Cell + Molec II Flashcards
What is mismatch repair? (MMR)
The process where mismatches made by DNA polymerase are fixed to mirror the template strand
Where does a mutation need to occur for it to be passed down vertically
In the Germ line
mutations in cell cycle, DNA repair-protein genes can lead to…
Cancers
Genome instability
Contributed to by mutations, leads to an increased chance for cancers
Silent Mutation:
changes codon but not a.a.
Source types of DNA Damage or errors
Endogenous and
exogenous;
source is w/in cell or from environment
Mismatch?
DNA pol inserts the wrong dNTP
depurination and deamination
Spontaneous loss of a purine (A or G)
or
Spontaneous loss of an amino group from Cytosine, causing it to turn into a uracil
Thymine dymerization
a thymine dimer (covalent) forming b/w two adjacent pyrimidines due to UV light interference
All types of DNA repair and damage
mismatch, depurination, deamination, thymine dimers, and single or double breaks
NER
Nuceotide excision repair;
for issues on one strand of DNA, area is excised and repaired by putting new dNTPs and ligase fixing it.
two types of repair for DS breaks
homologous recombination
nonhomologous end joining
nonhomologous end joining
nuclease cuts the ends of the broken DNA and ligase patches them back together; some nucleotide loss
homologous recombination steps
A. ds DNA break
B. resection of 5’ ends by nucleases
C. strand invasion, D-loop formation
– coordinated by recA in bacteria, Rad52 in yeast
D. repair DNA polymerase elongates invading
strand
E. invading strand released, pairs with
damaged DNA
F. repair DNA polymerase elongates damaged
DNA
G. ss DNA nick ligation by DNA ligase
RNA pol I and II?
I is for prokaryotes, II is for eukaryotes
gene
DNA sequence that codes for a functional product
Transcription
DNA sequence of coding region* copied into messenger RNA (mRNA)
Translation
sequence of mRNA specifies a sequence of amino acids in a polypeptide
DNA structure
-H on 2’ Carbon, DEoxyribose
adenine w Thymine pairing
stable in environment
typically ds
typically very long
doesn’t form into stems and loops
RNA structure
-OH on 2’ Carbon, RIBOSE
A with U
degrades quickly
typically ss
typically shorter
folds into stems and loops
all types of RNA we cover
mRNA; transcript
rRNA; ribosomal
miRNA; regulation of gene expression
tRNA; transfer RNA (adapter for mRNA codons)
snRNA; spliceosomes
What is the enzyme of replication?
RNA Pol
Amplication
Making more of a product from DNA information
Initiation in prokaryotes
Sigma factor binds to the promoter and allows RNA pol to bind
Elongation in prokaryotes
RNA adds complementary RNA nucleotides starting from transcription site (+1 from promoter)
sigma factor released
Termination in Prokaryotes
Terminator sequence causesRNA pol to dissociate
Transcription initiation complex
Multiple general transcription factors needed for RNA pol II to bind in eukaryotes
(includes the TATA box)
assembles at the promoter
TFIID
alongside TBP (TATA box binding protein)
is part of the transcription initiation complex and is essential for RNA Pol II to bind
What happens to mRNA as it leaves the transcription initiation complex?
The tail is phosphorylated
Where is precursor mRNA modified?
the nucleus
what is precursor mRNA?
pre-RNA. the primary transcript (unmodified mRNA)
what is done to pre-RNA as part of RNA processing?
5’ m7G cap is added on as mRNA is produced
polyadenylation happens: 100s of As added to 3’ tail
RNA splicing; splicing exons, removing introns
what removes introns?
spliceosomes
snRNPs
small nuclear ribonucleoprotein particles
are the product of snRNAs associating with proteins.
Alternative splicing
allows the making of multiple proteins from the same sequence being spliced differently (~95% of all human genes)
what else does RNA processing do>?
mark mRNAs for export out through the nuclear pore and not to be degraded
genetic code is…
correspondence b/w codons and a.a.s
tRNA parts
anticodon; complementary codons
amino acid bound
What is a ribosome made of?
ribosomal proteins and rRNA
(large and small subunit)
ribozyme
RNA enzyme
ex: ribosomes are 2/3 RNA by mass
what do the small and large ribosomal subunits do>?
small: binds to RNA
large: has 3 tNRA binding sites (EPA) and catalyzes the peptide bond thru em
aminoacyl-tRNA synthetase
an enzyme that attaches a.a. to tRNA
what is an RNA hairpin?
in ss RNA, its when the RNA folds back on itself
P site of ribosome
C-terminus of polypeptide uncoupled from tRNA and growing polypeptide stays here
E site function
spent tRNA is here and is then ejected
A site
charged tRNA with a.a.
initiator tRNA
methionine; has complementary anticodon to AUG start sequence in RNA
translation initiator factors and the rest of initiation
proteins match initiator, P-site of sm rRNA and 5’ cap of mRNA
then the small subunit searches for start codon in 5’-3’ direction
once found large subunit attaches
Termination
stop codons (UAA,UAG,UGA) have no tRNA associated with em
release factor; a protein that hydrolyzes peptide from last tRNA
ribosome disassembles
Start codon?
methionine AUG
Main contributor to redundancy?
almost all 3rd and some 1st codon positions can accept any nt of the same type
wobble:
some tRNAs pair with multiple codons
humans have 500 tRNAs but only 48 diff anticodons
housekeeping genes
almost unchanged across species; keep cell alive
Where can gene expression be regulated?
transcription, RNA processing, mRNA transport, mRNA degradation, translation, protein degradation, protein activity
how can a gene be regulated at TRANSCRIPTION
whether or not transcription initiation complex forms
how can a gene be regulated at NRA PROCESSING
no 5’ cap or poly-A tail, no export out of nucleus
how can a gene be regulated at mRNA TRANSPORT
ribosomes don’t have to associate with it
how can a gene be regulated at mRNA DEGREDATION
hydrolyzed before translation
how can a gene be regulated at translation
ribosome chooses to not translate?
how can a gene be regulated at Protein degredation?
hydrolyzing of protein
how can a gene be regulated at PROTEIN ACTIVITY
allosteric regulation, phosphorylation
promoter and operator
the operator is a regulatory sequence IN the promoter, heading a whole OPERON
what is a repressor protein
binds to OPERATOR to PREVENT transcription
Trp Operon process
if TRP high, repressor binds to operator, if TRP low the repressor is inactive and RNA pol can bind to transcribe
Lac Operon process
lac repressor inactivated by lactose (on if lactose)
CAP activator has CAP binding site for RNA pol IF cyclicAMP present (signal for low glucose here)
cyclic amp is low when glucose high, and CAP is inactive
Activator protein
helps RNA pol bind if active
CAP stands for what?
catabolic activator protein
What type of cells are enhancers in? what do they do?
eukaryotic cells
a regulatory DNA sequence bound by activators (transcription factors)
Mediator
complex of proteins b/w activators and the transcription initiation complex
if activator not present, no mediator, no bending of DNA to get RNA pol to bind
What happens when a repressors binds to the enhancer
stops transcription process from happening
what remodels chromatin so RNA pol can transcribe?
transcription regulators attract proteins that remodel chromatin, histone modifying enzymes attaches acetyl groups to histones to provide binding sites.
What is combinatorial control?
multiple TFs (transcription factors) needed for the mediator and each TF controls multiple genes
What is special about gene duplication?
the new copy isn’t essential, so it can evolve new functions
exon shuffling?
Swapping of protein domains (exons) from different genes, creates new proteins with new functions
transposition affects what
mobile genetic elements contribute to mutations
horizontal transfer
DNA passed form one cell to another
genome duplication
duplication of whole gnome
i.e. going from diploid to tetraploid etc
When do most mutations happen
cell division
germline cells are what
cells that become gametes
NOT somatic cells
Asexual mutations
all mutations are passed to offspring
sexual mutations
ONLY germline mutations passed on
All point mutations and what is means
when one nucleotide is replaced with another, like when a missmatch is replicated
examples; neutral mutations, silent mutations, missense mutations
Deleterious mutations
lower fitness
nonsense mutations
changes a codon t the STOP codon
Missense mutations
change the final product amino acid
what can point mutations in regulatory DNA do?
can change the expression of genes, like lactose-intolerance
Misalignment during homologous recombination leads to what?
gene duplications and deletions
can be caused by transposable elements
What can duplicated genes do?
diverge; either getting new functions or do nothing
orthologous genes
homologous genes descended from a common ancestral species
Paralogous genes
genes descended from a common ancestral GENE
example; globin family
Pseudogene
inactive gene disabled by mutations
gene family diversity example
lysozyme breaks down bacteria cell walls
alpha lactalbumin mammal milk protein
How many times did the whole genome get duplicated in early vertebrate evolution?
2 times
== FREE SPACE ==
== ==
conserved genes and conserved islands
high degree of similarity and are normally important, the islands are functionally important sections of a gene
Purifying selection
the fact that natural selection selects for conserving important genes
conserved synteny
recognizable blocks of genes shared among genomes
how much of human genome codes for what? (BIG)
2% -> proteins
20% -> introns
50% -> repetitive DNA
|—> 6% simple repeats
|—> the rest is transposable elements
DNA-only transposons vs retrotransposons
no RNA intermediate; code for protein transposase
and
with RNA intermediate (only in eukaryotes, more common)
what uses reverse transcriptase and what is it?
makes DNA from RNA template, and is used by retrotransposons
LINEs vs SINES
long and short interspersed nuclear elements
15 and 10% of genome respectively
short are too long to code for proteins, LINEs code for reverse transcriptase, SINEs don’t
where is a virus’s genetic code stored?
the capsid
Gene cloning
insert target gene into bacterial plasmid (making the DNA recombinant)
Transform bacteria with plasmid
let e. coli duplicate
restriction enzymes do what
cut DNA at restriction sites (normally palindromes)
leaving sticky ends, helpful because you can get new DNA in with same restriction sites, and the same restriction enzyme can later remove the amplified gene
How to isolate target gene from all of the others
gel electrophoresis
probe definition
labeled complementary sequence that will hybridize with target sequence
genomic library process
whole genome cut up with restriction enzyme
plasmid and DNA fragments mixed with ligase
plasmids mixed with competent DNA
(for cDNA library); reverse transcribe mRNA into DNA so you only have exons
what do you need for PCR
polymerase chain reaction requires; template DNA, DNA polymerase, DNA primers, lots of nucleotides.
What are primers?
a 15-20nt strand at the 5’ end of ea strandof target DNA
how long does PCR take?
30-35 cycles
steps of PCR
denature at 95 degrees
anneal (cool) primers hybridize
extend (warmer) DNA pol extends primers to make complementary strand
(after 3 runs, mostly making copies of target DNA)
Taq
archaea DNA pol from thermus aquaticus
Sanger Sequencing
takes product of PCR but adds dideoxynucleotides that are fluorescenty labeled and cause strand termination, gel electrophoresis organizes and lazer reads and makes base calls
Shotgun sequencing
lots of diff methods
divide genome into many small fragments amplify and sequence
assemble contiguous fragments
in situ hybridization
can show the locations of specific nucleotide sequences
use labeled probe complementary to target DNA
FISH
fluorescent in situ hybridization
probes modified with chemical groups recognized by antibodies with labels
immunofluorescence
created antibodies w fluorescent labels
-: you can only look at dead preserved cells
reporter gene
insert gene for easily visualized product
inserted after promoter
we used to learn genetics based off of genetic _____
knockout
transgenic organism
genome altered and new genes are inserted
RNAi
based on mechanism post-transcriptional control
use siRNAs to degrade foreign RNA
miRNA
have RNA-induce silencing complex to effectively degrade a specific protein in cells
What is CRISPR
a bacterial anti-viral system
clustered regularly interspersed short palindromic repeats
stem loops are encoded for Cas to recognize and guide them to cut DNA
Cas9 endonuclease
an engineered protein that can insert a transgene if there is guide RNA
conditional knnockout
insert 2 genes to regulate expression
recombinase
and a gene flanked by nt that recombinase can recognize
3 main components of PM
lipid bilayer, membrane proteins, carbohydrates
hydrophilic
can make hydrogen bonds with water
and are CHARGED or POLAR
hydrophobic force
minimizes interactions b/w hydrophobic and hydrophilic molecules
i.e. fat dropelets
components of a phospholipid
charged phosphate head and two hydrocarbon tails
kinked hydrocarbon tails;
unsaturated
scramblase
an enzyme that randomly flips phospholipidsto even them out
flippase
flip specific phospholipids to the cytosolic monolayer
types of membrane proteins and % of lipid bilayer
transporter, channel, anchor, receptor, enzyme. 50% of mass
if the membrane protein is facing the lumen of the golgi, where are they going to go?
first to a vesicle and then will be exposed on the non-cytosolic side of the PM
Where do membrane proteins start?
the ER
how to isolate membrane proteins
use amphipathic detergents
Peripheral proteins usually have what structure, and what is it called if its made up of the other secondary structure?
alpha helices
beta barrels
what is the glycocalyx
a layer of carbohydrates surrounding cells
involved with cell recognition and lubrication and protection
cell cortex
