Introduction to transcriptional machinery
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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

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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

  • ~100 fold variationin the binding rate of RNA Pol to different promoters in vitro.

  • Binding rates correlate with the frequencies of transcription in vivo.

T80A95T45A60A50T96


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 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)


Traditional View of Gene Expression


Contemporary View of Gene Expression


The Sister

Chromatids

of a Mitotic Pair


Chromatin Packing

2 nm

105mm

Double helix

11nm

“Beads-on-a-string”

~x7

30 nm fiber of

Packed nucleosomes

~x100

30 nm

Chromosomal loops

Attached to nuclear

scaffold

300 nm

Condensed section

of metaphase

chromosome

700 nm

~x104

Entire metaphase

chromosome

1400 nm

5-10 mm


GC Pairs Are Preferred

DNA

Histone Core

AT Pairs Are Preferred

Chromatin Structure

  • DNA accessibility – a major challenge in a chromatin environment

  • Nucleosomes –

    building

    blocks of

    chromatin


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-on-a-string”


View Along the Axis of One Turn of the 30nm Fiber

DNA

Histone

H1 Histone

Core


Side View of the 30nm Fiber

Histone core

11 nm fiber

30 nm fiber

DNA

Histone H1

Nucleosome

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 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: Recenthighlights of RNA-poly-II-mediated transcription Sims, Mandal & Reinberg


Histone Octamer

DNA

H2A-H2B

H3-H4


How this Helps Transcription?

Taken from: Recenthighlights of RNA-poly-II-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.


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