Transcription and RNA I Flashcards
Transcription
RNA synthesized using a DNA template, allows structural regulatory and translational info in DNA to be transferred to RNA where it can function throughout the cell
transcription as a process
transcriptional machinery localizes to specific sites in the nucleus (transcription factories) and it is accompanied by rapid changes in chromatin
mutations that alter transcription
associated with cancer, diabetes, developmental defects, autoimmune, neurological and cardiovascular diseases; most known are located within protein coding genes
cancer development
many cancer promoting oncogenes are transcription factors, alterations in transcription brought about by DNA methylation and histone modification patterns also associated
non-coding region
hundreds of thousands of potential transcriptional regulatory sequences located at non-coding regions some likely effect gene expression and potentially promote disease
transcription of protein coding genes
consists of 4 major phases
- Initiation
- Pausing
- Elongation
- Termination
RNA polymerase
3 types
RNA pol 1
RNA pol 2
RNA pol 3
RNA pol 1
RNA pol 1 -> Pre-rRNA -> rRNA
RNA pol 2
RNA pol 2 -> Pre- mRNA -> mRNA
RNA pol 2 -> ncRNA
RNA pol 3
RNA pol 3 -> pre-tRNA -> tRNA
RNA pol 3 -> pre-rRNA -> rRNA
RNA pol 3 -> small ncRNA
Ubiquitous constitutive genes
transcription factors and transcriptional machinery needed to transcribe these genes always present and active “house keeping genes”
Ubiquitious inducible genes
transcribed because transcription factors needed to transcribe them are activated in response to an inducing event
Cell type specific constitutive gene
Only expressed in specialized cells, but transcription factors and transcriptional machinery needed to transcribe these genes always present and active in those cells
Cell type specific inducible genes
only expressed in specialized cells when transcription factors needed to transcribe them are activated in response to inducing event
Constitutive vs inducible genes
constitutive always on inducible have to be turned on by inducing event
Major transcriptional regulators
DNA, nucleosomes, transcription factors, transcriptional coregulators, chromatin modifiers, RNA polymerase II preinitiation complex
Cataracts inheritability
TF heat shock factor 4 (HSF4) mutation which reduces level of the protein; HSF4 activates transcription of many genes required for proper lens development
Transcription factors
sequence specific DNA binding proteins which can directly or indirectly regulate transcription of genes
Transcription factor functional subregions
nuclear localization signal, DNA binding domain, TF interaction domains, most also contain protein interaction domains, some contain ligand binding domain, may contain transactivation or trans repression domains
protein interaction domains
allows transcription factors to recruit transcriptional regulatory protein complexes that can modify chromatin structure or activate or repress transcription
protein ligand binding domain
when occupied by ligand converts receptor to active transcription factor
transactivation and trans repression domains
can interact with and regulate activity of RNA polymerase II preinitation complex
Identified features HSF4
DNA binding domain, multifunctional domain (allows HSF4 to form trimers and interact with regulatory proteins), and transcriptionally activation domain
promotor
many DNA sequences that regulate transcription located here; consists of core promotor and proximal region; RNA pol II preinitiation complex binds to Tata box or equivalent DNA sequence just upstream of transcriptional start site
transcriptional regulatory DNA sequences
include response elements, enhancers, and silencers; regulatory sequences can also be found in distal sequences located upstream or down stream of TSS
Response elements
DNA sequences that transcription factors bind to, palindromic and non-palandromic
palindromic
reads same forwards one strand as other strand backwards; TFs (transcription factors) can bind to palindromic as homodimers or heterodimers
gene promotors and REs
gene promotors contain distance sets of REs but some genes may contain same REs so they can get cotranscribed
DNA of different cells in the body
cells in the body essentially contain all of the same DNA it is availability of transcription factors in a particular cell that dictates what genes get transcribed
Enhancers
TFs bind to REs in enhancer and interact and initiate transcription in a cell, tissue, or developmental specific manner
why do enhancers function in a cell/ tissue/ stage of development ?
Because TF that binds and interacts at the enhancer only expressed and activated in specific cell or tissue or during a specific developmental stage
enhancer location
can be upstream or downstream of core promotor and can act a a distance because distant regions of chromosomes can physically interact
topologically associated domains
chromosomes can fold into spatially distinct chromatin compartments; contain looped DNA structures held together at their base by cohesion; may represent functionally distinct regions of genome that are independently regulated
cohesion
multi-protein complex
intrachromosomal looping
enables distant enhancer bound TFs and associated coregulators to interact with core promotors to facilitate the assembly and activation of RNA pol II preinitiation complex; looping helps control expression of tissue specific genes
Enhancers and silencers relation to TAD
enhancers and silencers can only interact with and regulate genes in same TAD NOT those in adjacent TAD
Gamma crystalliin
regulated by HSF4; stable interactions btwn crystalline proteins enable tight packed configuration; mutated HSF4 leads to reduction of gamma crystallin levels leading to destabilization of lens protein matrix leading to cataracts
canine ectodermal dysplasia
autosomal semidominant leads to abnormalities in ectodermal appendages such as hair teeth nails sweat glands; Chinese crested hairless have this bc of a 7 basepair insertion resulting in FOXI3 protein being non functional
FOXI3
this is non functional in canine ectodermal dysplasia; this encodes a transcription factor that is expressed in developing hair and teeth and is required for proper development of ectodermal appendages; this TF is in many tissues but is kept inactive
Transcription factor activity or localiztion
signal’s from a cell’s internal and external environment regulate transcription of specific genes by controlling transcription factor activity or localization
extracellular signals
hormones, growth factors, differentiation factors, ECM
intracellular signals
DNA damage, organelle integrity, hypoxia, ROS, and pH
signaling intermediates
many signaling intermediates are protein kinases that when activated can phosphorylate TF proteins or other proteins that can affect TF activity
Transcription factors in the cytoplams
in the cytoplasm transcription factors are often sequestered by inhibitor proteins activated cell signaling kinases can promote release of or degradation of inhibitor so TF can go to nucleus and stimulate transcription
mechanism of transcription for FOXI3 gene
ectodysplasin secreted by outer enamel ectodermal cells binds to its receptor EDAR on tooth precursor cells, activated receptor leads to signaling cascade, degradation inhibitor IkB, enabling TF NFkB to enter nucleus stimulating transcription of FOX13 gene
Rdy
early onset rod-cone dysplasia in Abyssinians leads to aberrant photoreceptor development and degradation;bc of mutation to TF CDX (needed for photoreceptor cell development and survival); mutation leads to premature stop codon and CDX can’t stimulate transcription of target genes
Chromatin role in controlling transcription
chromatin determines if TFs and RNA pol II pre initiation complex can access DNA; chromatin closed state is densely packed nucleosomes with pos histone tails interacting with histones of other nucleosomes, chromatin open state is transcriptionally active, characterized by decreased nucleosome density and packing
regulating open and closed chromatin
done by protein complexes that regulate nucleosome positioning, density, and interactions;
to activate transcription of a gene
HAT (histone acetyltransferase) and activating ATP-dependent chormatin remodeling proteins are recruited
HAT
transfer acetyl group to basic amino acids neutralizing pos charge disrupting restrictive interactions between nucleosomes
Activating ATP dependent chromatin remodeling proteins
alter nucleosome density and positioning either by promoting nucleosome disassembly or by altering the location of nucleosome relative to DNA; resulting chromatin is depleted of nucleosomes allowing access and transcription
repress transcription of a gene
histone deacetyalase and repressing ATP dependent chromatin remodling protein restrict RNA pol II preinitiation complex and TF access to DNA
histone deacetylase
removes acetyl groups from histone tail lysine restoring positive charges and in turn interactions between nucleosomes
Repressing ATP dependent chromatin remodling protein
reestablish and maintain restrictive nucleosome density and positioning
Chromatin modifieres
Chromatin modifying protein complexes recruited by TFs and by specific epigenetic modifications like DNA methylation and posttranslation histone tail modification; epigenetic modifications good bc reversible
Histone modifictations writers
AA in N terminal tails of nucleosomal histones can be modified by methylation, phosphorylation, ubiquitination and acetylation; these are added by writer proteins
histone modifications readers
histone modifications recognized by readers which can recruit additional proteins and protein complexes to regulate transcription
histone modification erasers
remove specific histone modifications and allow new ones to be written
regulatory complex
histone readers, writers, and erasers together; readers direct to specific histone modifications erasers and writers modify nearby histone marks
Activating marks
associated with actively transcribed genes, enriched in chromatin of actively transcribed genes
repressive marks
associated with repressed genes, enriched in chromatin of non-transcribed genes
histone marks
can recruit proteins to modify chromatin structure to permit or inhibit transcription
cancer and epigenetics
genes encoding histones and DNA modifiying enzymes are frequently mutated in many types cancer these enable epigenetic modifications that promote tumorigenesis (increasing transcription oncogenes decrasessig tumor suppressor genes)
EZH2 misregulation
OVerexpression fo this gene or mutation lead to represses genes that would normally inhibit tumorigenesis
Preinitiation complex
RNA Pol II part of this along with core promotor initiation factors (general TFs)
RNA pol II
catalyzes RNA synth and contains c terminal tail which plays important role in regulation
TFII-D
contains TATA binding protein which positions RNA PIC just upstream of transcriptional start site
TFII-H
contains two enzymatic activities, helicase activity and protein kinase activty
TFII-H helicase activity
unwinds DNA to expose template strand resulting in transcriptionally primed open complex
TFII-H protein kinase activity
phosphorylates C terminal tail of Pol II
mediator
large multisubunit protein complex communicates regulatory signals from TFs and coregulators to transcriptional machinery; recruited by enhancer bound TFs, brought close to core promotor via chromosomal looping, stimulates assembly and activation of RNA PIC
RNA PIC assembly
after this template DNA is melted TFII-H helicase and polII initates transcription; TFII-H kinase phosphorylates c terminal tail enables transcription to continue and disentangles RNA pol II from RNA PIC (promotor escape)
creation of functional mRNA
phosphorylated Pol II C-terminal tail recruited chromatin remodling elongation and RNA processing factors that are needed to process primary transcript and process it into functional mRNA
RNA Pol II pausing
for many genes this pauses 30-60 nucleotides downstream of transcriptional start site; pausing mediated by inhibitory factors and by additional factors that stabilize its interaction with DNA; productive elongation restored after inactivation of pausing factors pausing allows for stable elongation complexes
