L9: Transcriptional Regulation Of Eukaryotic Gene Expression I Flashcards

1
Q

Eukaryotic Gene Regulatory Proteins (Transcription Factors)

A

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

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2
Q

Promoter

A

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

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3
Q

Proximal promoter region or elements

A

Eukaryotic gene control regions

Control regions within 100-200 bp of TSS

May be cell specific

Can be inverted and still stimulate transcription

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4
Q

Enhancer regions or elements

A

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

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5
Q

Mediator

A

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

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6
Q

Gene activator proteins

A

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

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7
Q

Order of steps in transcriptional initiation varies

A

From gene to gene

For same gene, depending on gene regulatory proteins bound at given time

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8
Q

Example of transcriptional initiation steps in same gene

A

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

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9
Q

Eukaryotic gene repressor protein operation

A

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)

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10
Q

Mechanisms in eukaryotic gene repressor proteins operation

A

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)

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11
Q

Gene regulator proteins

A

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

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12
Q

Deletion analysis

A

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

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13
Q

Steps of deletion analysis

A

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

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14
Q

DNA foot printing

A

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

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15
Q

Electrophoretic Mobility Shift Assay (EMSA)

A

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

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