🧬 Lecture 2: Eukaryotic Gene Expression + RNA Polymerases + Gene Promoters

Chromatin and Transcription in Eukaryotes

• Chromatin Structure
   Eukaryotic DNA is wrapped in chromatin
    Chromatin is DNA wrapped around proteins

• Requirement for Transcription
   Chromatin must open for gene activation
    Open chromatin allows transcription to proceed

• Type of Regulation
   Chromatin mediated regulation is eukaryotic specific
   It is part of epigenetic regulation
    Epigenetic regulation controls gene expression without changing DNA sequence

Euchromatin and Heterochromatin

• Heterochromatin
   Hetero means different
   Regions of chromosomes that are densely packed
   Rich in repetitive DNA
    Includes transposons centromeres and telomeres
   Not accessible to transcriptional machinery
    Transcriptional machinery is proteins needed to transcribe genes
   Inactive genes are found in heterochromatin

• Euchromatin
   Eu means true
   Less dense regions of chromatin
   Accessible to transcriptional machinery
   Active genes are found in euchromatin

Differences Between Prokaryotes and Eukaryotes Transcription Control

• Prokaryotes
   Relatively simple transcriptional control

• Eukaryotes
   Very elaborate transcriptional control
   Gene must first be located in open chromatin
    Open chromatin allows access to DNA
   Once accessible a variety of factors regulate expression
   Each individual gene is regulated separately

Eukaryotic RNA Polymerases

• Don’t memorize chart

• Know:

• Pol I transcribes rRNA

• Pol II transcribes mRNA

• Pol III transcribes tRNA

RNA Polymerase Structure

Crystal Structure
 The crystal structure of yeast RNA Polymerase II has been resolved at very high resolution
 This work was awarded a Nobel prize

Composition
 RNA Polymerase II consists of 12 polypeptides named RPB1, RPB2 … RPB12 (RP = RNA polymerase, B = 2)
 All other eukaryotic RNA polymerases share a very high level of similarity with yeast RNA Pol II

Key Features

Clamp Domain
 Located in RPB1
 Accommodates DNA during transcription
 After DNA is positioned the clamp closes via a bridge

Catalytic Center
 RNA synthesis occurs here
 Mg++ ions participate in the reaction

RNA Exit and Capping
 Newly synthesized RNA exits through a channel
 RNA is immediately capped with 7m Guanosine to protect it and prepare it for processing

Eukaryotic RNA Polymerases

Complex Composition
 Eukaryotic polymerases are complexes of multiple polypeptides

Similarity to Prokaryotes
 Prokaryotic β and β’ subunits are similar to eukaryotic RPB1 and RPB2

Structural Conservation
 All these enzymes share strong structural similarity across species

RNA Polymerase as a Molecular Machine

Key Features to Note
 The clamp and the bridge
 Position of DNA in the transcribing polymerase
 Channel through which the transcribed RNA is extruded from the complex
 Nascent RNA will be processed
  Processing includes splicing and other modifications

Carboxy-Terminal Domain (CTD) of RNA Polymerase II

Unique Feature
 CTD is a specialized domain of the RPB1 subunit (red arrow on image)
 Not found in any other polymerase, prokaryotic or eukaryotic

Functions
 Involved in multiple regulatory interactions
 Plays a key role in initiation, release, elongation, and processing of synthesized mRNAs

Structure
 In yeast, CTD contains 26 repeats of Tyr-Ser-Pro-Thr-Ser-Pro-Ser
 In mammals, CTD contains 52 repeats
 Ser residues are phosphorylated during transition from initiation to elongation
 CTD is intrinsically unstructured
  Not shown in crystallography-based slides because it cannot be analyzed by this method

RNA Polymerase II in Drosophila DNA

Image Details
 Drosophila salivary gland DNA
 Red shows phosphorylated CTD of RNA Pol II
 Green shows dephosphorylated Pol II

Key Points
 Dephosphorylated Pol II (green) is at the transcription start site
 Cannot start elongation yet

 Actively transcribed genes can initiate transcription at multiple points
  This produces multiple RNA copies from the same gene at the same time

 Phosphorylated Pol II (red) is ready to start elongation

Regulation of Genes Transcribed by RNA Polymerase II (Descriptions from ChatGPT)

Basal Promoter Elements
 Also called Core Promoter Sequences
 Conserved sequences where the transcription machinery assembles

Promoter-Proximal Elements
 Binding sites near the promoter for transcriptional activators
 Help increase the efficiency of transcription initiation

Distal Regulatory Elements
 Enhancers or repressors located far from the gene
 Enhancers increase transcription
 Repressors decrease transcription

Chromatin Structure
 Organization of DNA and histone proteins affects gene accessibility
 Open chromatin allows transcription
 Closed chromatin inhibits transcription

RNA Polymerase II Promoters and General Transcription Factors
 Promoters are recognized by general transcription factors
 These factors recruit RNA Pol II and help initiate transcription

Positions of Core Promoter Elements

BRE
 TFIIB recognition element
 Located around -37 to -32

TATA Box
 Located around -31 to -26

Inr (Initiator)
 Spans -2 to +4
 +1 is the transcription start site
 Drosophila: TCAST
 Mammals: YYANTYY

DPE (Downstream Promoter Element)
 Located +28 to +32
 Sequence example: GAGACAGTTC

Other Notes
 Core promoter elements are binding sites for general transcription factors (Ac, Me, IIH, IID, etc.)
 Mediator and activators interact with these elements to regulate transcription

Core Promoter Sequences in Eukaryotic DNA

TATA Box
 Tight consensus sequence
 Common in highly transcribed genes

Initiator (Inr)
 Less conserved element
 Some genes have Initiator but no TATA

BRE (TFIIB Recognition Element)
 Influences promoter activity

DPE (Downstream Promoter Element)
 Influences promoter activity

Transcription Start Site
 Defined point where transcription begins
 Usually an A on the coding strand

Polymerase Positioning
 Four elements (TATA, Inr, BRE, DPE) guide RNA polymerase to the promoter

RNA Polymerase II and Transcription Initiation

Promoter Recognition and Initiation
 RNA polymerase must recognize the promoter
 Initiate transcription at a very specific site
 Cannot do this alone

General Transcription Factors (GTFs)
 Assemble the preinitiation complex over core promoter sequences

Other Supporting Factors
 DNA helicases – help unwind DNA so polymerase can start
 Protein kinases – release polymerase to begin elongation
 Elongation factors – help polymerase move along DNA
 Chromatin remodelers – move nucleosomes out of the way

General Transcription Factors of RNA Polymerase II

RNA Polymerase I GTFs
 Labeled as TFI
 Examples: TFIA, TFIB

RNA Polymerase III GTFs
 Labeled as TFIII
 Examples: TFIIIB, TFIIIS

RNA Polymerase II GTFs
 Labeled as TFII
 Examples: TFIIA, TFIIB, TFIID, TFIIE, TFIIH