Eukaryotic Regulation

Chapter 17: Eukaryotic Gene Expression 1

Eukaryotic RegulationChapter 17

Sections:17.2, 17.3 - 17.7 &17.9


Eukaryotic Regulation Differs from Prokaryotic Regulation

Eukaryotes contain much greater amounts of genetic information

Many chromosomes Genetic information is segregated from nucleus to

cytoplasm; Prokaryotes use cytoplasm only Posttranscriptional Regulation Eukaryotic mRNA has longer half-life Eukaryotic mRNA is more stable


Types of Gene Regulation Control of Gene Expression

Chromosomal Organization Chromatin Remodeling

Transcription Promoters Enhancers (enhanceosome) Upstream Activating Sequences (UAS) Transcription Initiation Complex Activators


Control of Gene Expression (continued)

mRNA Degradation Translational Control

RNA Silencing RNAi

mRNA Processing Alternative splicing



Transcription Control


Transcriptional Control Why do you need a promoter?

Recognition site for binding of RNA polymerase Necessary for initiation of transcription Upstream from gene start site Several hundred nucleotides in length


Transcriptional Control Actual Promoter : TATA BOX (-25 to –35) Sequences within the promoter region that

function as enhancers are:

1. CAAT or CCAAT (cat box)

-70 to –80

2. GGGCGG (GC box) -110


Initiation Complex for Transcription

1. TFIID has 2 subunits : TBP and TAF

2. First, TBP subunit binds to TATA box

3. TAF promotes a conformational change in the DNA which allows other TF to bind (commitment stage)

4. Pol II leaves TATA box and transcribes (promoter clearance)




Enhancers1. Necessary for full level of transcription

2. Responsible for tissue-specific gene expression

3. Able to bind transcription factors by associating with RNA polymerase forming DNA loops


EnhancersDifferent from Promoters because:

1. No fixed position – upstream, downstream or within gene

2. Different orientation

3. Affect transcription of other genes if moved to another location



Positive Transcription Factors(True Activators)

A. Proteins with at least two functional domainsB. Functional Domains:

1. Bind to the enhancer (DNA binding domain)2. Protein-Protein interaction with RNA Pol or other transcription factors (trans-activating domain)



Positive Transcription Factors (True Activators)

DNA Binding Domains1. Helix-Turn-Helix (homeodomain) – 180 kb or 60 amino acids/ bind to major and minor grooves as well as backbone2. Zinc Fingers – Cys and His covalently bind zinc atom/bind major and minor goove Cys – N 2-4 - Cys – N 12-14 –His – N3 – His


Helix-Turn-Helix


Zinc Finger


Zinc Finger


Positive Transcription Factors (True Activators)

3. Leucine Zipper – 4 leucine residues spaced 7 amino acids apart and flanked by basic amino acids

- leucine regions form -helix

- leucine regions dimerize and and zip together


Leucine Zipper


Transcription Control


Transcription Control: GAL genes Galactose-utilizing genes Part of metabolic pathway to metabolize galactose in

yeast Follow the activation of genes GAL 1, 7, 10 that are

located near one another on the DNA Genes are made in response to the presence of

galactose Gal4p and Gal80p are regulatory proteins in the

process and UAS-G is the DNA sequence


Transcription Control: GAL genes


Transcription Control: GAL genes In the absence of galactose, GAL 80p is bound to

GAL 4p and GAL 4p is bound to the regulatory DNA sequence (UAS-G) Under these conditions, transcription of GAL 1, 7, 10 is

inhibited In the presence of galactose, a metabolite of

galactose binds to GAL 80p GAL 4p is then phosphorylated initiating a change in

conformation GAL 4p is now capable of activating transcription


Control of GAL Genes


Transcription Control: GAL genes

Fig. 17.5


GAL Genes


Transcription Control: Steroid Hormone

Not many changes in the external environment of cell in an animal

Hormones are secreted by cells in the animal and can signal changes from the environment

Peptide hormones bind to extra cellular receptors and steroid hormones bind to intracellular receptors





Steroid hormones often bind to cytoplasmic receptor and translocated to the nucleus where the complex acts

In the nucleus the complex binds to the DNA at a specific sequence

Hormones are potent regulators of gene expression, but only affect cells that produce the receptor that the particular hormone binds






Transcription Control: Steroid Hormone Steroid hormone control of gene expression

Important in development and physiological regulation

Because receptor is needed, have tissue or cell type specific effects

Specific for certain hormone receptor Usually found in a small number of cells Can affect tc, mRNA stability, mRNA processing



No hormone then the receptor is inactive and bound to a chaperone protein

Steroid hormone enters cell and binds to its specific receptor

Chaperone is displaced Hormone binds receptor = activation Complex is transported and acts in the nucleus



Hormone-receptor complex binds to specific DNA binding element Transcription activation or repression depending on

the complex Complex binds to the steroid hormone response

element (HRE) in the DNA HRE’s are in the enhancer region and in multiple

copies


Transcription Control Transcription of a gene is also affected by the

proteins bound to the DNA (histones) DNA is less compacted in regions where DNA is

transcribed Nucleosomes are not removed Generally physically inhibit gene transcription Transcription can occur in the presence of

nucleosomes when they are chemically modified DNA Methylation – CpG islands/X chromosome


Control of mRNA mRNA processing—regulation of production

of mature mRNA Alternative poly-A sites Alternative/differential splicing CALC gene employs both in different cell types


Control of mRNA

Fig. 17.7


Control of mRNA Evaluate gene expression of the human

calcitonin gene (CALC) in thyroid cells and neurons.

Thyroid cells Poly(A) signal after exon 4 is used Removed introns 1-4 and join exons 1-4 to make

calcitonin mRNA is translated.


Control of mRNA Evaluate gene expression of the human

calcitonin gene (CALC) in thyroid cells and neurons.

Neurons Poly(A) signal after exon 5 is used Remove all introns and exon 4 is removed as

well; join exons 1, 2, 3, 5 to make CGRP mRNA mRNA is translated.


Posttranslational modification Evaluate gene expression of the human

calcitonin gene (CALC) in thyroid cells and neurons. In both cell types the mRNA is translated into a

protein that needs processing—pre-hormone or pre-protein

This allows the protein to be synthesized and be present in the cell, but NOT be active.


Posttranslational modification When the proteins are needed, a protease cleaves

the pre-portion of the protein and the remainder of the polypeptide becomes active Calcitonin is produced in thyroid cells—hormone that

helps the kidney to retain calcium; Exon 4 encodes the active protein

cGRP is produced in neurons—found in hypothalamus and has neuromodulary/growth promoting properties; Exon 5 encodes the active protein


Control of Translation Shortened poly(A) tails prevent translation

Poly(A) tails are needed for translation initiation mRNAs that are ‘stored’ and prevented from

being translated have short Poly(A) tails (15-90 A’s long)


Control of Translation Shortened poly(A) tails prevent translation

Tails may be trimmed (deadenylation enzymes) or they may be short at synthesis.

Deadenylation enzymes recognize AU rich element (ARE) in the 3’ UTR of the mRNA and remove A’s from the tail

Other enzymes may recognize ARE in the 3’ UTR and lengthen the poly(A) tail when it is time to translate the mRNA


Control of mRNA mRNA stability—how long the mRNA is

found in the cell (RNA turnover) The longer the mRNA is found in the cell, the

more copies of protein are made. Stability of mRNA varies greatly from gene to

gene Important way to control gene expression



found in the cell (RNA turnover) Stability can be controlled by molecules present

in the cell Signals found in the 5’ or 3’ UTR Control when the mRNA is degraded



found in the cell (RNA turnover) 2 major pathways

Deadenylation –dependent decay pathway Deadenylation-independent decay pathway


Control by Protein Degradation Posttranslational control Controls how long the protein is present and active

in the cell Controlled by attachment of the protein ubiquitin to

the protein being targeted for degradation Signals for the protein to be degraded by the

proteasome N-terminus of the protein will determine its stability

by determining the rate that ubiquitin can bind to the protein

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Eukaryotic Regulation