Lecture 14-15 Transcription in Eukaryotes

Transcription in Eukaryotes is much more complicated than prokaryotes

• DNA-protein interactions

• Protein-protein interactions

• Chromatin structure

• Nuclear architecture

• Cellular compartmentalization

Gene expression is regulated at multiple levels

• at innitiation stage

• post-transcripton level: mRNA processing, mRNA stability, export and translation

General machinery of transcription

• Sequence-specific DNA-binding transcription factors.

• RNA polymerase II (RNA pol II)

• Coactivators and corepressors.

• Elongation factors

several RNA polymeras

Prok: 1 rna polymerase

Euk: 3 different rna polymerase

• Rna pol 1: rRNA gene (but not all rRNA)

• RNA pol 2: mRNA and MiRNA

• RNA pol3: tRNA and 5S rRNA

Pol II and transcription of protein coding genes

Transcription factors read genetic info and tell RNA pol 3 to react

turns on a particular gene is unique combination fo regulatory elements and trasncription factors

Gene and regulatory sequences

-1000 and -700 or more is lng range regulatory elements

-200 to -70 is promoter proximal elements

-31 to -26 is core promoter

The promoter elements

• Required for Pol II initiation

• Core promoter elements.

• Proximal promoter elements

has TATA box in core promoter box

Core promoter elements

• All core promoter elements, except for BRE, are recognized/bound by TFIID

• BRE bound by TFIIB

TATA box

• TBP (TATA binding protein) binds TATA box

• TATA box is only present in ~32% of potential core promoters.

• no TATA box →no transcription

PRoximal promoter element

CAAT box and GC box

• 5’ of core promoter

• inc transcription initiation

• Transcriotion factors recruit enhancer or silencers

• UAS(upstream activating sequence)-

  • rege TFID binding to core promoter

Structure and function of long-range regulatory elements

• Enhancers and silencers

  • 700-1000bp _ from transcription start.

  • can be up/downstrea or inside

  • promoter is always upstream

  • can mis-regulate other nearby genes

  • very close to genes

• insulators

  • Chromatin boundary markers: heterochromatin(left)/euchromatin. (right)

  • DNA is insulator

  • Histones modified by epigenetic factors

• LCR

  • Locus COntrol Region

    • oraganize and maintain active chromatin adn enhance trasncription of downstream genes

    • Long one direction local gene expression control

• MAR

  • Matrix attachement regions

    • organize genome into loop domains

    • attach to nuclear matrix

    • landing platform for transcription factors

The transcriptional machinery

• Transcriptional coactivators and corepressors

• The proteins promote or inhibit trasncription but NOT bind DNA

• co=NOt bind DNA

Components of the general transcription machinery

• RNA polymerase II→ make mRNA

• General transcription factors: TFIIB, TFIID, TFIIE, TFIIF, and TFIIH

  • shared and includes activators

• Mediators

  • Protein- protein interactions

RNA polymerase II

• Structure for Saccharomyces cerevisiae RNA polymerase II

  • Unstructured C-terminal domain (CTD) of Rpb1 is not seen by X-ray crystallography

RNA polymerase II C-terminal domain (CTD)

• 52 heptapeptide repeats (hydroxyl containing)

• c terminal needs to be phosphorylated

• dynamic phosphorylation of serine residues at positions 2 and 5 in repeats.

  • During initiation, CTD is unphosphorylated, and during elongation, CTD is phosphorylated

TFIIH also has cyclin-dependent kinase activity and helicase activity

• Transcription elongation requires a phosphorylated CTD

• TFIIH is the kinase that phosphorylates the CTD of RNA pol II.

H provides Kinase(for elongation) and helicase activity

Other transcription factors mediate genespecific transcriptional activation or

repression

• Repressors block the transcription machinery.

• Activators increase the rate of transcription

4 steps of trasncription initiation

1. Preinitiation complex assembly

   • TATA box

   • Then TFIID(bind in tata box) with TAF and TBP

   • TFIIB bind (TRB)

2. Initiation- promoter open, DNA denatured

   • bind 2 H

3. Promoter clearance elongation adn termination

   • closed comples, open complex and promoter clearence with 2H

4. reinitiation

   • promote transcription with TFIID

TFIID recruits the rest of the transcriptional machinery

• Binding of TFIID to the core promoter is a critical rate limiting step.

TFIIB orients the complex on the promoter

• TFIIB binds to one end of TBP and to a GC-rich DNA sequence after the TATA motif.

• TFIID-TBP-DNA complex determines the direction for the start of transcription

TFIIE, TFIIF, and TFIIH binding completes the preinitiation complex formation

• RNA polymerase II joins the assemblage in association with TFIIF and Mediator.

• Abortive initiation

Abortive initiation

RNA polymerase II synthesizes a series of short transcripts

Transcription factors are modular proteins

three major domains

• DNA-binding domain,

• transactivation domain

• dimerization domain.

bc 2 hybrid

Common DNA-binding domain motifs

• Helix-turn-helix

• Zinc finger

Dimerization domain (mediated by hydrophoobic interactions)

• Basic leucine zipper

  • leucine bind to leucine to create a zipper

• Basic helix-loop-helix

  • facilitate protein dimerization, homodimer or heterodimer

  • helix-helix interaction via a charge interaction

Transcriptional coactivators and corepressors

• Not binding DNA directly’

• Two main classes of coactivators and corepressors

  • Chromatin modification complexes.

  • Chromatin remodeling complexe

Chromatin modification complexes

Post-translational modification of histone N-terminal tails:

• The N-terminal tails of histones H2A, H2B, H3, and H4 are subject to a wide range of modifications.

• Function as master on/off switches for transcription.

• Recognition landmarks by other proteins

Four major types of modification

Acetylation of lysines

 Methylation of lysines and arginines

 Ubiquitinylation of lysines

 Phosphorylation of serines and threonines

Histone acetyltransferases

Histone acetyltransferase (HAT) directs acetylation of

histones at lysine residues.

• Histone deacetylase (HDAC) catalyzes removal of acetyl groups

Mechanism and functions of histone acetylation

The addition of an acetyl group neutralizes the positive

charge of histone proteins.

 Decrease the affinity of histone tails to the negatively

charged DNA.

 Acetylated lysines serve as binding sites to recruit

repressors or activators.

Histone methyltransferases

Histone methyltransferase (HMT) directs methylation

of histones on both lysine and arginine residues.

 Histone demethylase LSD-1 removes methyl groups

Mechanism and functions of histone methylation

The addition of a methyl group neutralizes the positive

charge of histone proteins.

 Histone methylation is linked to both activation and

repression of transcription.

Histone phosphorylation

Kinase adds a phosphate group to one or more serine or

threonine amino acids, adding a negative charge, thus

decreasing the affinity of histones to DNA.

Ubiquitinylation

A ubiquitin-conjugating enzyme adds one ubiquitin to a

lysine residue.

 Isopeptidase removes ubiquitin.

Linker histone variants

Mammals contain eight histone H1 subtypes

• H1a through H1e and H1’ in somatic cells

• Two germ-cell specific subtypes, H1t and H1oo.

Chromatin remodeling complexes

Nucleosome sliding

 Nucleosome displacement

 Nucleosome replacement

SWI/SNF causes nucleosome displacement

ATP dep process

ISWI chromatin remodeling complexes slide

histone octamers along DNA

Histone replacement with a variant histone by

the SWR1 chromatin remodeling complex

Swr1 can replace core histone proteins

NLS and NES for nuclear transport

Karyopherins mediate nuclear import and expo

Karyopherins that mediate nuclear import are called importins.

• Karyopherins that mediate export are called exportins.

Nuclear import

Cargo recognition and docking.

 Translocation through the nuclear pore complex.

 Cargo release and receptor recycling

Nuclear import against gradient

RanGDP bind to importin that goes to NPC complex and becomes RanGTP in nucleus

Facilitated by RanGEFm

Nuclear export pathway

Exportins bind to their cargo in the nucleus in the

presence of RanGTP.