Transcript Introduction to Transcriptional Machinery

Introduction to
Transcriptional Machinery
"DNA makes RNA,
RNA makes protein,
and proteins make
us." Francis Crick
Central Dogma of Molecular
Biology
RNA Polymerase of E. Coli
• Transcribes all mRNA, rRNAs and tRNAs
• 7,000 molecules per cell
• 5,000 molecules are synthesizing RNA at any
given time
• M.W. of the holoenzyme is ~465 Kd
RNA Polymerase of E. Coli
 Factor Controls Specificity
Holoenzyme & Core Enzyme
• Holoenzyme binds promoters with half
lives of hours - 1,000 time higher than
core enzyme.
• Holoenzyme has a drastically reduced
ability to recognize “loose binding sites” half life of <1sec – 104 time lower than
core enzyme.
Transcription Initiation
Promoter Elements in E. Coli
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-35: recognition domain
-10: unwinding domain
Seperating distances
UP element
Start Point: purine in 90% of the genes
16-19
First Level of Regulation
T80A95T45A60A50T96
• ~100 fold variation in the binding rate of
RNA Pol to different promoters in vitro.
• Binding rates correlate with the
frequencies of transcription in vivo.
E. Coli has several  Factors
 Factors Recognize Promoters
by Consensus Sequences
Termination
What was known in the 1960’s
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Jacob and Monod 1961 – genetic control
mechanisms in prokaryotes
Anticipation for Eukarotes…
Eukaryotes – genomic complexity –
reiterated DNA sequences
Lack of genetic approach
February 1969, Strait of Juan
de Fuca
“Eureka!”
Taken from: The eukaryotic
tarnscriptional machinery, Robert G
Roeder
3 RNA Polymerases
• Pol I localized within nucleoli – the sites of
rRNA gene transcription
• Pol II and Pol III
restricted to the
nucleoplasm
3 RNA Polymerases
• Roberto Weinmann - 1974
• Differential sensitivities to the mushroom
toxin  - amanitin
• Pol I – rRNA synthesis
• Pol II – adenovirus pre-mRNA
• Pol III – cellular 5S and tRNA
RNA Polymerases of
Eukaryotes
• Pol I - transcribes pre-ribosomal RNA
(18S, 5.8S, 28S)
• Pol II - mRNAs
• Pol III - tRNAs, 5S RNAs and some
specialized small RNAs.
RNA Polymerase II
• 2002 – RNA Pol II structure
• 2003 – transcription complex structure
(RNA Pol II + TFIIS)
• , ’, I, II,  - conserved in yeast and
bacteria – evolutionary
conserved mechanism
of transcription
Significant homology between
eukaryotic and bacterial RNA
polymerases in their structure
Transcription Mechanism
• RNA Pol II can catalyze RNA synthesis
but cannot initiate.
• Assembly
• Initiation
• Elongation
• Termination
Transcription
Mechanism
TBP
• Only GTF that creates sequence specific
contact with DNA
• Unusual Binding in minor groove
• Causes
DNA bending
TBP
• 80% conserved between yeast and man
• Large outer surface binds proteins
• Deformation of DNA structure, but no
strand separation
The transcriptional machinery
• Initiation begins with the formation of the first
phosphodiester bond and phosphorylation of
Ser5 on the CTD by TFIIH.
• mRNA passes through a positively charged exit
channel, and once the RNA is approximately
18n long it becomes accessible to the RNA
processing machinery.
• Consistent with the coupling of transcript
capping to early transcription events
Pre-mRNA Processing
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Addition of 5’ cap
Splicing – removal of intron sequences
Generation of 3’ poly-A tail.
3’ cleavage
RNA serveillance by the exosome
Packaging of the mRNA for export
Occurs (most efficiently) co-transcriptionally
Transcription Regulating
Elements
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GTFs - required at any Pol II promoter
Enhancers – sequences, increase transcription
Transactivators - bind enhancers
Co-activators - act indirectly, not by binding to
DNA, communication between transactivators
and RNA PolII + GTS
• Mediator - 20 proteins, Interacts with CTD
Major Differences between
Pro & Eu
• Prokaryotes RNA Pol has access to promoters
and initiates transcription even in the absence
of activators and repressors.
• Eukaryotes - promoters are generally inactive
in vivo
• Transcription in eukaryotes is seperated in
both space and time from translation
The CTD is
Phosphorylated
at Initiation
CTD
• Highly conserved tandemly repeated
heptapeptide motif (YSPTSPS)
• Platform for ordered assembly of the
different families of pre-mRNA processing
machinery
• Undergoes phosphorylation and
dephosphorylation during the transcription
cycle
CTD
• P-TEFb contains CDK9 and cyclin T
• It couples RNA processing to transcription
by phosphorylating Ser2 of CTD
• RNA Pol II is recycled through
dephosphorylation of Ser2 by the
phosphatase activity of Fcp1
CTD Phosphorylation During
Transcription
Splicing
(& Alternative Splicing)
Expansive role of
Transcription
• RNA surveillance – Exosome associates
with Spt6 EF
• Coupling of transcription to mRNA export
• 19S particle of the Proteosome recruited
to active promoters – important for efficient
RNA Pol II elongation
Translation and PostTranslation
• Bacteria – translation occurs as the
nascent transcript emerges from the RNA
polymerase
• It is assumed that in eukaryotes
transcription and translation are spatially
separated events
• Protein synthesis – solely a cytoplasmic
event? (1977 – Gozes et al, 2001 lborra et
al)
Traditional View of Gene
Expression
Contemporary View of Gene
Expression
The Sister
Chromatids
of a Mitotic Pair
Chromatin Packing
105 mm Double helix
2 nm
“Beads-on-a-string”
11nm
30 nm fiber of
Packed nucleosomes
30 nm
Chromosomal loops
Attached to nuclear
scaffold
300 nm
~x7
~x100
Condensed section
of metaphase
chromosome
700 nm
~x104
5-10 mm
Entire metaphase
chromosome
1400 nm
Chromatin Structure
• DNA accessibility – a major challenge in a
chromatin environment
• Nucleosomes –
GC Pairs Are Preferred
building
blocks of
chromatin
Histone Core
AT Pairs Are Preferred
DNA
Structure of the Nucleosome
•146 bp are wrapped around the histone core
1.75 times
• ~0-80 bp in the linker sequences between
nucleosomes
• Human genome (~6x109 bp) contains ~3x107
nucleosomes
• The histone core (octamer) consists of two copies of:
• Histones H2A, H2B, H3 and H4
• Histone H1 binds in the spacing linker sequence
The Nucleosome
DNA
H2B
Histone
Core
H2A
H4
H3
H3
H2B
H2A
Histones
•Highly conserved throughout eukaryotic
evolution
• Mutations in histones encoding genes are
often lethal
• Highly abundant (~60 million copies/cell)
• Additional non-histone proteins play a role in
the chromatin structure and function
Types and Properties of Histones
Interaction of DNA with Positively Charged
Residues in the Nucleosome Core
DNA
Red: The positively charged lysines & arginines
The DNA is wrapped along these residues
H1 Histone
• In the presence of H1,
166 bp
are protected from nucleolytic cleavage ->
full two tight loops (83 x 2 bp).
• When histone H1 is extracted, the
resulting structure is the 11 nm “beads-ona-string”
View Along the Axis of One Turn of the 30nm Fibe
DNA
H1 Histone
Histone
Core
Side View of the 30nm Fiber
Histone core
11 nm fiber
30 nm fiber
DNA
Nucleosome
Histone H1
Histone H1
“Chicken and Egg” Scenario
• heterochromatin and euchromatin
• How do TFs access the DNA in the first place?
• Example: GR, NF1 and MMTV gene (Di Groce
et al., 1999)
Histone Code Hypothesis
• language of covalent post-translational
histone modifications
• acetylation
• phosphorylation
• methylation
• ubiquitylation
• ADP-ribosylation and
• glycosylation
Regulation of Nucleosome
Stability
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Sequence elements
Post-translational modifications
Nucleosome remodeling complexes
Transcriptional Elongation
Nucleosome Depletion at
Promoters
Taken from: The transcriptional
regulatory code of eukaryotic cells,
Barrera & Ren
Dynamic Histone Methylation
• Histone methylation is irreversible!
• Methylation is dynamic - alterations in H3K4 and H3-K9 methylation – (Martinowich
et al. 2003)
• Required: a mechanism for removal of
long- term histone modifications!
Histone Variants
• H2AZ prevents spread of heterochromatin
and gene silencing in transcriptionally
active regions
• H3.3 enriched in histone modifications that
correspond to transcriptional activation
Histone Exchane
• SWI/SNF and the RSC exchange H2AH2B dimers
• FACT - EF that removes one H2A-H2B
dimer from the nucleosome
• SWR1 (ATPase) selectively exchanges
H2A histone variants
Histone Exchange
Taken from: Recent
highlights of RNA-poly-II-mediated
transcription Sims, Mandal & Reinberg
Histone Octamer
DNA
H3-H4
H2A-H2B
How this Helps Transcription?
Taken from: Recent
highlights of RNA-polyII-mediated
transcription Sims,
Mandal & Reinberg
Take Home Message
• Complexity of the transcription is the
rule, not the exception.
• Transcription is coupled to mRNA
processing, RNA surveillance and
export, among other cellular processes.
• Chromatin structure – transcription
regulatory code.