Regulation of Gene Expression

1
Regulation of Gene Expression

• Six steps at which eucaryotic gene expression
  can be regulated.

inactive mRNA
NUCLEUS
CYTOSOL
mRNA degradation control
DNA
mRNA
mRNA
RNA transcript
transcriptional control
RNA processing control
RNA transport and localization control
translation control
protein
protein activity control
inactive protein
2
Translational control

• In principle, every step required for the process
  of gene expression could be controlled.
• Predominant form Control of initiation of
  transcription.

Advantages of translational control
Rapid change in the concentration of the protein
without a rapid turnover of its mRNA fast
response Serves as a means to affect the level
of gene product in the absence of transcription
3
Conditional systems - principles

• Conditional systems may function on the basis of
• - regulatory proteins
• - aptamers
• - allosteric ribozymes
• - antisense RNA
• - RNAi

4
Conditional systems - Literature

• Regulatory proteins
• Oliveira CC, Goossen B, Zanchin NI, McCarthy JE,
  Hentze MW, Stripecke R. Translational repression
  by the human iron-regulatory factor (IRF) in
  Saccharomyces cerevisiae. Nucleic Acids Res. 1993
  Nov 2521(23)5316-22.
• Stripecke R, Oliveira CC, McCarthy JE, Hentze MW.
  Proteins binding to 5' untranslated region sites
  a general mechanism for translational regulation
  of mRNAs in human and yeast cells. Mol Cell Biol
  1994 Sep14(9)5898-909
• Ribozymes
• Soukup GA, Emilsson GA, Breaker RR. Altering
  molecular recognition of RNA aptamers by
  allosteric selection. J Mol Biol. 2000 May
  12298(4)623-32.
• An Allosteric Ribozyme Regulated by Doxycyline.
  Angew Chem Int Ed Engl. 2001
• Bramlage B, Luzi E, Eckstein F. HIV-1 LTR as a
  target for synthetic ribozyme-mediated inhibition
  of gene expression site selection and inhibition
  in cell culture.Nucleic Acids Res. 2000 Nov
  128(21)4059-67.
• Scherr M, Grez M, Ganser A, Engels JW. Specific
  hammerhead ribozyme-mediated cleavage of mutant
  N-ras mRNA in vitro and ex vivo.
  Oligoribonucleotides as therapeutic agents. J
  Biol Chem 1997 May 30272(22)14304-13
• Aptamers
• Werstuck G, Green MR. Controlling gene expression
  in living cells through small molecule-RNA
  interactions. Sience. 1998 Oct 9282(5387)296-8.
• Grate D, Wilson C.Inducible regulation of the S.
  cerevisiae cell cycle mediated by an RNA
  aptamer-ligand complex. Bioorg Med Chem. 2001
  Oct9(10)2565-70.

5
Proteins

• .

Iron-responsive regulation
IRE RNA element IRP RNA binding protein
IRE in 5UTR of mRNA encoding ferritin, IPR
binding KD 10-10 iron level in excess of
requirement leads to 100-fold reduced binding
Conditional translational regulation in yeast
reporter mRNA with IRE containing
5UTR, expressing IRP results in decreased
activity (regulatory factor 10) IRE-IRP are
sufficient components to create a regulatory
circuit fully active in a foreign cellular
environment
6
Ribozymes

• Different strategies

7
Ribozymes 1
Allosterical ribozymes as intrinsic part of
mRNA Ligand activated Breaker lab (FMN) Ligand
repressed Famulok lab (Doxycycline)

8
Ribozymes 1

• Selection strategies

9
Ribozymes 1

• SELEX results

10
Ribozymes 1
NOT ACTIVE IN VIVO !!
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Ribozymes 2
Endogenous addition of the ribozyme Hammerhead
ribozymes directed against LTR-HIV
G
U
X
12
Ribozymes 2
Endogenous addition of the ribozyme Hammerhead
ribozymes directed against LTR-HIV
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Aptamers
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Aptamers
Regulation by tetracycline binding aptamers
GFP expression in S.cerevisiae
3000
fluorescence / oD600
2000
1000
e-3
e-2
e-1
e0
e1
e2
c (tc) µM
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Aptamers
6
3
control
rel. luciferase activity ()
cap-prox
cap-dist
6
15
15
cap-dist
rel. luciferase activity ()
uORF
i_uORF
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Aptamers
Mechanism of translational inhibition
Sucrose gradient analysis in vitro
no tc
10µM tc
32P-capped RNA (cpm)
32P-capped RNA (cpm)
32P-capped RNA (cpm)
Fractions
Fractions
Fractions
Frations
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Summary