Eukaryotic Regulation

1
Eukaryotic Regulation

• Chapter 18
• Sections18.1 - 18.8

2
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

3
Types of Gene Regulation

• Control of Gene Expression
• Chromosomal Organization
• Chromatin Remodeling
• Transcription
• Promoters
• Enhancers (enhanceosome)
• Upstream Activating Sequences (UAS)
• Transcription Initiation Complex
• Activators

4
Control of Gene Expression (continued)

• mRNA Degradation
• Translational Control
• RNA Silencing
• RNAi
• mRNA Processing
• Alternative splicing

5
(No Transcript)
6
Transcription Control
7
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

8
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

9
Initiation Complex for Transcription

• TFIID has 2 subunits TBP and TAF
• First, TBP subunit binds to TATA box
• TAF promotes a conformational change in the DNA
  which allows other TF to bind (commitment stage)
• Pol II leaves TATA box and transcribes (promoter
  clearance)

10
(No Transcript)
11
(No Transcript)
12
Enhancers

• Necessary for full level of transcription
• Responsible for tissue-specific gene expression
• Able to bind transcription factors by associating
  with RNA polymerase forming DNA loops

13
Enhancers

• Different from Promoters because
• No fixed position upstream, downstream or
  within gene
• Different orientation
• Affect transcription of other genes if moved to
  another location

14
(No Transcript)
15
Positive Transcription Factors(True Activators)

• Proteins with at least two functional
• domains
• B. Functional Domains
• 1. Bind to the enhancer (DNA binding domain)
• 2. Protein-Protein interaction with RNA Pol
  or other transcription factors (trans-activating
  domain)

16
(No Transcript)
17
Positive Transcription Factors (True Activators)

• DNA Binding Domains
• 1. Helix-Turn-Helix (homeodomain) 180 kb or
  60 amino acids/ bind to major and minor grooves
  as well as backbone
• 2. 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

18
Helix-Turn-Helix
19
Zinc Finger
20
Zinc Finger
21
Positive Transcription Factors (True Activators)

• Leucine Zipper 4 leucine residues spaced 7
  amino acids apart and flanked by basic amino
  acids
• - leucine regions form a-helix
• - leucine regions dimerize and and zip together

22
Leucine Zipper
23
Transcription Control
24
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

25
Transcription Control GAL genes
26
Transcription Control GAL genes

• In the absence of galactose, Gal80p is bound to
  Gal4p and Gal4p 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 Gal80p
• Gal4p is then phosphorylated initiating a change
  in conformation
• Gal4p is now capable of activating transcription

27
Control of GAL Genes
28
Transcription Control GAL genes
Fig. 17.5
29
GAL Genes
30
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

31
Transcription Control Steroid Hormone
32
Transcription Control Steroid Hormone

• 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

33
Transcription Control Steroid Hormone
34
Transcription Control Steroid Hormone
35
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

36
Transcription Control Steroid Hormone

• Steroid hormone control of gene expression
• 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

37
Transcription Control Steroid Hormone

• Steroid hormone control of gene expression
• 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
• HREs are in the enhancer region and in multiple
  copies

38
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

39
Control of mRNA

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

40
Control of mRNA
Fig. 17.7
41
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.

42
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.

43
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 processingpre-hormone or
  pre-protein
• This allows the protein to be synthesized and be
  present in the cell, but NOT be active.

44
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 cellshormone
  that helps the kidney to retain calcium Exon 4
  encodes the active protein
• cGRP is produced in neuronsfound in hypothalamus
  and has neuromodulary/growth promoting
  properties Exon 5 encodes the active protein

45
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 As
  long)

46
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 As
  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

47
Control of mRNA

• mRNA stabilityhow 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

48
Control of mRNA

• mRNA stabilityhow long the mRNA is 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

49
Control of mRNA

• mRNA stabilityhow long the mRNA is found in the
  cell (RNA turnover)
• 2 major pathways
• Deadenylation dependent decay pathway
• Deadenylation-independent decay pathway

50
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