Lecture 9 Chapter 6
Gene expression and regulation
II
Neal Stewart
Focus questions
• How important are cis-regulatory elements
and trans-acting factors in gene
regulation?
• What are the control points that can
regulate gene expression?
Transcription revisited
Promoter elements not required for
transcription initiation
• CAAT box – usually located at -70 to -80
within the promoter
• GC box
• Other gene-specific elements (lightresponsive, nutrient-responsive, etc.)
• Enhancer elements
What are some biological roles of
transcription factors?
• Basal transcription regulation – general
transcription factors
• Development
• Response to intercellular signals
• Response to environment
• Cell cycle control
The CRT/DRE response element responds
to dehydration and cold-induced
transcription factors (CBF)
Figure 6.7
Transcription factors
Figure 6.8
Enhancer can work from downstream and
upstream region
Enhancers
•
Their location is not fixed. Location could be in
the upstream or downstream DNA, in intron,
exon or in the untranslated region.
•
They enhance transcription by acting on
promoter in cis (typically)
•
Each enhancer has its own binding protein.
These proteins are trans-regulatory activating
factors
•
Sequence of enhancers is variable.
•
Enhancers regulate tissue-specific and
temporal expression of genes.
TATA binding protein (TBP)
transcription factor
Wikipedia.com
DNA-binding domains allow
transcription factors to bind
directly to a cis-regulatory
element
Helix-loop-helix
Zinc finger domain
Leucine zipper
domain
Extreme trans-acting effectors of
transcription: TAL effectors
• From plant pathogenic bacteria
Xanthomonas
• Secreted by bacteria when they infect
• Transcriptional activator-like (TAL)
effectors bind with plant promoters to
express genes beneficial for the bacteria
http://www.sciencemag.org/content/333/6051/1843/F2.large.jpg
Repression of transcription
TFs that act as repressors
Some trans-acting elements
prevent transcription
Introducing RNAi
http://www.youtube.com/watch?v=
H5udFjWDM3E&feature=related
What is a microRNA (miRNA)?
Controlling gene expression post-transcriptionally.
microRNA is an abundant class of newly identified small
non-coding regulatory RNAs.
Major characteristics of miRNAs:
• 18-26 nt in length with a majority of 21-23 nt
• non-coding RNA
• derived from a precursor with a long nt sequence
• this precursor can form a stem-loop 2nd hairpin structure
• the hairpin structure has low minimal free folding energy (MFE) and high MFE index
Slide courtesy of Baohong Zhang, East Carolina Univ
miRNA regulates plant development
miRNA 156
increasing leaf initation,
decreasing apical dominance, and
forming bushier plant.
miRNA 164
stamens are fused together.
miRNA 172
sepal and petal disappeared.
miRNA 319
Leaf morphology
WT
miRNA Slide courtesy of Baohong Zhang, East Carolina Univ
Small interfering RNAs inhibit expression of a
homologous gene
Biogenesis of miRNAs
Plant
Animal
Bartel, 2004. Cell.
Mechanisms of miRNA-mediated gene regulation
Post-transcriptional gene regulation
Two major molecular mechanisms
Zhang et al. 2006. Developmental Biology
Slide courtesy of Baohong Zhang, East Carolina Univ
Mary-Dell Chilton
• Undergrad and PhD University of Illinois
• Postdoc with Gene Nester and Milt Gorgon Univ
Washington
• One of the first plant transformation Washington
University
• Career at CibaNovartisSyngenta
Pre-transcriptional gene
regulation by methylation of DNA
and acetylation of histones
Special proteins (e.g.
chromomethylases) maintain
methylation patterns
Switching a gene on and off through DNA
methylation and histone modification
Arabidopsis MET1 Cytosine Methyltransferase
Mutants
Kankel et al. 2003. 163 (3):1109 Genetics
Plants mutant for MET1 show
late-flowering phenotypes
Histone acetyl transferases and chromatin
remodeling allows promoters to be accessible to
RNAPII
Histone tails are modified and can
be studied easily
Figure 6.9
Some post-translational
modifications
•
•
•
•
•
•
•
•
•
Phosphorylation
Biotinylation
Glycosylation
Acetylation
Alkylation
Methylation
Glutamylation
Glycylation
Isoprenylation
• Lipoylation
• Phosphopantetheinyl
ation
• Sulfation
• Selenation
• C-terminal amidation
Phosphorylation is important for
intracellular signalling
http://www.scq.ubc.ca/wp-content/uploads/2006/07/phosphocascades.gif
Protein glycosylation in the ER
The central dogma revisited
•The order of the DNA template
or coding strand is 3’ to 5’
•This determines the order of the
mRNA strand (5’ to 3’) because
DNA template is complementary
to the mRNA strand.
Eukaryotic gene structure and
transcription of DNA into mRNA
TF
TF
RNA
TF
polymerase II
TF
AAAAA
TF
Figure 6.5
Manipulating gene expression
• Can be done at several levels
– Promoters, enhancers, transcription factors
– Post-transcriptional
– Translational
– Methylation
• Biotechnology typically manipulates
promoter
• Post-transcriptional gene silencing (RNAi)
increasingly important