BMB 3110 Lecture 37 Study Guide

BMB 3110 Lecture 37: Gene Expression in Eukaryotes

Lecture Structure

  • Content encompasses:

    • Eukaryotic RNA Polymerase (RNAP) Types

    • Pol II Initiation and Regulation

    • Nucleosomes and Transcription

Disclaimer

  • Notes intended for personal, educational, and noncommercial use of students enrolled in BMB 3110 at the University of Iowa.

  • Copyrighted illustrations from Biochemistry, A Short Course, 5th Ed.

  • Text and content developed by William Hacker © 2025.

  • Reproduction, distribution, or revision without prior written permission not allowed.

Learning Goals

At the end of this lecture, students should be able to:

  • Describe key differences between prokaryotic and eukaryotic transcription.

  • Identify types of RNA produced by eukaryotic polymerases.

  • Understand the structure of Pol II promoters.

  • Know the main steps in Pol II transcriptional initiation.

  • Explain the role of histones and their modifications in regulating transcription.

  • Recognize the significance of combinatorial control in cellular diversity.

Differences in Prokaryotic and Eukaryotic Gene Expression

Prokaryotes

  • **Single RNA Polymerase (RNAP):
    ** Only one type of RNAP responsible for transcription.

  • **Promoter Recognition:
    ** Promoters recognized directly by RNAP.

  • **mRNA Processing:
    ** Transcribed mRNA is generally unprocessed.

  • **Transcription and Translation:
    ** Occur simultaneously in the same cellular location.

Eukaryotes

  • **RNA Polymerases:
    ** Three different RNAPs (I, II, III) accommodate diverse RNA types.

  • **Promoter Recognition:
    ** Requires general transcription factors (GTFs) to recognize promoters.

  • **mRNA Processing:
    ** Transcribed mRNA typically undergoes significant processing (capping, splicing, polyadenylation).

  • **Transcription and Translation:
    ** Occurs in two separate cellular compartments (transcription in the nucleus, translation in the cytoplasm).

  • **Action on RNAP:
    ** Repressors and activators can act directly on RNAP and alter DNA accessibility.

Eukaryotic RNA Polymerase Types

Overview of RNAPs

  • RNA Polymerase I (Pol I):

    • Synthesizes rRNA (ribosomal RNA).

    • Produces a wide variety of RNA types with variable promoter elements.

  • RNA Polymerase II (Pol II):

    • Synthesizes mRNA and various non-coding RNAs.

    • Hundreds of copies of the same gene can be synthesized, with specific promoter elements located near the transcription start site.

  • RNA Polymerase III (Pol III):

    • Synthesizes tRNA, 5S rRNA, and other small RNAs.

    • Features elements downstream of the start site with two major classes of promoters.

Structural Comparison of RNA Polymerases

Prokaryotic RNAP

  • E. coli core RNA polymerase is composed of:

    • 2 Alpha-like subunits (α2)

    • 2 Beta-like subunits (ββ')

    • Omega subunit (ω)

    • B' domain involved in transcription.

Eukaryotic RNAPs

  • Each eukaryotic RNAP exhibits structural complexity with multiple subunits, primarily involved in specialized functions.

  • Eukaryotic RNAPs include a C-terminal domain (CTD) which plays crucial roles in transcription regulation and mRNA processing.

Pol II Promoters: Key Players

Cis-acting Elements

  • TATA Box:

    • Positioned near the -25 region of the promoter.

    • Sequence is homologous to bacterial -10 sequences, crucial for binding transcription factors.

  • Initiator (Inr):

    • Located near the +1 site.

    • Frequently defines the transcription start site; its variability is a critical feature.

  • Downstream Promoter Element (DPE):

    • Found near +30 position, often in the absence of a TATA box.

  • CAAT and GC Boxes:

    • Located within the -40 to -150 region, playing roles in promoter activity.

  • Enhancers and Silencers:

    • Can function at significant distances from the promoter, affecting transcription potential.

Trans-acting Factors

  • General Transcription Factors (GTFs):

    • Include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH.

  • Mediator Complex:

    • A multiprotein complex that assists in transcriptional regulation.

  • Histone Modifying and Remodeling Complexes:

    • Involved in the modification and repositioning of nucleosomes to facilitate transcription initiation.

Pol II Initiation: TFIID and Promoter Recognition

TFIID Functionality

  • TFIID is a GTF essential for discovering Pol II promoters.

    • Contains the TATA-box-binding protein (TBP) which binds to the minor groove of the DNA and induces a significant bend in the DNA structure.

    • Includes TBP-associated factors (TAFs) that help in identifying Inr and DPE in TATA-less promoters.

    • Prepares the promoter region for the subsequent assembly of additional Pol II GTFs.

Assembly of the Pre-initiation Complex

  • Ordered Assembly Steps:

    1. TFIID recognizes and binds the promoter.

    2. TFIIA and TFIIB bind to stabilize TBP-DNA interactions.

    3. TFIIB recruits RNA Pol II (associated with TFIIF).

    4. TFIIE binds, followed by TFIIH, culminate the formation of the pre-initiation complex.

TFIIH Enzymatic Activities

  • Helicase Activity:

    • ATP-dependent unwind the DNA at the promoter region.

  • Kinase Activity:

    • Phosphorylates Pol II CTD at serine 5, a crucial step in transcription initiation.

Pol II Regulation: The CTD

Structure and Function of the CTD

  • Carboxy-terminal Domain (CTD):

    • Part of the largest subunit of Pol II, critical for transcription regulation.

    • Composed of repeating elements of the form (YSPTSPS)n.

    • Stages of Phosphorylation:

    • Hypophosphorylated: During transcription initiation.

    • Hyperphosphorylated: During elongation.

    • Phosphorylation pattern is essential for recruiting mRNA processing factors:

    • First, Ser 5 is phosphorylated, followed by Ser 2 during elongation.

Pol II Regulation: Enhancers

Role of Enhancers

  • Enhancers are regulatory elements capable of influencing transcription from a distance, often thousands of base pairs away from the cycling promoter.

  • They are bound by specific transcription factors, often exhibiting tissue-specific activity.

  • Enhancers can activate transcription irrespective of their position or orientation relative to the promoter, encouraging DNA loops for effective communication with the core machinery.

Pol II Regulation: Coactivators

Functionality of Coactivators

  • Coactivators are non-DNA-binding proteins involved in modulating transcription:

    • Generally recruited after the binding of transcription factors.

    • Facilitate interactions between transcription machinery and Pol II, aiding in transcriptional activation.

Pol II Regulation: Mediator

Mediator Complex

  • Mediator is a large complex (up to 30 subunits) that serves as a bridge between transcription factors and Pol II.

  • It connects proteins at enhancers to those at the promoter and influences Pol II activity significantly.

  • When transcription factors activate the Mediator, it promotes the phosphorylation of Ser 5 on the Pol II CTD, enhancing the transcription potential.

Nucleosomes and Transcription Regulation

Structural Importance of Nucleosomes

  • Eukaryotic DNA is highly compacted; the DNA length in humans is approximately 3.6 meters, while the nucleus is roughly 5 µm in diameter.

  • Structural units of chromatin consist of DNA wrapped around histones (H2A, H2B, H3, H4) forming nucleosomes that manage DNA accessibility for transcription processes.

Post-Translational Modifications of Histones

  • Histone tails undergo various modifications (e.g., phosphorylation, acetylation, methylation) that influence transcription activity and DNA accessibility.

  • Acetylation: Carried out by histone acetyltransferases (HATs), increases DNA accessibility by neutralizing charges on lysines, thus promoting transcriptional activity.

  • Deacetylation: Reversed by histone deacetylases (HDACs).

  • Methylation: Adds methyl groups to cytosines, often leading to transcriptional repression by preventing the binding of necessary transcription factors.

Chromatin Remodeling Complexes

  • Essential for repositioning nucleosomes to expose promoters and facilitate transcription initiation.

  • Process to alter DNA accessibility involves the recruitment of factors that acetylate histones, which then recruit remodeling complexes to adjust nucleosome locations.

Transcriptional Regulation and Cellular Diversity

Combinatorial Control

  • Expression patterns differ across cell types even though all cells contain the same genes; developmental cues and external signals lead to specific gene expression strategies.

  • Transcription factors operate cooperatively within large, multi-subunit complexes allowing precise regulatory control.

  • The presence or absence of certain regulatory subunits can finely tune gene expression levels, shaping cellular identity and function.

Key Concepts Recap

  • Distinction between prokaryotic and eukaryotic transcription practices, including mechanisms like TFIID and TFIIH in Pol II initiation.

  • Classification of RNAs produced by different eukaryotic RNAPs and the related modifications and roles of histones.

  • Understanding the importance of DNA modifications (acetylation, methylation) in regulating transcription, as well as the role of various transcription factors, coactivators, and mediator complexes.

  • Insight into the structural features of Pol II promoters, including TATA box, Inr, and DPE configurations.

  • Overall, transcriptional regulation is vital for maintaining cellular diversity and function by controlling when and how genes are expressed based on specific cellular contexts.