Chapter 8 Flashcards
examples of housekeeping genes
RNA polymerase, ribosomal proteins, DNA repair enzymes, cytoskeletal proteins, etc.
techniques that compare protein composition between cells
mass spectrometry and quantitative proteomics
techniques to profile gene expression
sequencing RNA and mRNA molecules
cortisol causes for release and effects
hormone released by adrenal gland in starvation, stress, exercise
signals to liver to increase glucose production and upregulates gluconeogenesis genes
transcription factor/transcription regulators
proteins that bind to specific DNA sequences and regulate transcription and gene expression
general transcription factors
help RNA polymerase II locate promoter and initiate transcription
only in eukaryotes (prokaryotes use sigma factor)
specific transcription factors (regulators)
activators and repressors influence efficiency/rate of transcription initiation by promoting or blocking RNA polymerase recruitment
eukaryotes and prokaryotes
how do transcription regulators bind to DNA
surface features in the DNA can change based on nucleotide sequence
recognize specific 3D features
bind to major groove of double helix
how do transcription regulators usually bind to major groove in DNA
form tight noncovalent interactions (ionic, hydrophobic interaction, hydrogen bonding)
bind as dimers (or polymers/clusters) usually to increase tightness
4 common DNA binding motifs (in DNA binding proteins)
helix-turn-helix motif
homeodomain motif
zinc finger domain
leucine zipper motif
homeodomain motif
involved in regulatory control of developmentally important genes
consists of 3 alpha-helices (cylinders) which fit protein into major groove
helix 3 contacts with DNA bases
helix-turn-helix motif
two adjacent alpha helices separated by (90 deg) turn of several amino acids
homeodomain difference from helix turn helix
homeodomain has three helices
but action of homeodomain is mediated by helix turn helix motif
Zinc finger motif
alpha helix and beta sheet held together by zinc molecule
often found in clusters to allow alpha helix of each finger to contact DNA bases in each major groove (identification)
leucine zipper motif
bind as dimers -grip DNA like clothespin
formed by 2 alpha helices (from separate proteins)
benefits of transcription factors binding as dimers
doubles the area of contact
increases specificity and strength
operons
bacterial clusters of genes transcribed together into single (polycistronic) mRNA because they share the same promoter
tryptophan operon makeup
contains 5 genes which encode 5 different enzymes needed to synthesize tryptophan
expression of Trp operon controlled by regulatory DNA sequence (operator) within the promoter
regulation of Trp operon
negatively regulated by Trp repressor
in high Trp conditions (feedback inhibition)
Trp binds to Trp repressor
Trp repressor binds to Trp operator region
prevents RNA polymerase from binding to promoter
allosteric protein
regulated by binding of molecule to a site other than the active site (Trp repressor)
activator proteins
transcription regulators that facilitate binding of RNA polymerase to inefficient promoters
lac operon makeup and function
three genes and three enzymes to import and digest lactose
allows bacteria to use lactose as energy source when glucose is low
what happens to cAMP levels when glucose is low
increase
activator for Lac operon
catabolite activator protein (CAP)
how is CAP activated
binding of cAMP
purpose of lactose repressor
shuts down expression of Lac operon if no lactose is present
requirements for Lac operon activation
- glucose absent (high cAMP, binding of CAP)
- lactose present (no binding of repressor)
first gene is Lac operon
LacZ - encodes beta-galactosidase which breaks down lactose to galactose and glucose
order of components in Lac operon
-80: activator (CAP binding site)
-40: promoter (RNA pol. binding site)
1: operator (repressor binding site)
40: LacZ gene
when lactose present how does repressor release from DNA
allolactose binds to repressor and decreases its affinity for the operator DNA
function of chromatin remodeling proteins in eukaryotes
these proteins and enzymes covalently modify histones that are the core of nucleosomes
how do transcription activators activate expression of DNA
can recruit histone modifying enzymes and chromatin-remodeling protein complexes to the promoter to make chromatin more accessible to transcription factors and RNA polymerase
histone acetylases
add acetyl group to select lysine
makes DNA more accessible and enhances transcription efficiency
histone deacetylases
remove acetyl group and restores packaging
makes DNA less accessible and inactivates expression
gene activation in eukaryotes
activators bind to enhancers (regulatory DNA sequences that enhance transcription) located far away from promoter region
binding of activators causes DNA bending and positions activators closer to general transcription factors and RNA polymerase II
(activators interact with mediator -adaptor)
how do activators only interact with correct gene regulators and not loop in wrong direction?
chromosomes arranged in loops - TADs (topological associated domains) held together by protein clamps
factors in combinatorial control
general transcription factors: assemble at promoter, always the same
transcription regulators: location of binding sites and regulators themselves different
mediator: assembly place for factors, regulators, chromatin modifying proteins
COMBINED effect of all of these determines rate of transcription of a gene
how do eukaryotes control expression of more than one gene at once without operons
the same transcription factor can regulate the expression of multiple genes if the genes have regulatory sites that recognize the same transcription factor
e.g. cortisol binds to cortisol receptor which activates many different genes
how does cell differentiation occur during development
different combinations of gene regulatory proteins cause cells to differentiate
pluripotency
developmentally flexible- can give rise to different cell types (embryonic stem cells)
how many different types of cell types can an organism with three transcription regulators create?
2^3 = 8
(one with 0, 3 with 1, 2 with 2, 1 with all three)
master regulators
regulate all cells in an organ and regulate expression of other regulators
- produce a cascade of other regulators
Ey transcription master regulator in flies
control differentiation of cells that form the eye
-mutation in Ey -> no eyes
-some of genes regulated by Ey encode other transcription regulators for more genes
effect of expressing Ey in precursor leg cells in flies
cause formation of non functional eye structures on leg
iPCs
induced pluripotent cells- by set of 3 transcription factors that induce de-differentiation of fibroblasts
myoD
muscle specific transcriptional regulator
can specialized cells be induced in culture
yes
terminally differentiated cells
do not divide; skeletal muscles, neurons
differentiated cells that can divide
liver, skin
cell memory
pattern of gene expression profile responsible foe given cell identity (skin cell division gives more skin cells)
three ways a cell maintains its identity
- positive feedback loop (most prevalent)
- condensed chromatin
- DNA methylation
ALL epigenetic changes
epigenetic changes
no changes in DNA nucleotide sequence
positive feedback loop cell identity
master regulator activates its own transcription at every division generating a self sustaining circuit of gene expression
generates cell memory because protein produced stimulates its own production
e.g. MyoD directs cells to become muscle cells
condensed chromatin cell identity
chemically modified histones passed from parent to daughter cell
- modified histones are distributed randomly between daughter cells and enzymes fill in the blanks based on neighboring histones to restore parental modification
(same X chromosome in mammalian females inactive through many cell generations)
DNA methylation cell identity
covalent modification (methylation of cytosine) by maintenance methyltransferase maintains DNA methylation in parent to daughter cells: methylates only CG sequences that are base paired with methylated CG sequences
generally turns OFF transcription
types of post-transcriptional controls
operate after RNA polymerase has begun translation
- alternative splicing
- mRNA sequences that control translation (5’ and 3’ UTR)
- regulatory RNAs control gene expression
ribosome binding sites
place on the mRNA where ribosome binds; important for positioning the ribosome
bacterial translational control on ribosome binding sites
bacteria have proteins that bind to ribosome binding site and repress translation of specific mRNAs
proteins recruited by 3D structure of mRNA itself
thermosensor in L. monocytogenes
RNA sequence that controls translation of infectious genes due to temp.
elevated temp inside host changes 3D structure of mRNA (H bonds dissociate) and exposes ribosome binding site to translate infectious genes
microRNA
direct destruction of target mRNA
-precursor miRNA transcribed in nucleus and exported to cytosol where it forms mature miRNA with help of nuclease Dicer
- RISC protein binds with antisense single stranded miRNA and searches for match
- when match is found mRNA is degraded by nuclease within RISC and RISC released
RISC
RNA induced silencing complex
a “scanner”
RNA interference (RNAi)
type of gene silencing where siRNAs bind to mRNA and target degradation
-foreign double stranded RNA cleaved by Dicer to create siRNAs
-assemble with RISC proteins and sense strand discarded so that more foreign RNA can be destroyed
RITS
RNA induced transcriptional silencing
RNAi and transcriptional silencing
siRNA can assemble into RITS complex to search for complementary RNA emerging from RNA polymerase
- attracts histone modification proteins resulting in heterochromatin formation and transcriptional repression
-resembles adaptive immune response
CRISPR
clustered regulatory interspaced short palindromic repeats
CRIPSR system function
small noncoding RNA system that provides bacteria with immunity from viral infections by integrating viral DNA into bacterial genome and transcribing RNA and binding to Cas protein; now able to identify and cleave viral DNA
long noncoding RNA
roles are not clear
Xist (X-inactive specific transcript) initiates and maintains epigenetic silencing of one copy of X chromosome in females by promoting heterochromatin formation
some serve as scaffolds to bring proteins together that function in same processes
