Eukaryotic Transcription

Steps of Eukaryotic Transcription

1. Initiation- Transcription factors bind to the promoter and recruits RNA polymerase ll. Then DNA unwind, and transcription begins at the +1 site.

2. Elongation- RNA polymerase II moves along the DNA from 5' to 3' direction making a complementary mRNA strand. Then it adds nucleotides one by one and forms phosphodiester bonds to build the RNA chain.

3. Termination: Continues RNA polymerase does not stop immediately at the end of the gene. It keeps going 1,000–2,000 base pairs past the termination signal. Then the RNA transcript is cleaved (cut) and the mRNA is released.

Types of Eukaryotic RNA Polymerase

1. RNA polymerase l

2. RNA polymerase ll

3. RNA polymerase lll

RNA polymerase

• Found in the nucleolus

• Synthesizes rRNA

• Makes up the core structure and catalytic components of ribosomes

rRNA

A type of molecule that combines with proteins to form ribosomes, the cellular machinery responsible for making proteins

RNA polymerase ll

• Found in the nucleus

• Transcribes protein-coding genes (pre-mRNA). Then pre-mRNAs undergo processing before translation.

• Responsible for majority of transcription activity

RNA polymerase lll

• Found in the nucleus

• Transcribes tRNAs and small nuclear RNAs

• These molecules play roles in translation and RNA processing

Eukaryotic Promoter

• The promoter is a DNA sequence upstream (before) the transcription start site (TSS), which is labeled as +1.

• “Upstream” means in the negative direction (−) from the start site.

• This region is where RNA polymerase and transcription factors bind to begin transcription.

 TATA box (TATAAA): at -25 to -35 bp, A-T rich for easy unwinding

Eukaryotic Transcription Factors (TFs)

Basal transcription factors (TFIIA-TFIIJ) assemble at promoter

• ALL need to bind before initiation

• RNA polymerase ll then binds and forms transcription initiation complex

Eukaryotic Transcription Factors (TFs) Functions

• To bind to DNA

• Stabilize pre-initiation complex

• Unwind the DNA

Eukaryotic Initiation

• Transcription factors (TFs) bind first to the promoter (pre-initiation complex)

• TFs help recruit RNA polymerase ll to start transcription (initiation complex)

• RNA polymerase cannot bind DNA alone

• TATA box: Consensus sequence for eukaryotes

Elongation

• Same as prokaryotic elongation

• RNA polymerase II moves along the DNA from 5' to 3' direction making a complementary mRNA strand.

• It adds nucleotides one by one and forms phosphodiester bonds (links between the nucleotides) to build the RNA chain.

Termination

• RNA polymerase does not stop immediately at the end of the gene.

• It keeps going 1,000–2,000 base pairs past the termination signal.

• Then the RNA transcript is cleaved (cut) and the pre-mRNA is released.

Eukaryotic Pre-Transcriptional Regulation

• Chromatin status

• Basal and other transcription factors

• Enhancers- turn on transcription and this is where activators bind

• Silencers (Repressers)- turn off transcription and this is where repressers bind as well

DNA Structure

• DNA Double Helix: Composed of nucleotide base pairs (A-T and G-C)

• Chromatin: DNA wrapped around histone proteins → forms nucleosomes

• Chromosomes: High organized, and compact structures of chromatin found in the nucleus

• The structure of DNA determines accessibility for transcription to occur

Chromatin

DNA wrapped around histone proteins → forms nucleosomes

Chromosomes

High organized, and compact structures of chromatin found in the nucleus

2 Types of Chromatin status

Heterochromatin: “Closed” Chromatin

• Tightly packed

• No access to DNA= No transcription

Euchromatin: “Open” Chromatin

• Loosely packed

• Access to DNA= Transcription

• Transcription occurs only in euchromatic regions of chromosomes

Heterochromatin

• “Closed” Chromatin

• Tightly packed

• No access to DNA= No transcription

Euchromatic

• “Open” Chromatin

• Loosely packed

• Access to DNA= Transcription

• Transcription occurs only in euchromatic regions of chromosomes

Epigenetic Regulation of Transcription

Histone modifications: Chemical modifications to the histone proteins

• Histone Acetylation: Loosens chromatin → activates transcription

• Histone Methylation: Tightens chromatin → silences transcription

DNA methylation

• Adds methyl groups to the DNA strand

• Tightens chromatin → silences transcription

Other Transcription Factors

1. Transcriptional Activator Proteins

2. Transcriptional Repressor Proteins

3. Coactivators and Corepressors

Function of Transcriptional Activator Proteins:

• Bind to specific DNA sequence (often enhancers)

• Recruit or stabilize the transcription machinery

• Increase the rate of transcription initiation

Function of Transcriptional Repressor Proteins

• Bind to specific DNA sequences (often silencers/repressors)

• Block activator binding or inhibit RNA polymerase activity

Function of Coactivators and Corepressors

• Do not bind DNA directly

• Act as bridges between enhancers/repressors and the transcription complex

• Can modify chromatin structure

Enhancers

• DNA sequences that increase transcription efficiency

• Can be upstream, downstream, or within the gene

• Bind transcriptional activator proteins (TF) → DNA looping brings them to the promoter

• Helps stabilize RNA polymerase ll

• Can have multiple enhancers to control transcription

Silencers (Repressors)

• DNA sequences that can lead to transcription block

• Bind transcriptional repressor proteins → DNA looping brings them to the promoter

• Prevent activators or TFs from binding

• Can recruit histone deacetylases to tighten chromatin

Deacetylases

An enzyme that removes acetyl groups from histone proteins

Eukaryotic pre-mRNA Processing

Eukaryotic genes contain exons(coding) and introns(noncoding)

• Introns must be removed and exons joined to create a continuous coding sequence

mRNA processing increases RNA stability and lifespan: 

• Eukaryotic mRNA: lasts hours

• Prokaryotic mRNA: lasts seconds

Pre-mRNA is protected by RNA-stabilizing proteins during

Three Main Steps of mRNA Processing

1. 5’ Capping

2. 3’ Poly-A Tail Addition

3. Splicing (intron removal)

Together, these modifications produce a stable mature (spliced) mRNA

5’ Cap and 3’ Poly-A Tail

5’ Cap

• Added to the 5’ UTR (untranslated region) while transcription is still occuring

• Protects mRNA from degradation

• Helps ribosomes recognize and bind to the mRNA during translation initiation

3’ Poly-A Tail

• Poly-A polymerase adds 200 adenine nucleotides to the 3’ UTR (untranslated region)

• Protects mRNA from degradation

• Aids in nuclear export and translation

mRNA Splicing

Introns are removed and exons are joined by the spliceosome

• Spliceosome: Protein, enzyme, and RNA complex that removes introns and splices exons together

Alternative Splicing

Different combinations of exons are joined together (spliced) to produce multiple mature mRNA transcripts from a single gene