Lecture 2: Overview of Eukaryotic Gene Expression

Purpose of gene control in multi-cellular organisms

Execution of precise developmental and tissue specific program

Proper genes are expressed in proper cells at proper times

True for all cells but RBCs and repro cells

Start of transcription

Takes place on DNA that is wrapped chromatin

Chromatin needs to open for a gene to be activated and for transcription to proceed

Chromatin mediated regulation is a eukaryotic mechanism

One component of epigenetic regulation of gene expression

Organized state of chromatin influences transcription

Euchromatin

Less dense regions of chromatin

Active genes are found in euchromatin

Allows gene regulatory proteins and RNA polymerase complexes to bind to the DNA

Facilitating the active transcription of genes into mRNA

Heterochromatin

Regions of chromosomes that are densely packed

Rich in repetitive DNA

• Transposons

• Centromeres

• Telomeres

Not accessible to transcriptional machinery

Inactive genes are found in heterochromatin

Difference between Prokaryotes vs Eukaryotes

Very elaborate transcriptional control

Open a gene is found within open chromatin

Variety of factors regulate the expression of each individual gene

Pioneer TFs come in and a variety of other factors such as Pol. and GTFs help with transcription

Very common genes will be open

Every eukaryotic gene requires GTFs

Eukaryotic RNA polymerases

Polymerase I, II, III

Pre-rRNA

28S, 18S, 5.85 rRNAs

Ribosome compartments

Protein synthesis

RNA Pol I

mRNA

Encodes protein

RNA Pol II

siRNAs

Chromatin-mediated repression, translation control

RNA Pol II

miRNAs

Translation control

RNA Pol II

tRNAs

Protein synthesis

RNA Pol III

5S rRNA

Ribosome component

Protein synthesis

RNA Pol III

snRNA U6

RNA splicing

RNA Pol III

7S RNA

Signal recognition particles for insertion of polypeptides into the ER

RNA Pol III

Other small stable RNAs

Various functions

RNA Polymerase Structure → YEAST RNA pol II

RNA pol II consists of 12 polypeptides

• RPB1 - RPB12

All other eukaryotic RNA polymerases share very high level of homology with yeast RNA Pol II

RNA Pol Clamp domain

RPB1 accommodates the DNA

After positioning over DNA the clamp is closed by a bridge

Synthesis of RNA takes place at catalytic center with the participation of Mg++

Synthesized RNA exists through a “channel” and is immediately capped by 7m Guanosine

Complexes of multiple polypeptides: Yeast vs Bacteria

Prokaryotes

• Beta and beta prime subunits

Eukaryotes

• RPB1 and RPB2 subunits

Homologous in eukaryotes and prokaryotes

Structural similarity of the enzyme

Carboxy Terminal Domain (CTD) in RNA Pol II

Specialized domain not found in other polymerases

Prokaryotic and eukaryotic

Involved in multiple regulatory interactions and play a key role in initiation release, elongation and processing of synthesized mRNA

Yeasts: 26 repeats of Tyr-Ser-Pro

Mammals: 52 repeats of Tyr-Ser-Pro

Ser residues in CTD

Phosphorylated upon transition from initiation and elongation

Genes transcribed by RNA Pol II are regulated by

Conserved Basal Promotor Element (Core Promoter sequences)

Promoter proximal binding sites for transcriptional activators (GTPs)

Distal enhancers or repressors

Chromatin Structure

Core Promoter sequences in Eukaryotic DNA

Transcription starts at defined point: Initiation site

Usually an A (adenine) on the coding strand

Four elements direct the positioning of the polymerase at these promoters:

• TATA box

• Initiator

• BRE

• DPE

TATA Box

Tight consensus sequence

Prevalent in highly transcribed genes

Initiator

Less conserved element

Some genes contain initiator but no TATA

BRE

Influences activity of promoter

Most upstream

DPE

Downstream promoter element

Influences activity of promoter

Importance of RNA polymerases

Need to recognize promoter to correctly Initiate transcription

Several GTFs assemble preinitiation complex over Core promoter sequence

Other factors that help with preinitiation

DNA helicases help the polymerase initiate transcription

Protein kinases release the polymerase

Elongation factors facilitate movement of polymerase

Additional proteins move nucleosomes out of way

GTFs of RNA polymerase II

TFIIA, B, D, E, F, H