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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 • • • • • -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 • • • • 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 • • • • • • 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 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 • • • • 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.