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
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.
Promoter Elements in E. Coli
• -35: recognition domain• -10: unwinding domain• Seperating distances• UP element• Start Point: purine in 90% of the genes
16-19
T80A95T45A60A50T96
First Level of Regulation
• ~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.
What was known in the 1960’s
• Jacob and Monod 1961 – genetic control mechanisms in prokaryotes
• Anticipation for Eukarotes…• Eukaryotes – genomic complexity –
reiterated DNA sequences• Lack of genetic approach
3 RNA Polymerases
• Pol I localized within nucleoli – the sites of rRNA gene transcription
• Pol II and Pol III restricted to the nucleoplasm
• 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
3 RNA Polymerases
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
Transcription Mechanism
• RNA Pol II can catalyze RNA synthesis but cannot initiate.• Assembly• Initiation• Elongation• Termination
TBP
• Only GTF that creates sequence specific contact with DNA
• Unusual Binding in minor groove• Causes DNA bending
• 80% conserved between yeast and man• Large outer surface binds proteins• Deformation of DNA structure, but no strand separation
TBP
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
• 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
• 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
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
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 Post-Translation
• 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)
2 nm
11nm
30 nm
300 nm
700 nm
1400 nm
30 nm fiber of Packed nucleosomes
Chromosomal loopsAttached to nuclear scaffold
Condensed section of metaphasechromosome
Entire metaphasechromosome
“Beads-on-a-string”
Chromatin PackingDouble helix105 m
5-10 m
~x104
~x7
~x100
Chromatin Structure
• DNA accessibility – a major challenge in a chromatin environment
• Nucleosomes –
building
blocks of
chromatin
AT Pairs Are Preferred
GC Pairs Are Preferred
Histone Core DNA
•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
Structure of the Nucleosome
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
Interaction of DNA with Positively Charged Residues in the Nucleosome
Core
Red: The positively charged lysines & arginines The DNA is wrapped along these residues
DNA
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-on-a-string”
DNA
Side View of the 30nm Fiber
Histone core
Nucleosome Histone H1
30 nm fiber
11 nm fiber
Histone H1
• heterochromatin and euchromatin
• How do TFs access the DNA in the first place?
• Example: GR, NF1 and MMTV gene (Di Groce et al., 1999)
“Chicken and Egg” Scenario
Histone Code Hypothesis
• language of covalent post-translational histone modifications
• acetylation• phosphorylation • methylation • ubiquitylation • ADP-ribosylation and • glycosylation
• Sequence elements• Post-translational modifications• Nucleosome remodeling complexes• Transcriptional Elongation
Regulation of Nucleosome Stability
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 H3-K4 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 H2A-H2B 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
How this Helps Transcription?
Taken from: Recent highlights of RNA-poly-II-mediated transcription Sims, Mandal & Reinberg