Lec 1-2 Flashcards
What does a DNA molecule consist of?
2 complementary chains of nts, running antiparallel
What does a nt consist of?
phosphate, pentose sugar, nitrogen base
True or False: DNA is polar
True
What is on the 5’ and 3’ ends of DNA?
5’-P and 3’-OH
What bonds holds nts on opposite strands together?
hydrogen bond
How many hydrogen bonds are between C-G and A-T?
3 and 2
How is the space between the backbone fixed?
Pyrimidine needs purine to make up the space
How is eukaryotic DNA packaged?
set of chromosomes
What do chromosomes contain?
long strings of genes
How are prok chromosomes more efficient than euks?
don’t have genome-wide repeats (introns)
What does a DNA molecule that forms a linear chromosome must contain?
a centromere, 2 telomeres and replication origins scattered throughout
What is the purpose of a centromere?
keeps sister chromatids together
What do chromosomes consist of?
euchromatin (gene rich) and heterochromatin (less gene rich and never open for transcription due to being highly condensed)
Essential dogma:
DNA -> RNA -> protein
What are the parts of a euk gene:
- enhancer
- promoter
- start codon (ATG)
- ORF
- stop codon (TAA)
- addition site for poly A tail
What mutations are of interest?
Loss of function mutations
What genotype are most mutations?
Homozygous recessive
What are the types of mutations?
- point mutation: single nt change - null (total loss of function), silent (a.a. not changed), nonsense (RF shift and premature stop codon) or missense (a.a. changed)
- inversion: truncate gene
- deletion: mostly null
- translocation: breaks of segment and attaches to another
What is a conditional loss of function mutation?
Permissive and restrictive conditions
What are the methods for studying gene function?
i) Forward (classical) genetics - random mutagenesis (chemical mutagen + engineered DNA element to identify where it is)
ii) Reverse genetics (genome sequenced) - site directed mutagenesis (homologous recomb. + CRISPR/Cas 9 gene editing)
iii) Gene knockdown by RNAi
Describe transfection/transformation
transient (DNA will eventually be lost from cell) and stable (DNA integrated into host genome)
How can you use transgenes to study gene function?
i) overexpression + misexpression
ii) gene reporters
iii) epitope or GFP tag
iv) structure/function studies - delete certain domains to test function
How to clone gene with bacteria
- take gene sequence and insert into plasmid
- introduce ds recombinant plasmid dna to e.coli cell
- amplify by growing in cell culture
- purified plasmid isolated from lysed bacteria
How does bacteria protect itself against foreign DNA?
Restriction enzymes:
i) cut by same restriction nuclease produces sticky ends (complementarity)
ii) cut by different nucleases produces blunt ends
How is a gene sequence inserted into ds plasmid DNA?
- plasmid cut via restriction nuclease
- DNA ligase covalently links DNA fragment to be cloned with plasmid
- now have ds recombinant DNA plasmid
How does the DNA library work?
- human ds DNA is cleaved by restriction nuclease (all cut with same enzyme)
- DNA fragments are inserted into plasmids (ends are complementary to DNA fragments)
- plasmids introduced to bacteria cells
- now have mixture of plasmids and a source for every part of the genome
How are genes cloned in vitro via PCR?
- identified region of ds DNA to be amplified (known due to needing primers for DNA rep.)
i) heat to separate strands (denature H-bonds)
ii) cool to anneal primers (longer primer needs higher temp)
iii) DNA synthesis (needs DNA Taq. pol + all 4 nts)
What is the con of PCR?
doesn’t create genome library, therefore PCR and cloning go together
How do we get pure genes with PCR?
- isolate mRNA
- add 1st primer, reverse transcriptase and dNTPs to make RNA template that creates complementary DNA strand for PCR
- separate complementary DNA strand and add 2nd primer
- PCR occurs and end up with cDNA clones
What specifies a protein’s shape?
its a.a. sequence
What is the general formula of an a.a.?
- amino group (N-terminus)
- alpha Carbon
- side chain group
- carboxyl group (C-terminus)
What pH are both a.a. and carboxyl groups ionized?
7
What affects protein function?
size of side chain
What are the 4 protein structures?
i) primary: a.a. sequence read from N-term to C-term
ii) secondary: local 3D structure (alpha helix and beta sheet), involve h-bonds b/w non-sidechain groups on a.a.
iii) tertiary: 3D structure, globular form determined by interactions b/w side chains
iv) 3D structure of protein complex
Describe the alpha helix
ribbon structure due to carboxy of a.a. interacting with amino group of other a.a., every 4 a.a., side chains not involved
Describe the beta sheet
interactions b/2 a.a. on one strip with a.a. on other strip, side chain not involved
How do non-polar side chains react in water?
hydrophobic interactions, become sequestered together and away from water where hydrophobic core contains non-polar side chains
What causes the specific folding of proteins?
side chain interactions (electrostatic, H-bonds, Van der waals)
How do polar side chains react in water?
hydrophilic interactions, attracted to water and forms H-bonds to water
True or false: proteins fold into a conformation of lowest energy
true
What are protein families
proteins with the same domains and order of domains, with critical domains very similar amongst family members and their tertiary structure is similar along with function
What is a protease?
recognize other proteins and cleave them, belong to same family and have localized enzymatic activity (the a.a. there will be the same) ex. elastase and chymotrypsin both have NH2
True or false: some protein domains are found in many different proteins
true
How do domains impact complexity?
proteins with more than 1 domain increase in complexity
Describe a multidomain protein
if domains were removed, the other domain would still fold into its tertiary structure independent of the other domains
Summarize protein domains
i) they’re the functional units of a protein
ii) each domain relates to a protein’s specific function
iii) a domain can occur multiple times on a protein and can be found on many different proteins
iv) each domain within a protein can adopt its tertiary structure independent of other domains
v) new domains are a result of the addition/subtraction of domains (correspond to exons)
vi) domains are named after the 1st protein that was found to have that domain
True or false: larger protein molecules often contain more than 1 polypeptide chain
true, form a functional protein
How does the surface conformation of a protein determine its chemistry?
special a.a. side chains brought in proximity for specific interaction ex. rxn b/w Ser and His causes Ser to become supereactive, if another protein binds then Ser breaks the peptide bond
True or false: sequence comparisons between protein family members highlight crucial ligand-binding sites
true ex. SH2 domain confers on the protein the ability to bind to a phosphate that’s attached to another protein, the front has the most conserved a.a. which is similar to the front of the binding site of the phosphorylated protein
True or false: enzymes are not powerful and highly specific catalysts
false
What drives changes in proteins?
kinases that add phosphate
What largely carries out protein changes in euks?
protein phosphatases remove phosphate and protein kinases
How do kinases work
kinase binds to ATP, which transfers a phosphate to Ser, due to recognition motifs, now active. Cdk2 and Cdc2 recognize Ser, Thr.
How do phosphatases work?
recognizes phosphorylated sites and removes phosphate, now inactive
How do ubiquitous cell regulators (GTPase) work?
i) 2 states: GTP bound (active) and GDP bound (inactive) - conformational difference
1. start GDP bound, GDP is subject to nt exchange by GEF (Guanine Nt Exchange Factors), promotes nt exchange where GDP falls off and a new nt can attach where GTP comes in
3. now GTP bound
OR
1. start GTP bound (active)
2. GAP (GTPase Activating Proteins) promotes GTP hydrolysis, phosphate removed
3. now GDP bound (inactive)
- no phosphorylation, only exchange and hydrolysis
True or false: Many different covalent modifications combine to regulate protein function
true ex. some phosphorylation contributes to activation/ deactivation by occurring at many different sites and affects activity depending on site
