Lecture 10 Slides Flashcards
Lysosomes and vacuoles
Digestive organelles
Lysosomes digest proteins in organelles
Vacuoles we associate with plants
Proteozomes
Cytosolic proteins
How do I know if my gene is expressed? Where can I test for its expression?
Stages are DNA replication transcription MRNA Translation protein
You can test post transcription or post translation
Suppose you are looking for a specific protein? What test can you use to see if you’ve found it?
Western blotting
Why can’t you use PCR with mRNA?
It is not a template for DNA
What can you use to test for mRNA?
Microarray
Northern blotting
RTPCR?
PCR
Polymerase chain reaction Amplification DNA is copied Denaturion 95 degrees Annealing 55-65 degrees Extension 72degrees Repeat
RTPCR
Reverse transcriptase PCR
Detects RNA expression
Create cDNA, has no introns
Use RNA and enzyme to create cDNA, on which you can use PCR
Glyceraldehydethreephosphate (GAPDH)
Housekeeping gene used as reference, control
What is most important regulatory step
Transcription controls
BY DNA regulatory proteins
Which subunits do DNA subunits usually have
- Binds DNA
2. Activator/repressor domain
How does protein subunit bind dna
Usually via h bonding to bases, ionic bonds with backbone
How do protein subunits activate or suppress
Bind RNA polymerase II or other proteins (how modular transcription)
How does binding happen between protein subunits
Alpha helices with major groups in DNA
Why’s codon is at start site
ATG
Upstream regulatory element(URE)
A short sequence of 6-12 bps “cis”
Homeodomain
Part of protein that binds to specific regulatory regions of target genes.
Homeobox
DNA sequence found within genes that are involved in the regulatory patterns of anatomical development in animals, fungi, and plants
Benefit of having regulatory sequences and multiple conning sequences on genome
Reduces number of monomers you have to make
How do I know what DNA my transcription factor binds to?
Chromatin immunoprecipitation(CHIP seq) DNA sequencing
Primary tissue treated with formaldehyde to covalently cross link DNA and proteins
Protein specific antibodies are added to immunoprecipitates to target protein
Proteins are then unlinked and DNA is purified
DNA fragments are amplified and fluorescently tagged
A library is constructed
Fragments are sequenced
Sequences are aligned to a reference of genome and then can be visualized
How many proteins does a promoter bind to
Hundreds
How is regulation of prokaryotic transcription simple
Most controls are at or near promoter
Just one polymerase bacterium
What is a repressor? How does it work?
A protein that blocks RNAP from binding to promoter
What does repressor usually bind to
Operator sequence
What is activator? How does it work?
A protein that helps recruit RNAP to active site
How is regulation of eukaryotic dna complex
Eukaryotic DNA is flexible
UREs can be 50+ kb from +1
Many proteins are involved and interact together
TATA box
DNA sequence that indicates where a genetic sequence can be read and decoded. Type of promoter sequence. Says where transcription begins.
Example of how many proteins are involved in eukaryotic transcription
RnaII binds to TATA. A mediator binds around RNAII. If DNA is flexible and bent, two activators at UREs far away can recruit RNAII/mediator to promoter. Somewhere closer, URE can have a repressor on it so as not to express its sequence.
Three abilities of eukaryotic repressors
Can compete for activator binding site
Can inhibit activator interaction with RNAPII
inhibit RNAPII directly as well
Three ways to get rid of bound repressors
Conformational change Phosphorylation Ubi ligase (plants and animals)
Epi genetics
Heritable changes in gene expression that does not involve changes to the underlying DNA sequence; a change to phenotype without a change to genotype.
How can epigenetic mechanisms work
In cis -affect only one chromosomal copy
In trans - two self propagating mechanisms that work, affecting both chromosomal copies of a gene.
Epigenetic mechanisms that act in cis
DNA methylation
Histone modification
Epigenetic mechanisms that act in trans
Positive feedback loop by transcription regulator
Protein aggregation state
What do transcription activators recruit? 4
Histone acetyl transferase (HATs)
Histone kinases
Histone chaperones
Chromatin remodeling complexes
What do transcription repressors recruit?
Histone deacetylase
Histone methyl transferase
Chromatin remodeling complexes to create more heterochromatin and prevent gene expression
