Transcription Biology Review

Bios 691 – Systems Biology

January 2008

Outline

• Gene structure

• Chromatin structure & modifications

• Transcription apparatus

• Transcription factors and cofactors

• Elongation and termination

• RNA capping, splicing, and adenylation

• RNA processing and miRNA’s

Chromosome Organization

• Mammalian chromosomes tend to fill discrete regions within the nucleus

• An elaborate network of fibrils maintains these arrangements

• RNA ‘factories’ at distinct locations do most of the transcription work

• Nucleoli are factories for rRNA

Chromatin Structure

• Protein scaffolds anchor the DNA

• Within the scaffold there are loops

• Most transcription happens on the loops

• Much chromatin is wrapped in 30nm ‘heterochromatin’

Fine Structure of Chromatin

• Heterochromatin – inaccessible– Bound with many proteins– Centromeres; telomeres; some other areas

• Euchromatin – accessible– Still needs to be opened

Telomeric Heterochromatin and Sirtuins Euchromatin: 30 nm & open

DNA Packaging & Nucleosomes

Gene Structure – Exons & Introns

Exon Size distribution

Gene Structure – Initiation Sites

• Most (~2/3) genes have multiple promoters

• Most promoters are either ‘sharp’:– Very narrow range– Usually TATA + Inr– Often tissue specific

• or ‘broad’:– Typically 70 bp range– Rarely TATA / Inr– Often widespread

Histones and Modifications

DNA contacts histones on their tails Histone tails can be modified

Histones can stay loose or assemble tightly

Proteins Modify Histones

DNA Methylation

Adding a Methyl to Cytosine

Cytosine methylation is passed on to daughter cells

Controlling Transcription

DNA-Binding Proteins• All proteins interact weakly

with DNA• Proteins with projecting

amino acids interact with the DNA major groove

• Hydrogen bonds stabilize position of proteins on DNA

• Proteins that line up several amino acid contacts bind strongly to specific DNA sequences

Transcription Factor Families

• Several structures line up amino acids– Helix-turn-Helix

(Homeodomain)– Helix-loop-helix– Zinc Finger

• Mostly dimers• These families have

proliferated because of their role in attracting transcription apparatus

Cofactors

• Frequently the effect of DNA-binding proteins depends on co-factors

• E.g. ER sits on the DNA but requires estrogen as a co-factor to function

• Myc requires Max as a co-factor to stimulate transcription

• If Max is coupled with Mad instead, the genes are repressed

Kick-starting Pol II & Elongation• Mediator protein

bridges TF proteins and RNA Pol II

• Contains kinase domains – may phosphorylate CTD of RNA Pol II

Initiating Transcription

TBP on a TATA Box

RNA Polymerase II

RNA (red) copied from DNA (blue) by RNA Polymerase II

RNA Polymerase II Structure The cycle of adding nucleotides

Terminating Transcription

RNA Processing

RNA Processing Steps

• Nucleus– capped, – spliced, – cleaved, – polyadenylated

• Exported• Cytoplasm

– stored– translated– degraded

Capping mRNA

The RNA factory

RNA Splicing

Poly-adenylating RNA

•Poly-A Polymerase adds ~100-150 Adenines to 3’ end•After export to cytoplasm, nucleases chop off ~10-20 A’s at a bite•Nucleases compete with ribosomes for mRNA’s•When ~30 A’s left degradation speeds up

RNA Export

• RNA has to be passed through nuclear pores to show up in the cytoplasm (where we measure it)

Micro RNA’s

P-Bodies• Loci where RNA accumulates and is degraded• Have their own structural proteins

Implications for Systems Biology

• Levels of TF’s on a promoter may not predict levels of transcripts

• Rate of transcription may not predict level of mRNA in the cytoplasm

• Levels of mRNA in cytoplasm may not predict levels of protein