chapters 7,8 (control of gene expression, work with cells) Flashcards
what are transcriptional regulators and where are they located?
- proteins that bind to specific DNA sequences (cis-regulatory element) to regulate gene expression
- distributed unevenly within the cytoplasm providing positional information to distinguish between different parts of the embryo
- ex: glugocorticoid hormone bindind to its receptor can promote expression of multiple genes depending on starvation/stress
Syncytium
multiple nuclei contained in a common cytoplasm
What is combinatorial gene control?
- can make many cell types with few starting things through lots of combinations
- have to be together to elicit effect
- ex: 3 neuron-specific transcription regulators in cultured liver cells converted them into neuronal cells by activation of neuron-specific genes and repression of liver-specific genes
master transcription regulators
- Oct4, Sox2, Klf4
- enough to trigger a change in cell identity
even-skipped (Eve)
- a gene critical for embryonic development in Drosophilia Combinatorial regulation of Eve promoter region by transcription regulators will devetermine the stripe expression and position
- witout this gene, embryo will die early on
induced pulipotent stem cells (iPS cells)
- expression of the 3 transcription regulators Oct4, Sox2, Klf4 in fully differentiated cells will produce cells that adopt characteristics of pluripotent cells derived from embryo
- if you over-express the 3 genes in differentiated cells, you can turn them into iPS cells
- erases identity completetly for something new to be added
what is cell memory?
- a pattern of gene expression within a given cell that is responsible for maintaining that cell’s identity and is passed on to a daughter cell upon division
- follows positive feedback loop (ensures continous gene transcription in the absence of initital signal)
Mechanisms involved in maintaining cell memory
- DNA methylation/ dense DNA methylation
- CG islands
- x chromosome inactivation
- x chromosome dosage compensation
What are transcriptional circuits? What is an example of it?
- gene regulatory switches that produce different outcomes on cell function and development
- network motif: a type of gene expression switching arrangement (small, repeating pattern of connections within a complex network)
- positive, negative feedback loop, feed-forward loop, flip-flop device
what is DNA methylation?
- addition of methyl group to cytosine when in sequence with guanine (CG)
- for genomic imprinting (only 1 copy from 1 parent expressed, and the other one is silenced- methylated)
- can repress gene transcription
histone modification
trimethylation on lysine promotes nucleosome compaction and heterochromatin formation
dense DNA methylation process
- methylated Cs can interfere with binding of transcription regulators/factors for transcription initiation
- DNA methyl binding proteins + histone modifying enzyme+ DNA methylase enzymes = induce heterochromatin formation
if packed in heterocromatin region –> not transcribed –> not expressed
When does DNA demethylation occur? And what enzyme removes them?
occurs shortly after fertilization, where most methyl groups are removed by DNA demethylase
maintenance methyl transferase
- enzyme adds methyl group to C when in sequence CG that are base paried with methylated CG sequences
- allows methylation pattern to be inherited after DNA replication
- old strand methylated
- ensures methylation pattern is inherited
What is genomic imprinting?
either maternal or paternal inherited genes are expressed, while the other is silenced (methylated)
what is x inactivation?
- where one of the X chromosomes is silenced in mammalian females
- random producing females with a mosaic of clonal groups of cells with either maternal or paternal X silenced
CG island
- CG-rich regions unevenly distributed in the genome
- area of many gene promoters
- deamination of C turns into T
- throughout evolution, majority of methylated CG sequences were lost due to deamination of C to T
- remain unmethylated by binding specizalized proteins that methylate histone H3and repel de novo methylases
what is dosage compensation?
- a process to ensure similar levels of gene expression in males and females
- differes across multiple species
- mammals: random inactivation
- fly: increase expression on X to match levels in female
- marsupials: inactivation of paternal
- nematode: decreased of all
In mammals occurs when more than one X is present in somatic cell
what is x chromosome inactivation triggered by?
- synthesis of long noncoding RNA Xist
- synthesized by one of the 2 X chromosomes
- contains binding sites for multiple proteins like DNa methylases and histone-modifying enzymes, promoting fomraiton of compact DNA structure and silencing gene transcription
monoallelic gene expression
in a diploid organism, only one copy of the gene is expressed
epigenetic inheritence
heritable change in the cell’s phenotype that does not result in a change in teh nucleotidde sequence of DNA
Posttranscriptional controls
- transcription attenuation aborting gene expression
- riboswitches- abort transcription in response to a signal
- alternative splicing
- RNA cleavage and poly-A addition
- RNA nucleotides can be covalently modified
- regulation of nuclear transport
What are P bodies?
- membraneless aggregates of RNA proteins
- storage facilities for mRNA, which can be stored for later used or degraded
What are stress granules?
- membraneless organelle that forms in response to starvation or stress when translation is suddenly blocked
- mRNA accumulates in stress granules and are released to the cyctosol to be translated when stress is alleviated
Describe the mechanism by which the HIV virus promotes nuclear transport of its RNA into the cytosol
- transcription attenuation
- HIV virus DNA is integrated into the host genome and is transcribed by RNA polymerase. Normally, transcription terminates due to attenuation preventing viral genome transcription
- HIV protein Tat is translated and promoted transcription by using host cell’s normal mechanisms that RNA transcription will continue
transcription attenuation
- premature termination of transcription by RNA polymerase
- attenuation is removed by regulatory proteins that bind the newly made RNA chain to promote transcription
riboswitch
- a short RNA sequence near the 5’ end of mRNAs (the beginning of it)
- blocks trascription by RNA polymerase when bound to a regulatory molecule
- ex: control of purine synthesis in bacteria (when G is abundant, it binds the riboswitch, causing it to undergo conformation that forces RNA polymerase to stop transcription of genes needed for purine synthesis)
alternative splicing
- selective removal/retention of introns and extrons to produce different mRNA sequences out of the same transcript
- can be positively or negatively regulated
alternative cleavage and polyadenylation site
- selective truncation of mRNA at different sites to alter C terminal
- will impact alternative splicing if cleavage site is within an introl
what do RNA cleavage and poly-A addition do?
- alter C terminus of protein
- work on nonfuncitonal mRNA sequences
RNA covalent modificattion
- deamination of Adenine to produce inosine (A –> I)
- deaminatino of Cytosine to produce uracil (C–>U)
I-C
U-T
regulation of nuclear transport
- retention and degradation in nucleus
- viral protein Rev binds to RRE site on viral RNA to enable interaction with nuclear transporter and transport into the cytosol (viral RNA has lots of introns so it can’t leave the nucleus)
- in latent state, Rev is generated in low levels, which prevent viral RNA export
- goes back into nucleus, binds on transporters to let viral RNA pass out of the nucelus. Now must go to a specific part of the cell to make protein right there
controls once out of the nucelus (translational)
- localizing mRNA to specific compartments prior to translation
- mechanisms (Shine-Dalgarmo squence, temperate control, riboswitch, microRNA binds to RNA)
- RNA stability
localizing mRNA
- allows for concentrating specific RNAs at cellular compartments to generate asymmetry
- allows cell to independently control translation in different cellular compartments
- localization signal is often located at 4’ UTR
RNA interfernce (RNAi)
job is to reduce transcription of specific RNAs
miRNA
- in the genome
- downregulates translation of the mRNA or sequestration to P bodies
- cropped, cleaved in cytosol by Dicer, loaded onto RISC complex with Argonaute,
siRNA
- NOT in the genome
1. double-stranded RNA recuits Dicer, which clevaes RNA into siRNA,
2. siRNA loaded onto RISC to idnetify intruder RNAs and degrade them
3. loaded onto RITS comlpex to bind complementary sequences as they are transcribed and recruit enzymes to promote histone and DNA methylation to silence transcription of the viral DNA
piRNA
- coded in genome
1. fragmented RNAs are methylated at 3’ end and assembled with Piwi proteins
2. seek out RNA targets by base pairing
3. degrades transposon-coded RNA and promote heterochromatin formation
4. protects germ line cells from protential harmful effects of transposon movement
transposons
- can move within DNA and insert themelves into a new random location
- can cause genetic changes
crispr
special regions in bacterial genome where viral DNA is integrated to serve as template for subsequent defense against infection
Examples of noncoding RNA
- miRNA
- siRNA
- piRNA
- small (CRISPR, crRNA, Cas)
- long (lncRNA)
Shine-Dalgarno sequence
in bacteria, upstream of AUG is a site for repressor/promoter of translation
translational temperate control
- change mRNA structure and expose AUG
- affects 2nd structure of RNA
what does binding microRNA to RNA do?
- promotes degradation and reduces protein levels
- specficially target to degrade RNA
mRNA stability mechanisms
- if degraded too fast, no protein can be made
1. Poly-A shortening
2. endonuclease-mediated degradation of poly-A tails
3. P-bodies
4. stress granule
poly-A shortening
- caused by exonucleases
- happens as soon as mRNA is in the cytosol
- once poly-A tail reaches 25 nt, decapping occurs, followed by further degradation
endonuclease-mediated degradation of poly-A tails
- half-life is determined by mRNA nt sequence
- presence of binding sites on 3’ UTR for proteins that can slow down/hasten poly-A shortening
- translation efficiency
crRNA
small noncoding RNAs produced from CRISPR locus that will destroy the virus infected (act similarly siRNA)
Cas protein
binds crRNA and serves as complex to target and destroy viral RNA
long noncoding RNA functions
- act as scaffolds for proteins to coordinate their function (for biomolecular condensates)
- serve as guide sequences to bring proteins to specific DNA/RNA sequences
- can act as antisense RNAs and block translation of coding RNAs
longer than 100 nt
why isolate cells?
- to gain access to cell components for biochemical analysis
- to study specific type of cell outside of the organism
primary cultures vs immortalized cells
primary: prepared directly from the tissue of the organism (typically non-dividing)
immortalized: can divide indefinitely in culture; can be thawed and re-cultured after long term storage in liquid nitrogen; often derived from tumors
culture shock
- cells divide a lot and then stop naturally despite telomerase activity
- due to activation of protective mechanism due to excessive mitogenic stimulation in culture
(they freak out becaues theyre in a dish, even if they have all the nutrients they need)
methods to separate cellular components
- osmotic shock
- ultrasonic vibration
- grinding in a blender
- changing pH
- agents that solubilize the membrane
what do hybridoma cell lines do?
allow production of monoclonal antibodies (laboratory-made proteins that act like antibodies in the body’s immune system)
what does fusing a B lymphocyte from inoculated mouse with transformed B lymphocyte cell line generate?
cell that can multiply indefinitely and produce the antibody of choice
order of centrifugation pellet separation (bottom to top)
- ribosomes, viruses, large macro-molecules
- microsomes, small vesicles
- mitochondria, lysosomes, peroxisomes
- whole cells, nuclei, cytoskeletons
sedimentation
sucrose gradient
cesium chlorine gradient
sedimentation- allows finer degree of separation between organelles/ components of similar size
sucrose- allows separating components by size and shape
cesium chloride- allows separating components by buoyancy (highly sensity, can separate different isotopes)
methods to separate proteins from cells
- column chromatography
- HPLC
- immunoprecipitation
- Protein tagging
Methods to separate isolated proteins
- Protein gel electrophoresis
- Western blot
- Mass spec
- X-ray crystallography/ cryo-EM/ NMR
what are the reasons for cells to stop dividing in culture?
immunoprecipitation
- using antibody to “pull out” specific proteins from mixture
- antibody is attached to beads that can be separated from cell homogenate
advantages and disadvantages of tagging
ad: easier to purify, specific antibodies, columns
dis: protein structure is altered, may affect function, protein needs to eb introduced into the cell
western blot
- aka immunolgotting
- protiens get stuck on membrane (paper) and apply antibodies
gel electrophoresis and its components
- separating proteins based on their size
- polyacrylamide- polymer with different size pores through which proteins migrate
- SDS- coats protein with negative charge and unfold protein structure
- B-mercaphtoethanol - reduced S-S bonds (cleaves 3D structure)
- coomassie- blue dye that binds proteins and allows for visualization of bands
how does PCR work?
give examples to its usage
- used to amplify specific DNA fragments; open, primer binds, DNA polimerase binds
ex: to detect presence of virus in blood, to distinguish between individuals in forensics
what are restrictions enzymes used for?
to insert the DNA into vector at specific sites
genomic library
- for determining the sequence of entire genomes
- fractioning an entire genome into smaller parts, putting into expression vectors –> finding out what all the pieces are doing
whole genome library
digesting genome with restriction enzymes (or mechanical shearing) and cloning each DNA segment in a plasmid vector
methods to separate isolated proteins
- Protein gel electrophoresis
- Western blot
- Mass spec
- X-ray crystallography/ cryo-EM/ NMR
methods to detect protein interactions
- co-immunoprecipitation- use antibody to pull down protein of interest
- fluorescence anisotropy- when proteins interact with eachother, change how light hits them
- FRET- tag 2 proteins with 2 fluorescent molecules, if close enough it will excite 1, which will give energy to 2, and 2nd will extract the photon
methods to extract specific parts of DNA from genome
- restriction nucleases
- hybridization
- PCR
types of mutation
protein-coding genes
- purifying mRNA, making complementary cDNA (just the DNA that codes or proteins- no unecessary promoters, etc)
sanger sequencing
dideozyribonuclease triphosphates lack the 3’ hydroxyl group –> DNA polymerase cant add new nucleotide after that
next generation sequencing
- nt attached to fluorophore and contain reversible chemical modificaiton that block elongation
- add chemical to mask/unmask the 3’
In situ hybridization
provides spatial information about expression of a particular gene (to localize RNA transcripts)
genome annotaiton
finding regions of the genome that encode for proteins (regulatory elements, noncoding RNAs, etc)
open reading frame (ORF)
long stretch of nt that does not have a stop codon
genetic screen vs reverse genetics
screen- identifying behavioral/structural phenotypes and isolating the gene responsible
reverse- looking to see how a mutation changes behavioral/structural phenotype
lethal mutation
causes developing organism to die prematurely
conditional mutation
- restrictive, permissive, temperature-sensitive conditions
- if you have this mutation, will show itself
loss-of-function mutation
- reduces activity of a gene
- usually recessive
null mutation
type of loss-of-function mutation that completly abolishes the activity of the gene
gain-of-function mutation
- increases the activity of the gene or makes it active in inappropriate circumstances
- usually dominant
dominant-negative mutation
- dominant-acting mutation that blocks gene activity
- causes loss-of-function phenotype, even in the presence of normal copy of gene
suppressor mutation
- suppresses the phenotypic effect of another mutation, so that the double mutant seems normal
- intragenic supressor mutation- in gene affected by first mutation
- extragenic supressor mutation- in second gene
Snrps
Form splisosomes
de novo mutations
- spontaneously occur in the germline cells of parents
- cause disease regardless of heredity
methods to change gene expression
- reverse genetics
- Cre loxP system- method to generate tissue/cell-specific conditional transgenic animals
- crispr- to knockout genes of interest; dont need mice, just need guide RNA to bring Cas9 to the correct place
- RNAi- for gene silencing; to known DOWN genes of interest; can be used for genetic screening
reporter protein function
to detect where a gene is expressed, NOT to remove
qPCR
- quantitative real time PCR
- provides info about expression of a particular gene
cluster analysis
- RNA sequencing
- to determine sets of genes that are coordinately regulated
CHIP sequencing
identifies sites for DNA transcription regulators
The Sec61 translocator facilitates the translocation of proteins that contain a signal sequence. What organelle are these proteins transported into?
Endoplasmic reticulum
In contrast to transporters, the channel proteins in cellular membranes
can only mediate passive transport
A neuron’s repetitive firing rate is limited by an absolute refractory period, during which a new action potential cannot be generated. Which event is chiefly responsible for this limit?
Inactivation of voltage-gated Na+ channels
Choose the correct order to separate the following cellular components by ultracentrifugation, from low to high centrifugation speed required.
nuclei, mitochondria, microsomes, ribosomes
This enzyme cleaves double stranded RNA in the cytosol into ~22-23 nucleotide fragments, which create miRNA and siRNA.
dicer
What allows K+ channels to conduct K+ ions ten thousand times better than Na+ ions?
The Na+ ion cannot stably interact with the channel pore amino acid sidechains
Where in the mitochondria are the TOM translocator complexes located?
outer membrane
Misfolded proteins in the ER may actively undergo
- binding to chaperones such as BiP to allow unfolding and refolding
- being glycosylated and binding to lectins
- being transported back to the cytosol for degradation
- binding to chaperones such as calnexin to prevent aggregation
Imagine a soluble protein that has been engineered to contain a nuclear import signal, a nuclear export signal, and a canonical endoplasmic reticulum (ER) signal sequence. With all of these signals, where would you expect to find the protein after its synthesis?
ER
