Lecture 26 Flashcards
True or false: proteins are strictly units of an activation OR deactivation complex that acts on DNA.
False
the same protein can be a part of a repressing or activating complex
Describe the essential, and variable characteristics of a DNA transcription factor.
Essential:
a DNA binding module
An activation module (to turn on the gene)
Variable:
could have a dimerization module (to form a dimer with other protein subunits)
could have a regulatory module (to regulate the transcription factor)
true or false: gene repressors inhibit transcription when repressor and activator proteins compete for the same overlapping DNA binding site. explain
True
the more DNA binding sites that the gene repressor can occupy, the less available DNA binding sites there are for the activator proteins to bind to and trigger transcription
Describe the Helix-loop-helix type of Transcription factors/DNA binding proteins
Helix-loop-helix: is a dimer formed by 2 subunits that both feature a short alpha chain connected by a loop to a second longer alpha chain
can occur as either homodimer or heterdimer
has the 3 domains, similar to Leucine zipper transcription factors; DNA binding domain, dimerization domain, and activation domain
Describe the steps of CHIP (Chromatin Immuno-Precipitation) technique for identifying transcription factors
Add gene regulatory proteins so they bind to specific DNA sequences
cross-link proteins to DNA with formaldehyde
lyse cells and break DNA into small fragments
precipitate the DNA of interest that is bound to the gene regulatory protein using antibodies against the specific gene regulatory protein
reverse the formaldehyde cross-links (to remove the protein from the DNA of interest)
amplify the precipitated DNA of interest with PCR (you can identify the sequence after PCR if you wish)
Describe the Helix-turn-helix type of Transcription factors/DNA binding proteins
Helix-turn-helix: has 2 alpha helices connected by a short chain of AA’s that make a “turn” at a fixed angle
the longer helix is the “recognition helix” that fits into the major groove of the DNA when binding occurs
symmetric dimer versions of this transcription factor bind to DNA as dimers
the promoter is where ____ ____ and ___ ______ assemble in order to conduct transcription of a gene. The regulatory sequence is where regulatory proteins ____ and control the _____ of assemble process at the promoter.
transcription factors
RNA Polymerase II
bind
rate
what is the relationship between regulatory proteins and the Locus Control region of the beta globin genes?
regulatory proteins bind to the LCR
Describe the Zinc finger motif type of Transcription factors/DNA binding proteins
Zinc finger motif: has a DNA binding motif that includes a Zn atom and usually binds in “tandem clusters”
tandem cluster binding includes multiple zinc finger domain transcription factors with multiple contact points that helps stabilize that interaction with DNA
C2H2 (Cys-His) is an example of a zinc finger domain transcription factor
the sequence “GATA” is an example of what?
a recognition sequence for regulatory proteins
describe the different motifs of DNA regulatory proteins in terms of the types of bonds they may form with a gene regulatory protein (4 of them)
possible hydrogen bond donors
Possible hydrogen bond acceptors
Methyl groups
Hydrogen atoms
(typical interactions between DNA motifs and gene regulatory proteins involve 10-20 interactions)
transcription regulators bind to DNA in nucleosomes with ___ affinity and bind to naked DNA with ____ affinity
lower
higher
(because the surface of the nucleotide recognition sequence may be facing inward when attached to a nucleosome, which makes it less accessible to transcription regulators)
Beta Globin gene regulation is dependent upon a region of a gene that contains ___ beta globin genes and a ____ ____ ____. explain why the last blank is important
5
Locus Control Region (LCR) ; it may be upstream, but it is required for transcription
The mechanisms by which gene regulatory proteins regulate gene transcription (4 gene activator methods and 3 gene repressors methods)
gene activators
nucleosome remodeling
nucleosome removal
histone replacement
histone modification (such as acetylation)
Gene repressors
repressor conducts competitive DNA binding agains the activator at the binding site
both proteins bind to the DNA but the repressor binds to the activation domain of the activator protein
the repressor binds to DNA and blocks assembly of general transcription factors
List the ways in which transcription factors are identified and give a brief description of them
Gel Mobility shift assay (EMSA or electrophoretic mobility shift assay): detection of sequence-specific DNA binding proteins
Affinity Chromatography: isolate DNA binding protein and purification of sequence specific binding proteins
CHIP (chromatin immuno-precipitation): allows the Identification of the sites in the genome that a KNOWN regulatory protein binds to
Regulation by _____ is the fashion in which DNA activation/deactivation is regulated.
committee
what are gene regulatory proteins? how important are they?
gene regulatory proteins are transcription factors that bind and activate a gene (they bind to DNA binding motifs)
they are crucial to the regulation of protein synthesis
describe the ways (6 of them) in which repressor proteins inhibit transcription
Competitive DNA Binding: repressor competes with activator for the same DNA binding site
“Masking” of the activation surface: both proteins bind to the DNA but the repressor binds to the activation domain fo the activator protein
Direct interaction with the general transcription factors: the repressor binds to DNA and blocks assembly of general transcription factors
Repressor recruitment of chromatin remodeling complexes: chromatin remodeling complex returns the promoter to the “pre-transcriptional” nucleosome state
Repressor recruitment of Histone deacetylases: deacetylased promoters are much harder to remove from the histone
Repressor recruitment of Histone methyl transferase: methylated histones, via the methyl transferase, are bound to proteins that act to maintain chromatin in a transcriptionally silent form
list the 4 ways that gene activator proteins modify DNA in terms of it’s local chromatin structure. Why do these 4 processes make DNA “more activated”?
Nucleosome Remodeling
Nucleosome Removal
Histone replacement
Histone modification (acetylation to be specific)(not all histone modifications cause gene activation)
all of these methods made the DNA more “accessible”
what are gene regulatory proteins? how important are they?
gene regulatory proteins are transcription factors that bind and activate a gene (they bind to DNA binding motifs)
they are crucial to the regulation of protein synthesis
Describe the Helix-loop-helix type of Transcription factors/DNA binding proteins
Helix-loop-helix: is a dimer formed by 2 subunits that both feature a short alpha chain connected by a loop to a second longer alpha chain
can occur as either homodimer or heterdimer
has the 3 domains, similar to Leucine zipper transcription factors; DNA binding domain, dimerization domain, and activation domain
Describe a potential cure for sickle cell anemia that involves gene regulation. (this is all hypothetical as we are not there yet in terms of research/comprehension)
using gene regulation to “switch” from adult hemoglobin to fetal hemoglobin which is usually not made after birth.
list the 4 different transcription factor (DNA binding proteins)
Helix-turn-helix:
Zinc finger motif:
Leucine zipper:
Helix-loop-helix:
DNA _____ and a mediator complex allow the gene regulatory proteins to interact with the proteins that assemble at the promoter. define the mediator
looping
the mediator serves as an intermediary between gene regulatory proteins and RNA polymerase II
