Lecture 5 Flashcards
What do protein binding transcription control regions do?
Regulate expression of euk genes, they are located at multiple sites at various distances from the txn start site
What are the two types of protein binding txn control regions?
Promoter proximal elements and enhancers
What are promoter proximal elements and enhancers?
Gee and cell-type-specific regulatory elements on DNA
What are transcription activators and repressors?
Modular proteins containing a DNA binding domain and one or more activation or repression domains
How do we find gene regulatory sequences?
Link scanning reporter gene technique for identifying gene regulatory elements on DNA
What is linker scanning mutagenesis?
A way to find gene regulatory sequences.
What are the steps to linker scanning mutagenesis?
- Buy a plasmid that contains a reporter gene, usually a gene that encodes an enzyme
- The ORF of the reporter gene (tk) on the plasmid has no promoter
- Upstream of the tk ORF you clone a large piece of DNA that you know is controlling your gene (not tk) of interest
- Replace small pieces of the cloned regulatory DNA by a “linker”, produce multiple plasmids with linker subs
- Transform each individual plasmid in a cell culture and measure the activity of the reporter
- Loss of reporter activity means that you have hit a regulatory sequence
What is a reporter gene?
DNA sequence that encodes for protein sequence that we recognize
What do reporter plasmids contain?
- An ORF for an enzyme
- No promoter proximal regulatory sequences
- Multiple cloning site (MCS) for the cloning a regulatory sequence
- Translation initiation elements upstream of the reporter gene
- Poly A+ signal downstream of the reporter gene
What are some control elements that regulate gene expression in euk? TATA vs CpG islands
TATA promoter
Promoter elements: position RNA pol II to initiate txn at the start site, influence the rate of txn
Enhancers: can be upstream or downstream or in introns
Promoter-proximal elements: found upstream and downstream of txn start sites
CpG island promoter
Txn initiates at several sites in both the sense and antisense directions from the ends of the CpG-rich region
Sense direction transcripts are elongated and processed into mRNA by RNA splicing
Genes express mRNAs with alternative 5’ exons determined by the txn start site
Promoters contain promoter-proximal control elements
What are some characteristics of enhancers?
Can be far from the promoter, bend chromatin/DNA, orientation doesn’t matter, can be downstream or upstream, typically on the same chromosome
Enhancers are ___ regulatory elements
Distal
What are the main differences between enhancers and promoters?
Promoters function within a short distance while enhancers function over a long distance
Promoters are immediately upstream from the initiation site (RNA Pol II) while enhancers can be upstream, downstream from the start or within introns
Promoters are position-dependent which enhancers are position-independent
Promoters are orientation-dependent while enhancers are orientation-independent
What inactivates transcription?
Chromatin condensation blocks RNA pol and GTFs from interacting with promoters
What are the two things that repressor proteins can do?
Bind to txn control elements to inhibit txn initiation
Interact with multi protein co-repressor complexes to condense chromatin
What is are the functions of a pioneer transcription factor?
Binds to a specific regulatory sequence within the condensed chromatin
Interacts with chromatin remodeling enzymes and histone acetylases that decondense the chromatin making it accessible to rna pol II and gtfs
What is the function of activator proteins?
bind to specific transcription control elements in both promoter-proximal sites and distant enhancers
interact with one another and with the multisubunit mediator complex to assemble gtf’s and rna pol II on promoters
What is a domain?
part of a polypeptide with a specified function
What are four domains?
Transactivation domain, dimerization domain, dna binding domain, hormone binding domain
How are domains identified?
Deletion analysis of peptides
What are the steps to deleting mutants of the GAL4 gene in yeast?
- reporter gene containing lacZ reporter gene and TATA box ligated to UAS GAL (regulatory element) that contains Gal4 binding sites
- reporter gene construct and dnas encoding wild type or mutant gal4 introduced simultaneously into mutant gal4 yeast cells
- activity of beta galactosidase expressed from lacZ assayed (high activity if introduced gal4 DNA encodes a functional protein
- DNA binding site: eliminates binding to UASgal and beta galactosidase activity
- Activation domain: only the beta galactosidance activity eliminated
What is a motif?
conserved sequence in a protein found in a domain that is likely to produce a distinct 3D structure and a distinct activity in vivo
How many activation and dna binding domains do tf’s usually have?
TF’s may contain more than one activation domain but rarely contain more than one DNA binding domain
What domain does the Zn finger bind to?
DNA binding domain
What domain does the Leu zipper motif bind to?
Dimerization domain
What domain does the hormone binding motif bind to?
Hormone binding domain
C2H2 Zn finger characteristics?
- contains two conserved cysteine and two conserved histidine residues, whos side chains bind to one zn ion
C4 Zn finger characteristics?
- four conserved cysteins bind zn
- bind DNA as homodimer - one alpha helix in each monomer interacts with the dna
Leucine zipper protein motif characteristics?
- basic residues in th extended alpha helical regions on the monomers interact with the DNA backbone at adjacent sites in the major groove
- coiled-coil dimerization domain is stabilized by hydrophobic interactions between the monomers
- can form heterodimers
What are the characteristics of bHLH proteins?
- DNA-binding helices at N-termini of the monomers are separated by nonhelical loops from a leucine zipper-like region containing a coiled-coil dimerization domain.
- They can form heterodimers.
What is a distinct characteristic of DNA binding proteins?
work as dimers
If you have three monomers that can form dimers, you can have ___ combonations
6
What is an enhanceosome?
An enhanceosome is a protein complex that assembles at an enhancer region on DNA and helps to regulate the expression of a target gene
