Transcription activation in multicellular
organisms.
Liam.Keegan@ceitec.muni.cz
• Promoters
• Proximal regulatory elements
• Enhancers
25.7 Response Elements Are
Recognized by Activators
• Response elements may be located in promoters or enhancers. Proximal
regulatory elements were targets of the earliest studies.
Figure 25.11
Many gene regulators from mammalian cells were identified and purified using the
Bandshift assay or Gel mobility shift assay.
(also Gel Retardation assay, Electrophoretic Mobility Shift Assay (EMSA)).
Gel is non-denaturing and buffer is mild to avoid disrupting complex.
Gel matrix ‘traps’ protein DNA complex keeping components together to prevent disassociation.
Advantages of the bandshift assay.
• DNase1 footprinting required pure proteins but bandshift assays
could detect different scarce DNA-binding proteins even in
crude nuclear extracts.
• If the probe DNA fragment comes from an enhancer then a point
mutation that inactivates the enhancer should also prevent DNA
binding by the key regulatory protein in the extract. (Some
proteins bind DNA ends or other sites in the probe DNA)
• The specific bandshift provides a convenient assay to purify the
regulatory protein.
DNA-affinity chromatography using a specific oligonucleotide
target sequence can purify the binding protein in one step.
Oligonucleotide attached to affinity column
contains repeats of a DNA binding site for Sp1 prote
(5’-GGGCGGG-3’)N
Examples of important transcription regulators
identified after purification from nuclear extracts.
• Fos/jun, AP1. oncoproteins studied by Tom
Curran and others. Steve MacKnight showed
these proteins contain a leucine zipper.
• Nf-kappaB (Baltimore lab, MIT) Nuclear
factor binding the immunoglobulin kappa B
enhancer. Important protein in innate immunity
and response to viral infection.
• SRF (Maniatis lab and Richard Treisman in
London) Serum response factor activates genes
involved in growth of cells in tissue culture.
• SP1 (Tjian lab)
The simplest sequence-specific DNA recognition mechanism of all!
CAAT/Enhancer binding protein (C/EBP).
The mechanism of transcription activation by the genespecific
regulators.
• The primary idea is the same as for GAL4 in yeast.
• Recruitment of transcription inititation factors and
RNA polymerase to the promoter by DNA loop
formation between the enhancer and the promoter.
Mediator.
• In vitro transcription extracts were developed in
which Pol II and the General Transcription
Factors responded with increased transcription
when GAL4 or another activator was added.
• This allowed further purifications to isolate
additional components needed to mediate the
activation. Depending on what activator was
used this purified coactivators or mediator
complex.
• Some argue that the complex of Pol II, GTFs
and Mediator can exist as a ‘holoenzyme’ that
recruits to promoters as in E. coli.
25.2 Gene specific transcription factors (Activators/Coactivators)
are distinct from the General Transcription factors .
• The basal apparatus determines the startpoint for
transcription.
• Activators determine the frequency of transcription.
• Activators work by making protein–protein contacts
with the basal factors.
• Activators may work via coactivators.
• Some components of the transcriptional apparatus work
by changing chromatin structure. Coactivator p300/CBP
(CREB-binding protein) is a histone acetylase targeting
H4.
Figure 25.2
• Several factors in the basal apparatus are targets with
which activators or coactivators interact. Many
activators contact different TAFs. Acidic activators
contact TFIIB.
Figure 25.8
• An activator that does not have an activating domain may work by binding
a coactivator that has an activating domain. Strong viral activators like
Herpesvirus VP16 and Adenovirus E1A are recruited by binding other
proteins.
Figure 25.7
The IFN-β enhanceosome complex.
Vo N , Goodman R H J. Biol. Chem. 2001;276:13505-13508
©2001 by American Society for Biochemistry and Molecular Biology
Transcription activation in chromatin.
• RNA polymerase was not able to initiate transcription at
promoters and some sequence-specific DNA binding proteins
could not bind their sites if histones were added first (pioneer
transcription factors).
• Getting transcription initiation and later regulated transcription
on chromatin templates required extra factors that operate on
nucleosomes.
• Constructs with enhancers reintroduced in mammalian
chromosomes were often silenced. This turned out later to be
due to lack of regulatory elements like the globin gene Locus
Control Region (LCR).
Chromatin immunoprecipitation for genome-wide location analysis of
chromosomal proteins in living cells
5. Analyse which DNA fragments are enriched in the immunoprecipitated fraction.
6. Best done now by Next Generation sequencing (ChIP-Seq)
7. Compare reads of immunoprecipitated sequences back to the genome sequence to locate wh
the protein was bound.
Mapping Polycomb-repressed domains in the bithorax complex using in vivo formaldehyde cross-linked chromatin.
Orlando V, Paro R.
Cell. 1993 Dec 17;75(6):1187-98.
There is a contrast between a sequence-specific DNA-binding protein and the DNA packaging
nucleosome.
Histone amino acid side chains do not enter the major groove and ‘read’ the DNA sequence.
• … but nucleosomes are very crowded along the DNA. Tight wrapping of DNA in nucleosomes
means they have positional preferences to bind over more bendable A-T rich sequences first.
Very G-C sequences are covered last or not at all.
Most yeast promoters are relatively “nucleosome free” …many
mammalian promoters also….
Relatively nucleosome-free regions, DNAse hypersensitive sites and specific
histone modifications help identify enhancers in chromatin.
• Chromatin immunoprecipitation methods are easier to use with
abundant nucleosomes than with scarce sequence-specific
DNA-binding proteins.
• Modified histones associated with active (H3K4 Me1,Me3) and
inactive (H3K27 Me3) genes have been mapped extensively.
This is a proxy for mapping the regulatory proteins themselves
and allows likely enhancers and promoters to be identified.
• Promoters and enhancers also tend to be nucleosome-free.
Genome-wide locations of the coactivator P300/cEBP
identify enhancers.
Disease mutations in human enhancers.
• Until now human disease mutations in
noncoding sequences were very difficult to
identify.
• Once enhancers are identified across the human
genome they can be included with exons and
promoters in focused searches for diseasecausing
mutations.
Pre-mRNA processing in eukaryotes.
- Ribozymes and the RNA world-
Lecture outline
• Eukaryotic mRNA stability, 5’ cap and polyA
addition and splicing.
• Mechanism of pre-mRNA splicing and
alternative splicing. RNA editing.
• Self-splicing Group II introns, RNA catalysis,
Ribozymes and the The RNA World.
Capping and polyA tailing of pre-
mRNAs.
• Bacterial mRNA is unstable and has a half-life of
only a few minutes.
• Transcription in both bacteria and eukaryotes is
not very fast at 40 nucleotides/sec, 2.4 kb/min.
• Most bacterial mRNAs can be transcribed in a
few minutes whereas a human transcript of 100
kb would take 40 minutes.
Figure 7.14
The life cycle of a bacterial mRNA.
•hnRNA/pre-mRNA processing
• A typical eukaryotic mRNA:
•
• 5'cap..............________________________________.....................AAAAAAAAAn
• nontranslated coding long 3' trailer n =100-200
• leader <300nt often >1000nt
Figure 7.17
Pre-mRNA 5’ cap addition and 3’ polyadenylation and
splicing are completed before export from the nucleus.
The 5′ End of Eukaryotic mRNA Is Capped to
protect it against 5’ exonucleases.
• A 5′ cap is formed by adding a G to
the terminal base of the transcript
via a 5′–5′ link.
• The capping enzyme is guanylyl
transferase.
– 1-3 methyl groups are added to
the base of the new terminal
guanosine
– 2 methyl-riboses near cap.
– Base modification m6A in 5’
cap and also inside mRNAs at
RRACH consensus, especially
around stop codon.
Figure 7.18
3’ end cleavage and polyadenylation.
The cleavage and polyadenylation
specificity factor (CPSF) recognizes
AAUAAA (AtwoU, Athree).
Cleavage factors (CFI and CFII),
endonuclease cleaves RNA 30 bases
downstream.
Poly(A) polymerase add ~200 A
residues processively to the 3′ end.
The polyA tail stabilizes mRNA, facilitates
translation.
• Poly A tail is >200 nucleotides long and binds many
copies of Poly A-binding protein (PABP).
• PABP stabilizes the mRNA against degradation.
• PABP binds eIF4G (eukaryotic initiation factor 4G)
and facilitates translation of message by the ribosome.
• 5’ and 3’ ends are thought to contact one another by
forming a loop of the mRNA.
Removal of cap and polyA tails are required for
regulated degradation of mRNA.
• Degradation of yeast
mRNA requires removal
of the 5′ cap and the 3′
poly(A).
• One yeast pathway
involves exonucleolytic
degradation from 5′→3′.
Figure 7.24
• Another yeast mRNA degradation pathway uses a complex of
several exonucleases that work in the 3′→5′ direction The
exosome.
• The deadenylase of animal cells may bind directly to the 5′ cap.
Figure 7.25
The mechanism of eukaryotic premRNA
splicing.
Pre-mRNA splicing.
Figure 26.2
EM heteroduplex analysis of Adenovirus RNA transcripts annealed to
Adenovirus DNA showed formation of D (displacement) loops.
Figure 26.3
Consensus 5’ and 3’ splice sites.
The GU-AG rule
Protein coding sequence must be ‘in frame’ across the splice junction.
Remember GU…AG are splice junction sequences. Mnemonic;
GULAG maybe?
Intron sequences are generally not conserved even between closely
related genes.
Splicing must preserve the open reading
frame.
• 5’ and 3’ splice site have to be in the same reading
frame.
• Reading frames are 1, after first base of codon, 2 after
second base and 3 after third base, ie. between codons
• GU..AG consensus are the ends of the intron to be
removed, not in the actual coding sequence.
Splice Junctions Are Read in Pairs
• All 5′ splice sites are functionally equivalent, and all 3′ splice sites are functionally equivalent.
• Usually draw exons as boxes to emphasise them with splices joining box corners and introns as lines between the
boxes .
• Gene rearrangements within big introns have a good 1/3 chance to produce fusion proteins. (Exon shuffling in
evolution).
Figure 26.4
Figure 26.7
Alkaline lysis of ssRNA.
The reactive
2’ OH on ribose that
mediates alkaline
self-cleavage of
single stranded RNA
Splicing is catalysed by a series of five small nuclear ribonucleoprotein
particles (snRNPs, ‘SNURPs’). Conserved small structured RNAs with Sm
and other proteins bound.
SNURPs assemble sequentially on pre-mRNA to form a spliceosome.
U1 C
U1 A
U1 70K
Sm
U1 snRNP
Cryo-EM picture
(Stark et al. Nature 409, 539)
CAUUCAUApppGm3 5’
U1 C
U1 A
U1 70K
Sm
U1 snRNP
XXXXXXXXX
5’ Splice Site Recognition
GURA GU YNYURAY Y1 0 - 1 2 YA G
3’ Splice Site Recognition
65 35
U2AF
SF1/
BBP
branch
point
Py-
tract
3´
ss
GURA GU YNYURAY Y1 0 - 1 2 YA G
65 35
U2AF
3’ Splice Site Recognition
U2 snRNP
SF1/
BBP
GURA GU
A
Y1 0 - 1 2 YA G
65 35
U2AF
3’ Splice Site Recognition
UACUA C
AUGAU G
Sm
U2 snRNP
branch
point
The U4/U5/U6 tri snRNP arrives last and base pairs to U2.
Patel and Steitz, Nature Reviews Molecular Cell Biology (2001) 4,
960-970
Conformation changes bring splice junctions
together.
Pre-mRNA
spliceosome
RNA processing is coupled to transcription.
Splicing Is Connected to Export and
efficient translation of mRNA
Figure 26.16
Alternative splicing is an important
strategy to increase the number of
gene products produced from a
single gene.
Modes of alternative splicing.
Alternative splicing in Drosophila Dscam .
Inclusion of alternative exons may be promoted by exonic splicing
enhancers that bind serine/arginine-rich (SR) RNA-binding proteins.
Heterogeneous ribonuclear proteins (hnRNP proteins) coat pre-mRNAs
and mRNAs and often inhibit splicing events that SR proteins promote.
‘Ying-yang’ balance of these effects on many splicing events.
Splicing complexes Can be Formed by Intron
Definition or Exon Definition
• The direct, intron definition, way of forming an E
complex at short introns is for:
– U1 snRNP to bind at the 5′ splice site
– U2AF to bind at a pyrimidine tract between the branch
site and the 3′ splice site
• Another possibility, exon definition, for very long
introns is for the complex to form between:
– U2AF at the pyrimidine tract
– U1 snRNP at a downstream 5′ splice site
The importance of alternative splicing
It is estimated that 95% of human genes are
alternatively spliced from new sequencing information.
Alternative splicing creates protein diversity
26,000 human protein-encoding genes,
yet 90,000 proteins
Aberrant splicing is a frequent effect of human disease gene
mutations.
Base deamination RNA editing also occurs site-specifically in some
transcripts and changes codon meaning.
Adenosine deaminases acting on RNA (ADARs) edit 4% of Drosophila transcripts (972
sites), and ~250 known sites in human transcripts, including those encoding brain ionchannel
subunits, particularly GluR B, the dominant subunit of AMPA class glutamate
receptors.
APOBECs are C to U deaminases first identified in RNA ediding but more important as
DNA editors. Activation induced deaminase (AID) edits DNA in immunoglobulin genes to
initiate Somatic Hypermutation of antibody variable regions. APOBEC3G edits HIV and
interferes with virus replication.
RNA catalysis, Ribozymes and the
RNA World.
The RNA World.
Self-splcing intorns are small transposable elements that
remove themselves cleanly from the transcripts they are
inserted in.
Group I intron splicing in 26S rRNA of Tetrahymena is
catalysed by RNA in the absence of protein (Tom Cech).
The RNA World idea - RIBOZYMES.
• Ribosomes and spliceosomes function through RNA-RNA interactions. Before the
self-splicing introns were discovered it was assumed that catalysis would be caused
by the proteins in these complexes.
• The discovery that RNA alone is catalytic in the Group I self-splicing intron showed
that the RNA could be catalytic in ribosomes and spliceosomes also. RNA enzymes
are called RIBOZYMES and many artificial ones have been created.
• This contributes to our understanding of possible origins of life as RNA alone could
have carried out its own replication using natural ribozyme activities until translation
was invented as a way to make more versatile protein catalysts.
• DNA also may have been a later invention for use as a genomic library. It is more
stable to hydrolytic cleavage than RNA and more readable by proteins than dsRNA
to allow gene control.
The RNA World.
Group II introns also self-splice by RNA catalysis (Cech).
Figure 26.20
Figure 27.13
How introns might have spread.
Mobile Group II introns.
How spliceosomal introns might have evolved from
self-splicing Group II introns.
• Autosplicing introns may require maturase
protein activities encoded within the intron.
– They assist folding into the active
catalytic structure.
• Imagine that such an intron loses its maturase
but can use maturase provided by other
similar introns.
• This intron might eventually evolve to
consist of just the target splice junctions
GU..AG with all the catalytic activities
supplied by a trans-acting splicesome.
Figure 27.15
Summary.
• Eukaryotic mRNA stability, 5’ cap and polyA
addition and splicing.
• Mechanism of pre-mRNA splicing and
alternative splicing. RNA editing.
• Self-splicing Group II introns, RNA catalysis,
Ribozymes and the The RNA World.