8.16.16 Lecture Flashcards
Gene expression must be ___. This is the basis for what three things?
Regulated; Development, homeostasis, and pathogenesis
True or false - all cells have the same genome.
True
Different types of cells express different sets of ___ that determine the ___ and ___ of the cell type.
Genes; character; function
What is the transcriptome?
All mRNAs expressed by a cell
What is the proteome?
All proteins expressed by a cell
Genes can be regulated with respect to what three things?
Time, space, and quantity
What is the promoter region and what does it do?
Made up of the core promoter (-40 to +40) and the proximal promoter (-200 to +50); promotes transcription in an orientation and distance-dependent manner
What does the core promoter do?
Induces basal expression of a gene; the location of assembly of general TF and polymerase
What are regulatory elements and what do they do?
Enhancers and silencers; short DNA sequences that bind transcription factors/regulatory proteins that interact with the basal transcription machinery in a distance and orientation independent manner when outside of the proximal promoter region (can be upstream or downstream of start site, or within introns/exons). Note that when they are within the proximal promoter region, they are dependent on distance and orientation.
What affects the rate of initiation?
Regulatory elements
What are insulators and what do they do?
Short DNA sequences found at the ends of a gene unit, bind insulator proteins, contain signals in a unit, block influence of outside regulators, prevent heterochromatin migration
What allows regulators bound anywhere on a gene to interact with proteins assembled at a promoter? Many regulators act through ___. Others assemble directly at the promoter.
DNA looping; Mediator
True or false - each regulatory element may only bind one transcription factor.
False - multiple TF may be bound to one regulatory element
Alterations in chromatin structure are directed by eukaryotic ___.
Transcription activator proteins
Promoters are initially buried in ___.
Heterochromatin
At least one ___ is bound on the surface of heterochromatin to begin the process of transcription.
Transcription regulator
What are three possible alterations to chromatin structure that promote gene transcription by increasing the accessibility of DNA and facilitating binding of RNA polymerase and general transcription factors?
- ATP-dependent chromatin remodeling complex causes nucleosome sliding to open up access to DNA
- Histone chaperones - removing histones, creating nucleosome-free DNA, or replacing histones with variants to favor euchromatin
- Histone-modifying enzyme - specific modification patterns destabilize heterochromatin and attract components of transcription.
What is one potential order of gene activation? Note that this differs from gene to gene.
- TF binds to chromatin
- Chromatin remodeling occurs via the remodeling complex
- Covalent histone modification occurs via histone modification enzymes
- Additional activator proteins bind to the gene regulatory region
- Assembly of pre-initiation complex at the promoter (Mediator, general TF, RNA polymerase)
- Transcription initiation (other gene activator proteins, rearrangement of proteins in pre-initiation complex)
Describe acetylation and methylation of Lysine.
- Acetylation removes the positive charge of Lysine, which breaks its interaction with negatively charged DNA.
- Methylation creates binding sites for proteins.
How do histone acetylation and deacetylation occur?
Histone acetyl transferase (HAT) catalyzes acetylation, generating euchromatin. Histone deacetylase (HDAC) catalyze deacetylation, generating heterochromatin.
What does histone methylase do?
Add CH3 to Lys and Arg
What does histone kinase do?
Phosphorylate Ser
Each histone modification attracts ___ that specifically bind to the modified site. What are the two major types?
Proteins; Bromodomain (binds to acetylated Lys and Arg, and phosphorylated Ser) and Chromodomain (binds to methylated Lys and Arg)
Give an example of successive histone modifications during transcription initiation. Note that these steps vary in various genes.
- Transcription activator protein and HAT bind to DNA.
- HAT acetylates Lys.
- Histone kinase binds and phosphorylates Ser.
- Further acetylation
- TFIID and chromatin remodeling complex bind chromatin (recognize acetylated histone tails through bromodomain)
- Remainder of transcription machinery assembles; transcription begins.
* Note that coactivation occurs throughout
What do transcription factors do?
Induce transcription
What are the properties of transcription factors?
- Modular designs with three domains
- DNA binding domain contains a structural motif with amino acids that interact with a unique DNA element exposed by the major and/or minor groove of the helix.
- Commonly homo- or hetero-dimers
- Dimers bind to a palindrome in the DNA, which contain a symmetry appropriate for dimer binding to two successive turns in the DNA helix.
What are the three domains typically found in transcription factors?
- Activation (or repressor) domain
- Dimerization domain
- DNA binding domain
TF generally interact with the ___ groove of DNA. Why?
Major; it has more exposed information (more available bases)
What are the four common motifs found in DNA binding proteins?
- Helix-turn-helix (HTH)
- Helix-loop-helix (HLH)
- Zinc finger (Zn finger)
- Basic Leucine zipper (b-ZIP)
What is the recognition helix of HTH?
C-terminal alpha-helix; participates in sequence-specific recognition of DNA (contains DNA binding domain); fits into the major groove.
What is the support helix of HTH?
N-terminal alpha-helix; structural component that helps position the recognition helix (via hydrophobic interactions)
How does HTH bind DNA?
As a dimer in which the two copies of the recognition helix are separated by exactly 1 turn of the helix; both helices fit into the major groove; bind at a palindromic DNA site
The DNA binding protein dimers are typically ___, not ___. What does this mean?
Synergistic; not additive (work better than the sum of the two parts)
What is a pseudopalindrome?
An imperfect palindrome
Describe a helix-loop-helix motif.
Short alpha helix, connected by a flexible loop to a longer alpha helix; binds to DNA and to the 2 helix structure of a second protein to create a homo- or hetero-dimer.
How does HLH interact with the major groove?
2 alpha helices extend from the dimerization interface to contact the major groove at a palindrome.
The dimerization region of HLH is usually generated by ___.
A leucine zipper (leucine edge is hydrophobic and can dimerize)
Each monomer of HLH contains a ___ joined by a loop to a second helix which contains ___.
Recognition helix; a leucine zipper
What is an example of HLH motifs?
Myc, Mad, Max
Describe the zinc finger motif.
Zn holds an alpha helix and beta sheet together. These are often found in a cluster with an alpha helix of each finger contacting the major groove of the DNA.
In a Zn finger, the Zn is typically coordinated by ___.
2 Cys and 2 His, or some combination of the two 2-3 amino acids apart; there is also an intervening polypeptide chain of 12-15 amino acids present
Describe the bZIP motif.
Two alpha helices (one from each monomer) join to form a short coiled-coil; bind as hetero- or homo-dimers where the two long alpha helices are held together by interactions between hydrophobic amino acid side chains that extend from one side of each helix.
The bZIP dimer grips the double helix like a ___.
Clothespin
Homodimers bind to symmetric DNA sequences. Heterodimers bind to ___ DNA sequences.
Hybrid
Describe the process of DNA footprinting.
- Label DNA fragments with 32P on one end.
- Cleave DNA with a nuclease that makes random single-stranded cuts.
- Denature to separate strands.
- Separate fragments on a gel, detect by autoradiography.
- When DNA binding protein is present, it protects the nucleotides at the binding site and protects phosphodiester bonds from cleavage.
- Those fragments that would terminate the binding site are missing, leaving a gap (footprint) in the gel pattern
Does DNA footprinting tell us the DNA sequence or protein identity?
No
Describe the process of a gel-mobility shift assay (EMSA).
- Mix extract of an antibody producing cell lines with radioactive DNA fragment containing a regulatory sequence from a gene encoding the light chain of the antibody made by cell lines.
- Analyze the mobility of DNA with PAGE and autoradiography.
- Free DNA migrates to bottom; fragments with DNA bound are slowed down.
Describe the process of chromatin immunoprecipitation (CHIP) assay.
- Covalently cross-link proteins to DNA with formaldehyde.
- Lyse cells.
- Break DNA into small fragments.
- Precipitate DNA using antibodies against gene regulatory protein.
- Reverse formaldehyde cross-links with acid; remove protein.
- Amplify precipitated DNA.
- Hybridize to microarray containing entire genome. This gives the precise genomic location of gene regulatory protein binding sites.
Describe the process of reporter assays.
- Insert potential enhancer from gene of interest into a plasmid with Luciferase.
- Transcription
- Translation
- Lyse cells
- Add Luciferase substrate
- If light: Luciferase was expressed, the element promotes transcription.
- If no light: element does not promote transcription
