Unit I Flashcards
What are the different forms of energy and their sources?
Kinetic (radiant, thermal, mechanical, electrical) Potential (chemical bonds, concentration gradients, charge separation, redox pairs)
What is the 1st law of thermodynamics?
Energy is always conserved. It can be converted, but not created/destroyed
What is the 2nd law of thermodynamics?
Entropy of the universe always increases
Gibbs Free Energy equation
del_G = del_H - T*del_S
Negative del_G means _____
reaction is favorable. Will occur spontaneously.
Positive del_G means ______
reaction is unfavorable. Will not occur spontaneously.
Rate of a reaction is determined by _____
activation energy
Gibbs Free Energy (redox)
del_G = -n*F*del_E
2 classes of high energy bonds
Thioester & Phosphate (phosphoanhydride)
In hydrolysis of high energy bonds, the free energy of products is _______ than that of reactents
less
As electrons pass from compounds with low to high E, free energy is _____
released
Purines
Adenine & Guanine
Pyrimidines
Thymine & Cytosine
Solubility of nucleotide components
nucleotide > nucleoside > base pyrimidines > purines
Diseases related to nucleotide solubility
Gout & Lesch-Nyhan (accumulation of purines)
Phosphodiester bonds are between _____ and _____ of nucleotides
5’ phosphate and 3’ hydroxyl
Polarity of DNA/RNA is ______ to ______ because ______
5’ to 3’; Replication/transcription are 5’ to 3’
DNA was established as genetic material through experiment involving _____
live-nonvirulent bacteria and DNA from dead-virulent bacteria placed in solution. This resulted in live, virulent bacteria.
Chargaff’s Rule
–> purines = pyrimidines
i. e. : (A+G) = (T+C) and in base pairing: G=C, A=T
- The ratio of G+C / A+T is different for each organism
Describe DNA structure
- right-handed double helix strands are anti-parallel
- The sugar/phosphate is on outside of the helix
- bases are inside of helix geometry, this allows only A:T and G:C base pairs
How is DNA structure stable?
H-bonding between base pairs and stacking energy (hydrophobic interactions) balances the negative charge from phosphates
_____ G:C content increases the stability of DNA because _____
- higher
- G:C base-pairs have 3 H-bonds
What are 7 types of DNA damage?
Methylation, Deamination, Depurination, UV, hydroxylation, alkylation, intercalation
What drug acts like an alkylating agent?
cisplatin
What drug is an intercalating agent (alters structure of DNA)?
Actinomycin D
UV light damages DNA by creating _____
thymine dimers
Deamination of 5’-methylcystosine results in _____
Thymine
Deamination of cytosine results in _____
Uracil
What factors affect melting temperature of DNA?
- salt concentration (direct)
- pH extremes
- DNA chain length (direct)
- G:C content (direct)
RNA is more prone to hydrolyzation because _____
2’ hydroxyl is prone to nucleophilic attack
RNA is usually _____ stranded
single
What is puromycin and how does it work?
it is a nucleotide analogue that mimics tRNA acceptor stem (3’ end), thus it inhibits translation.
The _____ of a ribosome does not contain proteins.
the core
Translation occurs in _____ of ribosome
the large subunit
During translation, mRNA slides along the _____ of the ribosome
small subunit
What are the classes and types of RNA?
- Structural (rRNA, tRNA, snRNA, snoRNA)
- Regulatory (miRNA, siRNA)
- Informational (mRNA)
Regulatory RNAs are made from _____ parts of genome
-non-coding
Replication origins are recognized by _____
origin binding proteins
DNA is melted/unwound by _____
DNA helicase
Torsional stress is continually relaxed at the replication fork by _____
DNA gyrase
What do SSB proteins do?
protect unwound, single-stranded DNA during replication
The RNA primer used for replication is made by _____
Primase
Elongation of DNA during replication is done by _____
DNA Pol III
What are the functions of DNA pol I?
–> 5’ to 3’ exonuclease (remove RNA primer)
–> 5’ to 3’ DNA synthesis
–> 3’ to 5’ exonuclease (proofreading)
What does DNA ligase do?
-Joins Okazaki fragments together after the RNA primer has been overwritten by DNA pol I
DNA pol III is processive because _____
it holds on to DNA via a sliding clamp (high replication rate)
Describe steps of DNA replication
- origin binding proteins bind to replication origin (AT rich sequence)
- DNA Helicase unwinds DNA
- SSB proteins and DNA Gyrase bind to each DNA strand.
- Primase synthesizes RNA primer
- DNA Pol III extends leading and lagging strands
- DNA Pol I replaces RNA primer
- DNA Ligase ligates DNA fragments together
What is the error rate of DNA pol?
10^-6 ~ 10^-8
Telomerase is a _____ dependent _____ polymerase (aka reverse transcriptase)
RNA; DNA
How does DNA Pol know which base to incorporate into the growing DNA chain?
-The presence of correct H-bonds between bases & common geometry of base pairs
Telomerase is only active in _____ cells
stem cells and cancer
Direct reversal repairs what kind of DNA damage?
thymidine dimers alkylation strand break
What repairs DNA strand breaks?
DNA Ligase
what repairs thymidine dimers?
-photolyase–> these are DNA repair enzymes that repair damage caused by exposure to ultraviolet light.
What repairs guanine alkylation?
MGMT (methylguanine methyltransferase)
How does excision repair work?
- recognize damage
- endonuclease cuts the strand on each side of the damage
- endo/exonuclease removes the damage
- DNA pol I/III replaces it with correct bases
- DNA ligase reattaches the strand
What types of damage does BER repair?
base lose
deamination
alkylation
base oxidation
How does BER work?
glycosylase recognizes the damage and cleaves the base from the sugar. Then endonucleases make the repair.
What types of damage does NER repair?
-Thymidine dimers, insertion/deletion, chemical adducts, crosslinks
How does NER work?
- recognize damage
- protein complex binds
- unwind DNA
- endonuclease removal of damage
- DNA pol I/III replaces with correct bases
- DNA ligase reattaches strands
_____ NER recognizes damage anywhere in genome and defects lead to _____
Global Genome Cancer (Xeroderma pigmentosum)
_____ NER recognizes damage in transcribed genome and defects lead to _____
- Transcription Coupled
- CNS disorder (Cockayne syndrome)
What types of damage does MMR repair?
-mismatch, alkylation, oxidation
How does MMR work?
- recognize damage by MSH protein complexe
- Cleavage by MLH protein complex
- DNA helicase unwinds strands
- DNA pol I/III replaces with correct bases
- DNA ligase reattaches strand
How does MMR recognize daughter strand instead of parent strand?
- In prokaryotes, the daughter strand is not yet methylated
- In eukaryotes, daughter strand has more nicks
Mutations in MMR machinery cause _____
HNPCC (colorectal cancer)
How does TLS repair work?
“Translesion synthesis”–> When damage is too great for other repair mechanisms, Bypass-DNA-pol (with less specificity) synthesizes new strand from damaged template strand.
How are bypass DNA pol different from regular DNA pol?
-Don’t have 3’ to 5’ exonuclease activity (proofreading) thus the bypass DNA pol is more error-prone
How are double strand breaks repaired?
NHEJ: no homology required, inaccurate.
HR: requires homology, accurate
_____ regulate the DNA damage checkpoint
checkpoint kinases
Primary mRNA transcripts have _____ at their 5’ end
3 phosphates that are connected 5’ to 5’
mRNA primary transcript is _____ to DNA template strand and _____ to the DNA coding strand
–> complementary; similar
The initiation codon for transcription is _____
ATG
RNA pol I is in the _____ and transcribes _____
nucleolus; rRNA ( the most abundant)
RNA pol II transcribes _____
- mRNA (informational)
- snRNA, miRNA (regulatory)
RNA pol III transcribes _____
tRNA (the smallest RNA)
Describe steps of transcription
- RNA pol binds to the promoter on duplex DNA
- RNA pol melts DNA and forms the open complex (isomerization)
- RNA pol elongates the transcript 5’ to 3’
- At stop site, RNA pol releases and transcription complex dissociates
RNA pol uses a ____, which makes it processive
sliding clamp
How does alpha-amanitin work?
binds to the bridge-helix on RNA pol II and halts transcription
What drug blocks the RNA exit channel on bacterial RNA pol and halts transcription?
rifampicin
What sequence is conserved in promoters?
the TATA box
What are the transcriptional control elements on DNA?
Core promoter Promoter proximal elements Enhancer/Repressors
What diseases result from mutations in TFIIH?
Xeroderma pigmentosum; Cockaynes syndrome; Trichothiodystrophy
What are the components of RNA pol pre-initiation complex?
- RNA pol
- Mediator Transcription factors (TFIID/H)
- Promoter DNA
How is a 5’ cap added to pre-mRNA?
- Triphosphatase cleaves gamma phosphate from the 5’ end of pre-mRNA
- Guanylyltransferase adds guanosine+P
- Guanine 7 methyltransferase adds methyl group to 7’ of guanosine
The functions of 5’ cap are _____
- stabilizes RNA
- nuclear export
- translational regulation
- splicing
The conserved sequence at 5’ splice site is _____
GU
The conserved sequence at 3’ splice site is _____
AG
The nucleotide at the splicing branch point is _____
A
_____ attaches to the 5’ splice site
U1 snRNA
_____ attaches to the splicing branch point
U2 snRNA
What are the 5 types of alternative splicing?
Exon inclusion/exclusion
Intron inclusion/exclusion
Mutually exclusive exons
5’ Exon truncation/extension
3’ Exon truncation/extension
_____ is an example of a genetic disorder caused by splicing defects
spinal muscular atrophy (SMN1)
How is 3’ polyA tail made?
- endonuclease cleaves part of the 3’ end of pre-mRNA
- polyA pol adds a string of A to 3’ end
What are the 2 functions of polyA tail?
- stabilizes mRNA
- stabilizes translation on the ribosome
How is polyA tail synthesis associated with termination of transcription?
polyA tail formation triggers termination of transcription
The conserved sequence in polyA tail is _____
AAUAAA
_____ is an example of how alternative polyA sites can lead to multiple proteins from a single gene
IgM (membrane-bound vs. secreted forms)
List 3 diseases that are due to defects in DNA control elements
- Beta-thalassemia
- Hemophelia Beta-Leyden
- Fragile X syndrome
Describe domains of sequence-specific DNA binding protein
DNA binding domain (conserved) Activation domain flexible protein domain
List major families of sequence-specific DNA binding proteins
- Homeodomain
- Zinc finger
- basic leucine zipper
- Helix Loop Helix
List 3 diseases that are due to defects in sequence-specific DNA binding proteins
craniosynostosis, AIS, Waardenburg syndrome
_____ is when sequence specific DNA binding proteins homo/heterodimerize to regulate gene expression
combinatorial control
what are the 2 classes of chromatin modifying factors?
DNA-dependent ATPase
HAT/HDAC
How do HAT/HDAC work?
HATs acetylate K on histones, remove positive charge, activate transcription. HDACs reverse this process.
specificity of transcriptional regulation is due to _____
binding of transcription factors to DNA control elements
List 5 ways sequence-specific DNA binding proteins are regulated
- ligand binding 2. nuclear entry 3. amount of TF 4. DNA binding 5. phosphorylation
how is tamoxifen involved in breast cancer therapy?
binds to estrogen receptor and recruits HDACs (instead of HATs that bound estrogen recruits)
Describe mechanism of Nf-kB
TF that is normally in cytoplasm and bound to IkB. IkB normally masks the NLS on NF-kB. During inflammatory response, IkB is degraded, so Nf-kB migrates into nucleus
_____ controls (lowers) the amount of p53 and regulates its function (DNA damage control)
MDM2
Give an example of how DNA binding activity of a sequence-specific DNA binding protein can be regulated
Ebox proteins homodimerize due to presence of basic domains and promote cell differentiation. Id proteins form heterodimers with Ebox and prevent DNA binding due to absence of basic domains
Phosphorylation of _____ promotes transcription by allowing it to bind with a HAT
CREB
Other than transcriptional regulation, how else is gene expression regulated?
- mRNA nuclear export 2. mRNA degradation 3. translational regulation 4. miRNAs 5. protein degradation
_____ describes multiple codons coding for a single amino acid
degeneracy
_____ describes when the first 2 nucleotides determine which amino acid is coded by the codon and the third is insignificant
wobble
describe prokaryotic translation initiation
IF1+IF3 bind mRNA to small subunit at SD sequence. IF2 brings first tRNA to P site. GTP hydrolysis dissociates initiation factors and binds large subunit.
describe eukaryotic translation initiation
5’ cap recruits initiation factors that recruit small subunit. small subunit scans mRNA for AUG in 5’ to 3’ direction and binds to it. initial tRNA is loaded into P site. large subunit binds
Why is cap-independent (IRES-driven) translation important in eukaryotes?
can shut down translation machinary when invaded by virus, but still maintain translation of certain, specific mRNA.
_____ of eIF-2alpha due to environmental stimuli prevents delivery of initial tRNA to P site.
phosphorylation
Describe the function of interferon
when virus invades cell, cell releases interferon that signal neighboring cells to turn off translation
_____ is an example of an mRNA that is specifically edited to encode 2 different proteins depending on cell type.
apoB
The strength of a start codon in eukaryotes is related to how much the sequence matches the _____
Kozak sequence
What does rapamycin do?
halt translation by binding/sequestering eIF-4E
When iron is _____ in the cell, _____ binds to _____ on _____ and prevents degradation of the mRNA and thus allows for coding of _____, which brings iron into the cell
low IRE-BP IRE 3’ UTR Transferrin Receptor
When iron is _____ in the cell, _____ binds to _____, so that the mRNA is exposed and degraded.
high IRE-BP Iron
When iron is _____ in the cell, IRE-BP binds to IRE on 5’ UTR and prevents translation of _____
low ferritin
When iron is _____ in the cell, IRE-BP binds to iron, so _____ is made and iron is sequestered
high ferritin
When pH is _____ than pKa, the protonated acid form of amino acids dominates
less
Under _____ conditions C forms disulfide bonds that form inter/intra chain links
oxidizing
List post-translational modifications and diseases/drugs associated with each.
- Hydroxylation (scurvy) 2. Carboxylation (bleeding) 3. Glycosylation (CDG) 4. Acetylation/Methylation (HDAC inhibitors) 5. Phosphorylation (Gleevac/leukemia) 6. Ubiquitination (proteasome inhibitor/myeloma)
Primary protein structures are formed by _____ bonds, while secondary, tertiary, and quatrenary structures are formed by _____ bonds
covalent non-covalent (H-bond, ionic/hydrophobic/Van der Waal interactions)
H-bonds between NH on _____ amino acid and CO on _____ amino acid form alpha helix.
1st 5th
_____ and _____ are alpha helix formers, while _____ and _____ are helix breakers
A & L P & G
_____ allows hemoglobin to be an effective oxygen transporter
cooperativity
What can denature proteins?
heat, pH extremes, chemicals
The ribonuclease refolding experiment showed that _____
protein primary structure determines its function
_____ help proteins to fold
chaperones
The two main classes of chaperones are _____ and _____
Hsp (bind hydrophobic areas of unfolded proteins to prevent degradation) GroEL/GroES (hydrolyzes ATP to fold/refold proteins. nonspecific)
_____ help with protein folding by replacing improper disulfide bonds with correct ones.
protein disulfide isomerases
_____ help with protein folding by changing the configuration of P residues
peptide prolyl isomerases
Protein misfolding causes _____
prion disease Alzheimer’s Parkinson’s amyloidosis
Describe what happens in prion disease
PrP misfold (beta sheet instead of alpha helix) causes it to become resistant to protease activity
What are the 7 ways proteins can be purified?
- Gel filtration chromatography (size) 2. Ion exchange chromatography (charge) 3. Affinity chromatography (ligand binding) 4. SDS-PAGE (size) 5. Mass spectrometry (size/charge) 6. Edman degradation (N-terminal sequencing) 7. Western blot (immunology)
miRNA are derived from _____
imperfectly matched dsRNA (dicer and drosha dependent)
When miRNA have an imperfect match to their target mRNA, the target is _____
translationally repressed
When miRNA have a perfect match to their target mRNA, the target is _____
degraded
What is the mechanism of action of miRNA?
RISC/Argonaute mediated cleavage deadenylation/decapping block translation initiation/elongation
siRNA are derived from _____
perfect match dsRNA (dicer dependent)
siRNA always _____ match their target RNA
perfect
piRNA is derived from _____
ssRNA (dicer independent)
siRNA functions relate to _____
defense/immunity (mRNA degredation)
piRNA functions relate to _____
gametes and control of mobile elements
What are the functions of lncRNAs?
scaffold regulate alternative splicing transcriptional repression regulate miRNA DNA mimicry
Describe the mechanism of action of ASOs
bind to target mRNA and mediate cleavage by RNase bind to splice site and force translation of alternative protein siRNA: RISC mediated cleavage anti-miR: inhibit miRNA
What are the 3 general features of cloning vectors?
restriction sites origin of replication selectable marker
In gel electrophoresis for DNA separation, the sample is loaded at _____ end
cathode (negative)
_____ blotting is used to detect DNA
southern
_____ blotting is used to detect RNA
northern
What are the 4 characteristics of hybridization probes?
Length (determines Tm) Sequence (determines target) Marker (for detection) Quantity (has to be in excess)
Describe how microarrays work
The array is composed of immobilized, unlabeled probes. target cDNA/DNA is put on array. Fluorescence measured.
In PCR, denaturation occurs at _____ degrees, annealing at _____, and elongation at _____
95 55 72
Describe how sickle cell anemia can be diagnosed using RFLP
make probe to whole gene. Add restriction enzyme. do southern blot. abnormal sample will be missing a restriction site (only 1 fragment). Normal sample will have 2 fragments.
Describe how DNA fingerprinting works
make probes to VNTRs on multiple samples. compare on southern blot.
Prion strains are due to _____
different misfolding patterns
BSE is a _____ compared to vCJD
different prion strain
_____ CDK activity during _____ phase allows for _____ of pre-RC, but not its _____
low G1 formation activation
_____ CDK activity during _____ phase allows for _____ of pre-RC, but not its _____
high S activation formation
What is the function of the G1 checkpoints?
cell growth (w/o division) cell differentiation check for DNA damage
What is the function of the G2 checkpoint?
check new DNA for damage
What is the function of the M checkpoint?
check function of spindle for correct DNA distribution into daughter cells
The read length of Illumina is _____ and its error rate is _____
50-250 bases 0.1%
The read length of PacBio is _____ and its error rate is _____
2500 bases 15%
What 3 criteria must be considered when using next-gen sequencing for SNP identification?
coverage (i.e. overlap of fragments) error rate ploidy (i.e. heterozygosity)
Exome sequencing is useful when a mutation is _____ and follows _____
rare mendelian inheritance
Since exome sequencing only involves _____, it cannot be used to diagnose mutations in _____ parts of DNA
exons non-coding
List 5 causes of DSBs in DNA
immune system rearrangements SSBs during replication meiosis radiation medical imaging/treatments
homologous recombination occurs only during _____ phases of cell cycle
S & G2
loss of heterozygosity in homologous recombination occurs due to _____
use of homolog instead of sister chromatid for DSB repair (depends on Holliday junction)
_____ regulates HR, while _____ regulates NHEJ
BRCA1 53BP1
Describe the 3 classes of proteins involved in DSB repair
sensors (ku) transducers (ATm/ATR) repair complexes (BRCA1/53BP1)
The difference between cofactors and coenzymes is that _____
coenzymes are consumed cofactors are NOT consumed
List 5 ways that enzymes can lower activation energy
binding energy (w/ S to form ES) increase effective concentration of S metal ion catalysis (cofactor) acid/base catalysis (w/ transition state) covalent catalysis (w/ S)
irreversible inhibitors _____ change the enzyme structure/function
permanently
competitive inhibitors bind to _____ and function by _____
enzyme compete with substrate for binding to active site
uncompetitive inhibitors act as _____ regulators and bind to _____
allosteric ES (enzyme-substrate complex)
mixed inhibitors bind to _____
E or ES
What are the 4 mechanisms of enzyme regulation?
allosteric regulation covalent modification proteolytic cleavage protein binding