10B_Euk_Transcription_SP25_e7c96e38c30e671db5a5576a2c5a1c18

Eukaryotic Transcription Overview Transcription is a fundamental process in gene expression, enabling the synthesis of RNA from a DNA template. Eukaryotic transcription is more complex than that in prokaryotes and involves additional regulatory mechanisms that ensure precise gene expression.

Objectives of the Lecture

• Compare and contrast promoter elements in prokaryotes and eukaryotes.

• Compare transcription processes in prokaryotes and eukaryotes.

• Describe the roles of general eukaryotic transcription factors.

• Understand the 3D structure of RNA and its implications for function.

Introduction to Eukaryotic Transcription

Mechanisms of transcription are fundamentally similar across cells, but eukaryotic transcription includes additional complexity such as chromatin structure and a variety of transcription factors that interact with different regulatory elements.

Key Differences from Prokaryotic Transcription

• Location: Eukaryotic transcription takes place in the nucleus whereas in prokaryotes, it occurs in the cytoplasm. This compartmentalization allows for additional regulatory processes in eukaryotes.

• Chromatin: Eukaryotic DNA is packaged in a complex called chromatin, which needs to be modified (e.g., via histone acetylation or methylation) to allow transcription access.

• RNA Polymerases: Eukaryotes possess multiple types of RNA polymerases (I, II, III) responsible for synthesizing different RNA molecules (mRNA, rRNA, tRNA, etc.).

• Transcription Factors: Numerous general transcription factors are needed for transcription initiation, in contrast to prokaryotes where only a single sigma factor is required.

• mRNA Processing: Eukaryotic mRNAs undergo extensive processing after transcription, including 5' capping, polyadenylation at the 3' end, and splicing out of introns.

Types of Eukaryotic RNA Polymerases

• m: messenger RNA Polymerase

• mi: micro RNA Polymerase

• lnc: long non-coding RNA Polymerase

• t: transfer RNA Polymerase

• r: ribosomal RNA Polymerase

• sn: small nuclear RNA Polymerase

• sc: small cytoplasmic RNA Polymerase

Nuclear RNA Polymerases

Eukaryotic RNA polymerases are composed of 12 to 17 subunits; nine of these subunits are conserved across different species. Interestingly, five subunits have similarities with bacterial RNA polymerase found in E. coli, highlighting a shared evolutionary origin. An example of a well-studied eukaryotic enzyme is Yeast RNA Polymerase II.

Transcription Factors (TFs)

• General Transcription Factors: Required universally at all promoters to facilitate the binding of RNA polymerase.

• Regulatory Transcription Factors: Control expression at the individual gene level and can activate or repress transcription. Approximately 10% of all human genes encode transcription factors, illustrating their critical role in regulating gene expression.

General Transcription Factors Overview

Key functions of general transcription factors include:

• They orient RNA polymerase on the promoter region of the DNA.

• They unwind the promoter DNA, providing access to the transcription machinery.

• They do not directly influence the transcription rate but set the stage for effective transcription initiation.

Core Promoters

Definition: The core promoter is the minimal sequence required for RNA polymerase (RNAP) and transcription factors (TFs) to initiate transcription. This promoter region functions across various cell types but operates at lower efficiency and lacks universal elements, being tailored to specific gene requirements.

Binding of General TFs to Core Promoter Elements

Important subunits in this process include:

• TBP (TATA-binding protein): The initial factor that binds to the TATA box in the promoter.

• TFIID: A complex that includes TBP and is crucial for recruiting other transcription factors.

• TFIIB: Helps to accurately position RNA polymerase II on the promoter.

Pol II Transcription Factors

• TFIIB, TFIID, TFIIF: These factors assist RNA polymerase II in binding to the core promoter.

• TFIIE: This factor recruits TFIIH and regulates its activity, which is essential for promoter clearance.

• TFIIH: This complex unwinds the DNA helix, which is critical for opening the transcription complex and phosphorylating the C-terminal domain (CTD) of RNA polymerase II, facilitating transcription progression.

C-terminal Domain (CTD) of RNAP II

The CTD features tandem repeats of the amino acids Tyr-Ser-Pro-Thr-Ser-Pro-Ser, with variations in the number of repeats across species. Phosphorylation of the CTD is required for:

• Promoter clearance, allowing transcription to proceed.

• Recruitment of elongation factors and mRNA processing components essential for post-transcriptional modifications.

RNAP II Transcription Initiation Process

The formation of the preinitiation complex (PIC) is mandatory for initiation and consists of RNA polymerase II alongside six general transcription factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Among them, TFIIH plays a dual role by not only opening the DNA helix for transcription to commence but also by phosphorylating the CTD of RNAP II to allow processivity during elongation.

RNAP II Transcription Elongation

The mediator complex links RNAP II to regulatory transcription factors. The phosphorylation of the CTD by TFIIH allows for the release of the transcription factors and the binding of elongation factors and RNA processing components necessary for synthesizing mature mRNA.

Transcription Termination

Different pathways exist for terminating transcription based on the type of RNA being produced (coding vs. non-coding). Key determinants for pathway specificity include:

• Phosphorylation status of the Pol II CTD.

• RNA sequence signals and the involvement of termination factors.

Models for RNA Polymerase II Termination

• Allosteric Model: Proposes that termination is regulated based on the conformational state of the RNA polymerase complex.

• Torpedo Model: Involves the degradation of RNA downstream of the polyadenylation site by exonucleases, which leads to termination through the destabilization of the elongation complex.

Conclusion: Key Concepts

Eukaryotic transcription, in comparison to prokaryotes, is notably more complex. The presence of multiple RNA polymerases, various general transcription factors, and extensive post-transcriptional processing represents distinctive features of eukaryotic transcription mechanisms that facilitate precise regulation of gene expression.