15.3 Eukaryotic Transcription

Eukaryotic Transcription

Eukaryotic transcription converts the information stored in DNA into RNA, serving as the first major step in gene expression. Unlike prokaryotic transcription, the eukaryotic version requires a large cast of proteins working together at precise moments. This section covers the three stages of transcription (initiation, elongation, termination), the different RNA polymerases, transcription factors that regulate gene expression, and the RNA processing events that happen alongside transcription.

Steps of Eukaryotic Transcription

1. Initiation

Before RNA polymerase II can start transcribing, a large protein assembly called the pre-initiation complex (PIC) must form at the gene's promoter region. Here's how that happens:

1. TFIID arrives first. Its subunit, the TATA-binding protein (TBP), recognizes and binds the TATA box (consensus sequence TATAAA), located about 25–30 base pairs upstream of the transcription start site.
2. TFIIB binds to TBP and serves as a bridge to recruit RNA polymerase II to the promoter.
3. TFIIF associates with RNA polymerase II and helps stabilize its interaction with the growing complex.
4. TFIIE and TFIIH join last to complete the PIC. TFIIH is especially important because it has helicase activity: it uses ATP to unwind the DNA double helix around the start site, creating a transcription bubble where RNA polymerase II can access the template strand.
5. Once the bubble is open, RNA polymerase II begins synthesizing RNA, and transcription enters the elongation phase.

The other general transcription factors (TFIIA) play supporting roles in stabilizing these interactions.

2. Elongation

• RNA polymerase II moves along the template strand, synthesizing pre-mRNA in the 5' to 3' direction by adding ribonucleotides complementary to the template.
• Elongation factors (such as ELL and Elongin) travel with the polymerase, helping it maintain speed and stability so it doesn't stall or fall off the DNA.
• The polymerase continues until it encounters signals that trigger termination.

3. Termination

Termination of RNA Pol II transcription is tightly coupled to cleavage and polyadenylation of the pre-mRNA:

1. As RNA polymerase II transcribes past the end of the gene, the polyadenylation signal sequence (typically AAUAAA) appears in the pre-mRNA.
2. CPSF (cleavage and polyadenylation specificity factor) recognizes and binds this signal.
3. CstF (cleavage stimulatory factor) binds a GU-rich sequence located 20–40 nucleotides downstream of the polyadenylation signal.
4. A cleavage complex cuts the pre-mRNA 10–35 nucleotides downstream of the AAUAAA signal.
5. Poly(A) polymerase then adds a poly(A) tail of roughly 200–250 adenine residues to the newly created 3' end.
6. RNA polymerase II and its associated factors dissociate from the DNA, ending transcription.

Function of RNA Polymerase II

RNA polymerase II is the enzyme responsible for transcribing protein-coding genes into pre-mRNA. It catalyzes the formation of phosphodiester bonds between incoming ribonucleotides, building the RNA chain one nucleotide at a time.

Beyond simply linking nucleotides together, RNA Pol II:

• Maintains accurate base pairing with the DNA template strand throughout elongation
• Interacts with general transcription factors, activators, repressors, and elongation factors to fine-tune transcription speed and accuracy
• Produces the pre-mRNA transcripts that will be processed into mature mRNA and ultimately translated into proteins

Think of RNA Pol II as the central enzyme of gene expression for protein-coding genes. Without it, no mRNA gets made, and no proteins get built from those genes.

Roles of RNA Polymerases I, II, and III

Eukaryotes have three nuclear RNA polymerases, each dedicated to transcribing different classes of RNA:

The key takeaway: RNA Pol II handles protein-coding genes, while Pol I and Pol III specialize in non-coding RNAs that are essential for the translation machinery itself.

Transcription Factors in Gene Regulation

Transcription in eukaryotes is heavily regulated. Several categories of proteins control whether, when, and how much a gene is transcribed.

General Transcription Factors (GTFs)

These are the baseline factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH) required to assemble the PIC at any promoter. They interact with core promoter elements like the TATA box, the initiator element, and the downstream promoter element to position RNA Pol II correctly. Every protein-coding gene needs GTFs for transcription to begin.

Activators

Activators are proteins that increase the rate of transcription initiation. They bind to DNA sequences called enhancers, which can be located upstream, downstream, or even within introns of the gene they regulate. Activators work by recruiting co-activators (such as CBP/p300) and the mediator complex, which stabilize the PIC and help RNA Pol II get started more efficiently. Examples include Sp1, CREB, and NF-κB.

Repressors

Repressors do the opposite: they decrease transcription. They bind to DNA sequences called silencers and recruit co-repressors (such as Sin3 and NCoR) that destabilize or block PIC assembly. Examples include REST, YY1, and MeCP2.

Chromatin Remodeling Factors

DNA in eukaryotes is wrapped around histone proteins, forming chromatin. If chromatin is tightly packed, transcription factors can't access the DNA. Chromatin remodeling factors change this packaging:

• Histone acetyltransferases (HATs) add acetyl groups to histone tails, loosening chromatin and making DNA more accessible. This generally promotes transcription.
• Histone deacetylases (HDACs) remove those acetyl groups, tightening chromatin and reducing transcription.
• ATP-dependent remodeling complexes (like SWI/SNF and ISWI) use energy from ATP to physically slide or eject nucleosomes, exposing DNA for transcription factor binding.

Transcription and RNA Processing

A distinctive feature of eukaryotic transcription is that RNA processing happens co-transcriptionally, meaning it occurs while the pre-mRNA is still being made. The three major processing events are:

1. 5' Capping: A 7-methylguanosine cap is added to the 5' end of the pre-mRNA shortly after transcription begins. This cap protects the RNA from degradation and is later recognized by the ribosome during translation.
2. Splicing: Introns (non-coding sequences) are removed, and exons (coding sequences) are joined together. This is carried out by the spliceosome, a complex of snRNAs and proteins.
3. Polyadenylation: The poly(A) tail (described in the termination section above) is added to the 3' end. It protects the mRNA from degradation and aids in nuclear export.

These three modifications convert the pre-mRNA into a mature mRNA that's ready to be exported from the nucleus and translated by ribosomes. The coordination between transcription and processing is efficient because processing factors associate with the tail of RNA Pol II itself, so they're in position to act on the RNA as soon as it emerges from the polymerase.