L9: Transcriptional Regulation Of Eukaryotic Gene Expression I Flashcards
Eukaryotic Gene Regulatory Proteins (Transcription Factors)
Bind specific DNA sequences in control region of gene ->
Determine where transcription will start
Activate or repress transcription
Bind multiple regulatory 10s or 1000s of bp ‘upstream’ of gene (opp direction of transcription) or ‘downstream’ of gene (same direction as transcription) -> integration of dozens of signals converging on single promoter to produce appropriate levels of transcripts (in prokaryotes: 1 or few signals) and allows expression of gene in different cell types during development
Promoter
Eukaryotic gene control region
TATA box: position -25 to -35 relative to transcription start site (TSS). Sequence rich in As and Ts
General transcription factors (GTFs) & RNA polymerase II assemble:
GTFs- proteins required for transcription initiation of all RNA pol II transcribed genes.
TFIID: distorts DNA at promoter- landmark for active promoter. TATA binding protein (TBP)- a subunit.
TFIIH- helicase activity unwinds DNA- access for RNA pol II.
Most GTFs dissociate when elongation of transcription starts
Proximal promoter region or elements
Eukaryotic gene control regions
Control regions within 100-200 bp of TSS
May be cell specific
Can be inverted and still stimulate transcription
Enhancer regions or elements
Eukaryotic gene control regions
Control regions >200bp from TSS; can be 1000s of bp away
Can be inverted and still stimulate expression
Can be upstream, downstream, within an intron, downstream of final exon of gene and still stimulate expression
Often composed of multiple individual gene control elements. Each binds specific gene regulatory protein. Cooperative binding
Mediator
Mediator of transcription complex
Multiprotein complex ~30 subunits. Genes encoding subunits are conserved in multicellular organisms
Binds RNA pol II
Subunits binds activation domains of some gene regulatory proteins. May bind several simultaneously
1 subunit has histone acetylase activity. Promoter in hyoeracetylated state. Favours transcription
Directly regulates assembly of transcription pre-initiation complex
Gene activator proteins
Direct local alterations in chromatin structure
Recruit the following to promoter:
chromatin remodeling complexes (-> change interactions between DNA and histones), histone chaperones (remove and replace histones) and histone-modifying enzymes (change groups on histone proteins)
-> DNA becomes more accessible to transcriptional machinery (GTFs, RNA pol II, Mediator) & additional gene regulatory proteins
Order of steps in transcriptional initiation varies
From gene to gene
For same gene, depending on gene regulatory proteins bound at given time
Example of transcriptional initiation steps in same gene
Gene activator protein binds to chromatin (+ chromatin remodeling complex) -> chromatin remodelling (+histone modification enzymes) -> covalent histone modification (+other activator proteins) -> additional activator proteins bound to gene regulatory region (+mediator & general transcription factors RNA polymerase) -> assembly of pre-initiation complex at promoter (other gene activator proteins, rearrangement of proteins in pre-initiation complex) -> transcription initiative
Eukaryotic gene repressor protein operation
Multiple mechanisms. Many repressor proteins work through -> one mechanism on single gene
Repression of gene activity (e.g important during development. Genes must only be expressed at appropriate times)
Mechanisms in eukaryotic gene repressor proteins operation
Activator & repressor proteins compete for same regulatory DNA sequence
Activator and repressor proteins bind DNA but repressor binds activation domain of activator
Repressor protein prevents assembly of GTFs or RNA pol II release from GTFs
Repressor protein recruits chromatin remodelling complex -> returns chromatin to pre-transcription state
Repressor protein recruits histone deacetylase to promoter -> decreased transcription
Repressor protein attracts histone methyltransferase. -> recruitment of other DNA-binding proteins and gene silencing (silencing may be at single gene or region of chromatin)
Gene regulator proteins
Acts as parts to build complexes whose function depends in final assembly (combinatorial control)
Each on their own have weak affinity for DNA. Increased affinity together
Some create environment promoting other proteins to bind. Other activators & repressors bind promoter directly. Co-activators & co-repressors that do not bind DNA directly
May be involved in gene activation or repression complexes. Dependent on particular DNA regulatory sequences and other regulatory proteins present
Deletion analysis
Identification of eukaryotic gene regulatory regions
Isolate upstream region of gene (contain regulatory sequences)
Use restriction enzymes or exonuclease-> series of deletions of upstream region
Insert into vector containing reporter gene
Transform e.coli to amplify and isolate individual plasmids
Transfect cultured cells with individual plasmids
Assay reporter enzyme activity in transfected cell extracts
Identify parts of upstreams region responsible for reporter gene expression
Steps of deletion analysis
Recombinant DNA techniques -> 5’ deletion series & plasmid vector with reporter gene -> ligate into vector carrying reporter gene. Transform e.coli & isolate plasmid DNAs -> 5’ deletion mutants -> transfect each type of plasmid separately into cultured cells -> reporter plasmid. MENA, enzyme -> prepare cell extract & assay activity of reporter enzyme -> reporter-gene expression
DNA foot printing
Identification of eukaryotic gene regulatory regions
Isolate upstream region of gene
Label 5’ end of DNA segment (e.g using polynucleotide kinase and labelled ATP)
Cleavage of DNA by nuclease digestion (cant occur where regulatory protein is bound)
Protein removed, DNA made ss and separated on gel (electrophoresis)
DNA protected by protein leaves ‘footprint’ -> isolate region & determine sequence -> identification of regulatory sequences
Electrophoretic Mobility Shift Assay (EMSA)
Aka gel shift assay
Isolate and label (e.g radioactively) DNA of regulatory region
Add cell extract (DNA alone = control)
DNA bound to proteins in cell extract -> show different mobility on polyacrylamide gel than free DNA. Shift in size depending on size of bound protein
To identify regulatory proteins: fractionate cell extract -> use aliquot of fractions in gel shift assay -> go back to fractions of extract that gave shift and purify individual regulatory proteins
