage affecting multiple base pairs, bulky adducts and pyrimidine dimers
107
Nucleotide Excision Repair Steps
1. Damage detection
2. Strand separation (helicases)
3. Incision (endonuclease/nickase)
4. Excision (20bps)(nuclease)
5. Polymerization (DNA replication factors and polymerases)
6. Ligation (DNA ligase)
108
Xeroderma Pigmentosum (XP)
Individuals with XP are sensitive to sunlight.
the cells of individuals with XP are deficient in the repair of UV-induced damge to DNA.
NER pathway impaired.
Individuals with XP may develop skin cancer or neurological abnormalities
109
Nucleotide Excision Repair genes
XPA, XPB, XPC, XPD, XPE, XPF, XPG
110
Mismatch repair
Conserved from bacteria to eukaryotes.
Active during DNA replication
Loss of MMR leads to a 100 fold increase in mutation frequency due to replication errors.
111
Mismatch repair steps
1. Detection
2. Incision
3. excision
4. Synthesis
5. Ligation
112
Hereditary non-polyposis colorectal cancer (HNPCC)
Defects in MMR because of mutations in Msh2/Msh6/Mlh1 and Pms1
Autosomal dominant disorder
Increases predisposition to several cancers because of defects in DNA repair.
Tumor arises when the wildtype copy is lost from heterozygous tissues
113
Double stranded break repair
Most cytotoxic damage to DNA
Affects both DNA strands
Cannot use template to repair
repair is critical: can lead to chromosomal abnormalities or cell death.
Also used in meiosis
Two types: homologous recombination, non-homologous end joining
114
Mutations in DSB repair genes
lead to a serioes of hereditary neurodegenerative, developmental disorders, immunodeficiencies and cancer predispositions
115
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
116
Non homologous end joining (NHEJ)
DNA strands joined independent of compementarity.
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.
117
NHEJ steps
1. Detection
2. Strand resection
3. Polymerization
4. Ligation
118
HR steps
1. Detection
2. Strand resection
3. Strand exchange/invasion
119
Synthesis dependent strand annealing pathway (SDSA)
Reconstitution of original strand-no crossovers
Excision, polymerization, and ligation all occur together: DNA helicase breaks invading off the homologous template after polymerization, original strnds re-annea
No chance of strand exchnage- no crossovers
120
Polymerase chain reaction
PCR
"making and isolating DNA"
An in vitron system to amplify DNA fragments.
121
PCR steps
1. Add reagents together in a tube.
2. Denature: heat to seperate DNA strands-> 95
3. Anneal: cool slowly, allowing primers to bind- 60
4. Extend: heat to working temperature of Taq DNA polymerase- 72
5. Repeat
6. Produce exponentially more target
7.....
8. Profit
122
Measuring DNA
Agarose gel electrophoresis
123
Agarose gel electrophoresis
A system to measure and seperate DNA fragments.
DNA is negatively charged: migrates towards the positive pole: running an electric current through the gel moves DNA.
DNA fragments will seperate from each other based on size (bp): long DNA fragments migrates slower through dense agarose matrix.
Ladder is run along side to determine band size.
Can be used to detect the presences of specific DNA. can be used for genotyping.
124
Plasmid vectors
Cicular, double stranded DNA molecules present in bacteria.
Range from 1 kb to over 200 kb.
Possible foreign/recombinant DNA insert up to 10kb
Replicate autonomously
Many carry antibiotic resistanct genes, which can be used as selectable markers.
125
In vivo
in life
126
Gene cloning
isolation and amplification of a given gene; introducing a desired DNA molecule into a viable host molecule
Creates a recombinant molecule that can be introduced and propagated in vivo inside a host cell
Can be extracted and purified
127
Recombinant DNA molecule
two or more different DNA strands joined together
128
Essential assets of an engineered plasmid for gene cloning
1. Ori
2. Amp^R
3.Polylinker/multiple cloning site
129
Ori
origin site of replication.
Allows plasmid replication independently of bacterial chromosomal DNA
Must replicate in live bacteria
130
Amp^R
Selective marker
Allows for selection of only bacteria that carry it
Ampicillin resistance
must be selected on live bacteria
131
POLYLINKER/multiple cloning site
Specific location for inserting DNA of interest.
Unique restriction digest sites.
Must allow for several options for safe cloning of inserts.
132
Phages
modified viral DNA
132
Plasmids
modified small bacterial non-chromosomal DNA
133
Growth Hormone Deficiency (GHD)
Pituitary makes insufficient amounts of gorwth hormone.
Children with GHD have short stature and delayed sexual maturity.
Treament requires supplementation with Human growth hormone (HGH).
Used to use human cadavers until 1980s.
Synthetic HGH produced in genetically modified bacteria
134
Producing human growth hormone in bacteria requires
1. Precise (directional) cloning of the coding sequence of the human gene (cDNA)
2. Downstream of a bacterial promoter and sequences required for translation
135
Selections Strategies in Cloning:
(1) selection for plasmid: ensure that the only bacteria with the plasmid survives, antibiotic resistance
(2) Selection for insert: identify bacteria with recombinant plasmids (has insert), blue/white selection
136
Genetic engineering
The use of recombinant DNA technology to alter an organism's genotype
137
Transgene
genetically engineered DNA to be introduced to a genome
138
Transgenic organism
Organism that contains a transgene
139
Genetically modified organism (GMO)
organism that is genetically engineered. Used more frequenctly in the context of commercial organisms.
140
Genetic engineering can be used for
Biotechnology
Research
141
Bioinformatics
Annotating whole genome sequencing
142
Homologous genes
Share a common ancestor and display significant sequence conservation
143
Orthologs
Homologous genes that are synthetic
Located in the same genetic locus in closely related species
144
Paralogs
Homologous genes that evolved as duplicates
Genes often duplicate as genomes evolve
Arose from a gene duplication in a common ancestor
145
Pseudogenes
relics that reveal evolutionary ancestry
146
Catabolic pathways
Breakdown substrates, induction of enzyme synthesis
147
Anabolic pathways
Builds products
Repression of enzyme stimulus
148
Inducible systems
off by default
149
Repressible systems
on by default
150
The lac operon
Is transcribed only in the presence of lactose and cAMP-CAP.
Normally inactivated-off
Saves energy
Lactose ins the inducer: induces transcription, inactivates repressor
151
The trp Operon
A repressible system
Synthesizes the amino acid tryptophan
If there is no trp, then the trp codons can't get translated
152
Enhanceosome
large protein complex that acts synergistically to active transcription
153
Insulator
cis-element that restricts enhancers: sometimes called a silencer
154
Chromatin remodeling
the process of changing histone position
Helps transcription by moving histones blocking the prmoter, enhancers, etc.
155
Epigenetics
is heritable traits excluding DNA sequence: based on 'parental' 'environment=lamarckian
156
Population
A group of individuals on the same species in the same place and time
157
Population genetics
Studies how alleles "flow" down generation in a population.
Attempts to make sense of evolutionary trends.
158
Haplotypes
The combination of alleles from multiple loci that segregate together
159
A gene pool
The sum of all alleles present in the breeding members of a population at a given time,
160
Hardy-Weinberg principle (HWP)
Expresses a mathematical relationship between allelic and genotypic frequencies
Can predict genotypic frequencies in an "ideal" population based on the allele frequencies and vice versa.
161
Ideal populations are
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)
Allele frequencies will not change
Only exist in theory
162
Positive mating
like attracts like
163
Disassortative mating
Opposites attract
164
