Exam 3: Lecture 3 Flashcards Preview

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Flashcards in Exam 3: Lecture 3 Deck (22):
1

Enhancer Elements

-provide spatial and temporal control of gene transcription in metazoans

-typical eukaryotic gene is expressed at different times during development and within multiple tissues

-most circumstances an individual enhancer element will control expression of a gene in a single tissue

-gene that is expressed in multiple tissues will contain several enhancers

-consider ddp gene of Drosophila: contains several individual enhancers, each one directing the expression within a different developing tissue.

2

Enhancer Elements Location

-most upstream of transcriptional start site.

-also quite common to find them within intronic sequences

-sometimes even located downstream of transcriptional terminal signal

-anywhere along gene

3

Enhancer Element Range

-Enhancers can direct transcription at close range (0-10kb) or from distant locations (100kb)

-distantly located enhancers are brought into close proximity to the core promoter by the proteins that will bend or loop DNA

4

Order of Control

-expression of gen in individual tissue is under control of three different types of enhancers: primary enhancer, secondary enhancer and rarely a shadow enhancer

5

Primary Enhancer

-deletion of this enhancer will lead to loss of expression of gene within given tissue

-i.e. primary eye enhancer deleted from Drosophilia eya gene

-if eya is no longer expressed in eye, cpd eyes are eliminated

6

Secondary Enhacers

-several genes contain these -usually located between 5-10kb from TSS

-usually insufficient to maintain gene expression in absence of primary enhancer as their activity is dependent upon primary enhancer

-can often be experimentally activated by forced expression of upstream transcriptional activators

-i.e. forced expression of Sine Oculis transcriptional co-activator can activate secondary enhancer

-act as booster to complete transcription once you have initiation via primary enhancer

7

Shadow Enhancer

-in rare circumstances expression of some genes will also be under the control of this enhancer

-often located 50-100kb away from TSS

-often regulated by environmental factors such as temperature and humidity

8

Structure of Enhancers: Binding Proteins (graphic)

-eukaryotic enhancers bound by different DNA binding proteins

-some enhancers contain single recognition sites for multiple unique transcription factors

-mutations in any one of these sites can inactivate the entire enhancer and gene expression can be inhibited

-other enhancers can contain multiple recognition sites for several different DNA binding proteins

-loss of a single site within these classes of enhancers usually lowers the transcriptional levels of the gene of interest.

-decrease in transcriptional output is often severe enough to result in phenotypic defects

-transcriptional output of a gene is often dependent upon the number of transcription factor binding sites that are found within an enhancer

9

Structure of Enhancers: Binding Sites Quality (graphic w/ examples)

-quality of binding sites can also affect the transcriptional output of an enhancer

-quality refers to the whether or not the DNA sequence is optimal for the DNA binding protein

-check example in notes

10

Sequence-Specific DNA Binding Proteins

-developmentally regulated transcription factors influence transcription by binding to sequence-specific sites within enhancer elements

-regions of transcription factor that interacts with nucleic acids is called DNA binding domain.

-over evolutionary time, several different types of DNA binding domains have evolved (i.e. homeodomain, basic helix-loop-helix domain, zinc finger domain, and leucine zipper domain)

11

Commonality Between Domains

-while they have significant differences, each DNA binding domain consists of at least one alpha helical structure that is inserted into the major groove.

-amino acids (approx. 20) of recognition helix recognize and interact with specific sequence of bases within the major groove

-some transcriptional factors including those with a homeodomain can bind to DNA as a monomer while others containing basic helix-loop-helix motifs must bind to DNA as homo or heterodimers.

12

Transcriptional Factor Structure

-developmentally regulated transcription factors exert temporal and spatial control over expression of genes

-these DNA binding proteins are recruited to target genes through interactions with sequence specific sites within enhancer elements

-transcription factors activate gene expression by also physically interacting with general transcription factor machinery and RNA Pol II

-effect of these interactions is phosphorylation of C-terminal tail of RNA Pol II

-once RNA Pol II is phosphorylated it is released from promoter and free to initiate transcription

13

DNA Binding Domain (DBD)

-some transcription factors contain DBD and an activation domain (AD)

-in other cases these two domains are distributed amongst two different proteins

-i.e. Eyes Absent protein which contains activation domain and Sine Oculis which contains DBD so these proteins bind to each other forming a composite transcription factor

14

Binding Sites Location

-binding sites for enhancers can be located at considerable distances from core promoter

-in this situations DNA bending proteins will alter the conformation of the local DNA double helix such that the transcription factor will be brought into contact with either RNA Pol II or the general transcription factor machinery

15

Mediator Complex

-eukaryotic cells contain additional transcriptional co-activator called mediator complex

-binds to C-terminal tail of RNA Pol II and acts as bridge between developmentally regulated transcriptional factors and RNA Pol II

-recent studies have shown that mediator complex phosphorylates RNA Pol II

-this is a signal for the polymerase to leave the promoter and begin transcribing the template into mRNA

16

Modifications in Chromatin Structure

-developmentally regulated transcription factors can also influence gene expression through modifications of chromatin structure (in addition to directly influencing RNA Pol II)

-if promoter region free of nucleosomes, then core promoter sequences will be accessible to general transcription factors and to RNA Pol II

17

Histone Acetyl Transferase (HAT)

-some transcriptional activators can directly recruit these

-addition of acetyl groups to N-terminal tails of histones gives them a more negative charge and will repel DNA double helix

-allows for core promoter to become accessible to general transcriptional machinery and RNA Pol II

-these types of modifications can open up several kilobases of DNA

18

Chromatin Remodelers

-some transcription factors interact with these -do not alter charge of histones

-alter structure of double helix that is bound to histone core

-also makes core promoter binding sites accessible to general transcription factors and RNA Pol II

-changes in DNA structure are more localized

19

Molecular Dissection of Transcription Factors: GAL4 (What?)

-DNA binding protein encoded by the yeast genome, is one of the most extensively studied transcription factors

-contains a DNA binding domain and an activation domain

-binds as a homodimer to a 17bp site called an Upstream Activator Sequence (UAS)

-GAL4 activates the expression of the GAL1 gene by binding to a cluster of 4 UAS sites located 275bp upstream of the transcriptional start site

20

Molecular Dissection of Transcription Factors: GAL4 and lecZ (Process)

-synthetic enhancer/promoter construct was generated containing UAS sites, the core promoter and the bacterial lacZ gene.

-if wild type GAL4 added to this construct, it binds to the UAS sites and activates transcription of the lacZ genes

-if a modified GAL4 protein lacking the activation domain is mixed with the synthetic enhancer/promoter construct it is insufficient to activate transcription of lacZ even though it can bind to the UAS sites -indicates that the DNA binding domain is insufficient to activate transcription

21

Molecular Dissection of Transcription Factors: Lex A and GAL4 and lecZ (Process)

-Lex A transcription factor has a structure similar to that of GAL4.

-if a synthetic construct containing LexA binding sites, a core promoter and the lacZ gene is mixed with LexA protein then expression of the lacZ gene can be activated.

-if the activation domain is removed transcription of lacZ does not occur

22

Molecular Dissection of Transcription Factors: Chimeric Protein (Process)

-if chimeric protein containing the LexA DNA binding domain and the GAL4 activation domain is incubated with the synthetic construct containing LexA binding sites, the chimeric transcription factor can bind to DNA and can activate transcription of lacZ

-indicates that activation domains are interchangeable

-if chimeric protein is mixed with a synthetic construct containing GAL4 sites then chimeric protein can neither bind DNA nor activate transcription.

-indicates that the DNA binding domain confers binding specificity