Molecular genetics & gene
expression
Mat Halter and Neal Stewart
2016
Discussion questions
• What are the differences between DNA
and RNA?
• Describe the main parts of a gene and its
functions.
• What role do cis-regulatory elements and
trans-acting factors play in gene
regulation?
• What is responsible for the wide diversity
of protein structure found in nature?
• In what different ways can gene
expression be regulated?
http://www.yellowtang.org/cells.php
A chromosome is composed of nucleosome-bound DNA called chromatin
Molecular Structure of Nucleic Acids:
A Structure for Deoxyribose Nucleic Acid
April 25, 1953
Francis Crick
http://www.stern.de/_content/50/44/504453/watson_crick_500.jpg
James Watson
Figure 6.2
Nucleotide base pairing
A pair with T
G pair with C
Nucleotide base pairing occurs
through “hydrogen bonding”
Strands have directionality from
5’ to 3’ and when paired strands
are in “antiparallel” orientation
DNA and RNA are structurally
similar
Some differences of RNA and DNA
Difference between thymine and uracil
Discussion questions
• What are the differences between DNA
and RNA?
• Describe the main parts of a gene and
their functions.
• What role do cis-regulatory elements and
trans-acting factors play in gene
regulation?
• What is responsible for the wide diversity
of protein structure found in nature?
• In what different ways can gene
expression be regulated?
The Central Dogma
Transcription
DNA
RNA
Protein
Eukaryotic gene structure
Figure 6.5
Transcription of DNA into mRNA
TF
TF
RNA
TF
polymerase II
TF
AAAAA
TF
mRNA = messenger RNA
Fig 6.6
Eukaryotic genes contain introns which are
spliced to form mature mRNA
mRNA structure differs between prokaryotes
and eukaryotes
Polycistronic prokaryotic message = several mRNAs are
regulated by one promoter
Monocistronic eukaryotic message
Discussion questions
• What are the differences between DNA
and RNA?
• Describe the main parts of a gene and
their functions.
• What role do cis-regulatory elements and
trans-acting factors play in gene
regulation?
• What is responsible for the wide diversity
of protein structure found in nature?
• In what different ways can gene
expression be regulated?
Cis- acting regulatory elements
• Promoter region- Located immediately upstream of the transcription
start site and serves as a binding site for the RNA -polymerase II
complex.
• Enhancer region- Promotes transcription by recruiting histonemodifying enzymes that open the chromatin structure, making the
sequence more accessible—sometimes acts in trans.
• Insulators- Sequences that have the ability to protect genes from
inappropriate signals emanating from their surrounding genome.
Vascular TissueSpecific Promoter
GUS gene
Terminator
http://www.forschenistkunst.at/bildbewertung/objekt35.php?id=35
Transcription factors: proteins that bind to
promoters to affect transcription
• Transcriptional activators- Recruits the RNA polymerase complex to
the transcription start site by binding to either sequences in the promoter or
distant cis-acting elements to increase transcription.
• Transcriptional repressor- Prevents transcription of a gene by interfering
with RNA polymerase activity on a promoter
Promoter
LacZ
LacY
LacA
RNA
Polymerase
mRNA
VIDEO
Transcription of DNA into mRNA
TF
TF
RNA
TF
polymerase II
TF
AAAAA
TF
mRNA = messenger RNA
The TFs in this case are transcriptional activators
Discussion questions
• What are the differences between DNA
and RNA?
• Describe the main parts of a gene and
their functions.
• What role do cis-regulatory elements
and trans-acting factors play in gene
regulation?
• What is responsible for the wide diversity
of protein structure found in nature?
• In what different ways can gene
expression be regulated?
https://www.youtube.com/watc
h?v=D3fOXt4MrOM
Transcription and translation, the
movie.
The Central Dogma of
Molecular Biology
Translation
DNA
RNA
Protein
http://www.yellowtang.org/cells.php
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.
Transfer RNA (tRNA)
Amino
Acid
AUG
UCG
ACA
GGC
UGA
Initiation of translation
The start codon is found by scanning downstream
from the 5’ end of the mRNA
VIDEO
Amino acid backbone
20 amino acids
TABLE 6.1 The 20 Amino Acids Commonly Found in Proteins
Amino Acids
Alanine
Arginine
Asparagine
Aspartate
Cysteine
Glutamine
Glutamate
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
Three-Letter Abbreviation
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Try
Tyr
Val
One-Letter Abbreviation
A
R
N
D
C
Q
E
G
H
I
L
K
M
F
P
S
T
W
Y
V
Figure 6.11
Figure 6.11
Polypeptide structure.
The building block of a
polypeptide is the
peptide bond formed
between amino acids.
Peptide bonds
connect amino acids
to create a polypeptide
chain. Proteins are
formed through the
association of
individual polypeptide
chains that may be
identical to each other
or unique in sequence.
Figure 6.12
Figure 6.12 The genetic code gives rise to either overlapping or non-overlapping
reading sequences. A codon consists of three consecutive nucleotides that code for
an amino acid. The nucleotides in a codon may give rise to multiple amino acids
depending on the reading frame.
Discussion questions
• What are the differences between DNA
and RNA?
• Describe the main parts of a gene and
their functions.
• What role do cis-regulatory elements and
trans-acting factors play in gene
regulation?
• What is responsible for the wide
diversity of protein structure found in
nature?
• In what different ways can gene
expression be regulated?
Gene expression regulation
• Transcriptional level:
– Transcription factors (environmental,
physiological)
– Alternate splicing
• Translational level:
– Elongation factors
– RNA interference
Discussion questions
• What are the differences between DNA
and RNA?
• Describe the main parts of a gene and
their functions.
• What role do cis-regulatory elements and
trans-acting factors play in gene
regulation?
• What is responsible for the wide diversity
of protein structure found in nature?
• In what different ways can gene
expression be regulated?
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
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.
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 transformed plants 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
Figure 6.9
Some post-translational
modifications
•
•
•
•
•
•
•
•
•
Phosphorylation
Biotinylation
Glycosylation
Acetylation
Alkylation
Methylation
Glutamylation
Glycylation
Isoprenylation
• Lipoylation
• Phosphopantetheinyl
ation
• Sulfation
• Selenation
• C-terminal amidation
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