7 Transcription

From DNA to RNA to proteins

• double strand vs single strand

• DNA is stable and needs to be protected

• RNA needs to be open and available (for transcription and folding)

• Sigma factor initiates the prokaryotic transcription: recognises the consensus sequence (-35, -10=TATA)

  It recruits the RNA polymerase to the Promotor

  RNA polymerase unwinds DNA and begins transcription

• Elongation: the Polymerase loses interaction with Sigma and promoter after appr. 10 nukleotids

• Termination: Signal is encoded in the DNA and causes a structure in the transcript which destabilises the Polymerase

• transcribes 3ˋ-> 5ˋ

• A gene can be transcribed in both directions

Yes

• Initiation: the transcription factor and the TBP (transcription factor binding protein: for distortion of the DNA) bind together to the TATA box-> this forms the pre-initiation complex

  Then a variety of additional TF bind to the complex

Pre-initiation: the assembly of basal transcription factors and the recruitment of the RNA Polymerase2

• opens the DNA and positions the RNA Polymerase 2 for transcription

Initiation /promotor clearance: beginning of the transcription

Elongation: Production of the transcript

Termination: ˋstoppingˋ of transcription

The mRNA transcription is the Moabit regulatory step in the expression of most eukaryotic genes

• First, TF2D (incl. TBP +TAF) binds to TATA

• TF2A +TF2B stabilise binding of TF2D. They complete the platform for RNA Pol2 to bind

• TF2F directly interacts with RNA-Pol 2 and guides it to the promoter

• TF2E helps open Pol2 and enables movement down the DNA-Helikase-activity

• TF2H: helical activity (unwinds the DNA) and CTD kinase activity

• Mediator (a large multiprotein complex) is recruited by enhancers (upstream or downstream) to regulate Pol II activity

• CDK 7 phosphorylates Ser5 of the CTD inducing the dissolve of the RNA-Pol 2 from the platform

• The ser5 phosphorylation allows for the binding of specific proteins involved in promoter release (as well as proteins involved in other processes such as capping)

• several transcription elongation factors bind to the initiation complex

• P-TEFb (positive transcription elongation factor b): consists of the CDK9 kinase that associates either with cyclin T1, T2a, T2b or K

  CDK9 phosphorylates Ser2 of the CTD

• The Ser2 phosphorylation serves as a docking site for other proteins involved in various steps of transcription and mRNA processing

Enhancers are clusters of DNA binding sites for transcription independent of their orientation and at distances as great as 85kb from start sites.

Sequences that can repress promoter activity in an orientation- and position -independent manner. These may function by altering the chromatin structure over a larger region. It can decrease the expression of multiple genes within a specific region.

A group of enhancers. Like silencers, LCRs may affect the transcription of multiple genes

Insulators serve as a barrier between different elements. For example, an insulator can serve as a barrier between a silencer and an enhancer, thus allowing the enhancer to activate gene expression.

A Mediator is a large multi-subunit protein complex which aids in the recruitment and release of RNA Pol 2.

Many different transcription factors interact with the mediator to regulate the transcription.

• Prokaryotes

  • bi- and Multi- cistronic

• Eukaryotes

  • Introns

    alternative splicing produces multiple RNAs from a single gene

  • 5’ capping

  • 3´ processing (polyadenylation)

  • Transport from nucleus into Cytoplasm

Bind to DNA without unwinding it (mostlyhydrogen bonding in major groove)

Specific AA of TF interact with the DNA

TF recognise defined DNA sequences

Helix-Turn-Helix

• Binds to major groove

• often dimerize -> more specifity

Homeodomain proteins

• contacts major and minor groove

• Incl helix-turn-helix domain

Zinc finger

• often multiple fingers

ß-sheets

• DNA recognition through a two-stranded ß sheet

• Feedback mechanism

Leucine zipper

• dimerisation -> Y shape

  homodimerization + heterodimerization -> various interactions

• Can provide hydrophobic interaction

• Coiled coil interaction between two alpha-helices

• AA side chains determine DNA binding specifitiy

• AA side chains can provide specific interactions with the DNA bases to provide sequence specific recognition

• Primarily occurs in the major groove

• Changes in AA sequence of uinc fingers determines sequence recognition