Flashcards in Lecture 10 Slides Deck (36):
Lysosomes and vacuoles
Lysosomes digest proteins in organelles
Vacuoles we associate with plants
How do I know if my gene is expressed? Where can I test for its expression?
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?
Why can't you use PCR with mRNA?
It is not a template for DNA
What can you use to test for mRNA?
Polymerase chain reaction
DNA is copied
Denaturion 95 degrees
Annealing 55-65 degrees
Reverse transcriptase PCR
Detects RNA expression
Create cDNA, has no introns
Use RNA and enzyme to create cDNA, on which you can use PCR
Housekeeping gene used as reference, control
What is most important regulatory step
BY DNA regulatory proteins
Which subunits do DNA subunits usually have
1. 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
Upstream regulatory element(URE)
A short sequence of 6-12 bps "cis"
Part of protein that binds to specific regulatory regions of target genes.
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)
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
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
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
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
Ubi ligase (plants and animals)
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
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)
Chromatin remodeling complexes
What do transcription repressors recruit?
Histone methyl transferase
Chromatin remodeling complexes to create more heterochromatin and prevent gene expression