Transcript How exercise may regulate transcription

Molecular Exercise Physiology
What is Molecular Exercise Physiology?
Presentation 1
Henning Wackerhage
Important
The online presentations on Molecular Exercise Physiology may
be used for self-teaching purposes for Molecular Exercise
Physiology-SIG members. However, the must not be used for
teaching students without prior authorisation.
If we wish to use some of these slides for your presentations to
students, please contact:
Dr Henning Wackerhage
Senior Lecturer in Molecular Exercise Physiology
University of Aberdeen
E-mail: [email protected]
Learning outcomes
At the end of this presentation you should be able to:
• Define the term Molecular Exercise Physiology and give
examples for research questions in Molecular Exercise
Physiology.
• Explain how a gene is transcribed and translated into a
protein.
• Explain how exercise-activated signal transduction pathways
may regulate transcription and translation of proteins.
Introduction
Part 1
What is Molecular Exercise
Physiology?
Definition
Prof. Frank Booth was one of the first researchers in
Cellular and Molecular Exercise Physiology. See:
Booth FW: Perspectives on molecular and cellular
exercise physiology. J. Appl. Physiol, 65: 14611471, 1988. Molecular exercise physiology is a
shortened version of the term used by Booth. A
narrow definition of the term “molecular exercise
physiology” is given below:
Molecular exercise physiology is the study of signal
transduction and genetics in relation to exercise. Molecular
exercise physiologists aim to characterise the mechanisms that
are responsible for the adaptation of cells and organs to exercise
and to identify the genetic determinants of athletic talent.
Applications of molecular exercise physiology
Possible applications are:
• Investigate the molecular basis of muscle adaptation to
exercise.
• Detect the signal transduction and gene regulation changes
in states such as diabetes mellitus, muscle unloading or
ageing and investigate whether exercise can reverse these
changes.
Why study Molecular Exercise Physiology?
• Because it can explain adaptations that have been described
previously.
• Because the research benefits from and advances other fields
such as cancer and ageing research.
• Major advances in knowledge occur at the moment while
most other exercise research is not that novel.
Classical approach
Exercise
?
“Black box”
Adaptations
Molecular exercise physiology approach
Molecular
Exercise
Physiologist
Exercise
Signal
transduction
& gene
regulation
“Black box”
Adaptations
Examples
Classical exercise physiologists found that endurance exercise increases
the number of capillaries in a muscle. Molecular exercise physiologists
try to identify the exercise-activated signal transduction pathways that
are responsible for the growth of capillaries.
Classical exercise physiologists have described the growth of muscle
fibres in response to resistance training. Molecular exercise
physiologists have identified how exercise may activate regulators of
translation/protein synthesis.
Classical exercise physiologists have discovered that exercise makes
hearts grow (cause the athlete’s heart). Molecular exercise physiologists
have identified candidate signal transduction pathways that may
regulate the growth of heart muscle cells.
There is much more to discover!
Introduction
Part 2
DNA and all that…
Definition
A gene is DNA that codes for a protein.
Please note: Not all DNA is coding for proteins. There are large
non-coding regions and some genes code just for RNA (will be
explained later).
Task: What is the difference between RNA and DNA? What is
mRNA and tRNA? Find out.
DNA: The model
Watson and Crick used these
and other data to model DNA
structure: bases occur in CG
and AT pairs, double helix.
X-ray of DNA (first by Wilkins, Franklin
and colleagues at King's College, London)
DNA is coiled, supercoiled and packed
DNA double helix
Nucleosome
filament
Chromatin
filament
DNA
Nucleosome
Human chromosomes (23 pairs)
Human beings have 23 pairs of chromosomes which are densely
packed DNA. However, most of the time DNA is unravelled.
Genome information
Some facts about the human genome:
• Human genome size: about 3,200 Mb (mega
bases).
• Estimated gene numbers: human: 31,000,
yeast: 6000, fly: 13,000, worm: 18,000, plant:
26,000.
• Only 1.1 to 1.4 % of the human sequence
encodes protein. The rest is non-coding.
• 28 % of the sequence is transcribed into RNA (5
% of this is translated into proteins).
• Only 94 of 1,278 protein families are
specific to vertebrates.
How does DNA code for proteins?
DNA is a blueprint for proteins. The coding alphabet consists of four
“letters” (i.e. bases plus one more in mRNA).
Purine bases:
Adenine (A)
Guanine (G)
Pyrimidine bases:
Cytosine (C) Uracil (U, RNA)
Base pairs:
Adenine- Thymine
Guanine – Cytosine
Uracil (instead of T in RNA)
Thymine (T, DNA)
How does DNA code for proteins?
The bases are connected and form long DNA chains. Here is a DNA
sequence downloaded from the Ensembl genome browser:
AGCTTATTCTGCATAATTAGAAAAGAAAGACACCAAGCCATTTAAACATAATT
TATGTACTTTATGGCTTTATACAATTATAGCAAAGATTGTTCTTGTGTCTGTAA
GTACATCAACATCAGGCACTTCTCAGAGTATCGGAACAAGAACGTGGAATC
TGCACTGTTACTAAACTCGGGTAGCGAAATGCAGGAGGCATGACTACGTCC
TGATGGGACTTACATGGCCACCCCTGGCCACACTGCCAGGCTGTGC
Gene expression: how it works
Reading a gene and producing a protein is a two-step process:
First, a gene (red) is transcribed into messenger RNA (mRNA;
orange) and second, mRNA is translated into a peptide/protein.
Gene
DNA GTCTTTCAAATATTGAATATGACAAAGATGTTTACTGTACCAGATTG
Transcription
mRNA
UAUAACUUAUACUGUUUCUACAAAUGA
Translation
Peptide/protein
Peptide sequence: Met (start) Asn Leu Tyr Cys Phe Tyr
Lys (termination)
How transcription works
Packed DNA
TF
Step
1.
DNA
is
unravelled and activated
transcription factors (TF)
bind to the DNA.
Step 2. RNA polymerase
II (Pol II) is recruited by
the active, DNA-bound
transcription factor.
Step 3. RNA polymerase
transcribes gene (shown
in green) into mRNA
(shown in red).
TF
TF
Pol II
Pol II
How translation works
Ribosomes are located in
the cytosol which is where
mRNA is transported to.
The mRNA (shown in red)
is
translated
into
a
polypeptide (shown as a
chain of ovals).
How to transcribe DNA into mRNA and how
to translate mRNA into protein?
If you know a DNA sequence then you can use the genetic code
to first transcribe the gene into mRNA and second to translate
mRNA into an amino acid (protein) sequence. Some programmes
on the internet help you to achieve that. See task on next page.
Task
Task: Copy the DNA sequence below and convert it first into
mRNA sequence and then a peptide. Use the following website:
www.nitrogenorder.org/cgi-bin/nucleo.cgi.
TGTCTTTCAAAAAAATGTGAAAACACTTTAATATAACTTATACTGTTTCTACAAATTAGA
TGTAAGAAATAATTTCATTTAGTCATAGTACAATAAATTTGATTAACAAAATCCCAATTT
ACAAAACAGAAGTAAATAAATGCAGAAATATATTCATTATCAAATTATAAAATAAAAGTA
ACAGTTATTGTTGAGATAGCATCAGTTTATTCTTCATTTCAGATAATAGAGTCAATTATT
TTGGTATACTTAGTAATGTTTTTGCAAGTATTAAAATAATGGAACGTTGAGATTTAAACA
CAATGACAGTAAACCATTGAAATTTCAAATTCACTTTATACAGCCATCATGAATCCATAA
GTGAATGTTAATCATGTAAAAAAATATAAAATGATTGTAATATAACCATCTAATCATTAA
CATATGGAGTTTTAAGACCACTATTTAATCTGCTAATTTGCTCTGAAATATAAGTAGTTG
CTTTTCTGTGATGCATGACATGTCTTTGTGCCTTAGTACTTAATTTGAAATGTCCTTAGT
GTAAAAATATACCAAAGTAAATAAAAAAGGAGACACTTATTTACAAAACAATATTGTATA
CATATTATATAAATGCATTGTTTCAGTCTTTTTATACAATATTGATAGAGTCGATCATTT
Important
You should have learned about transcription and translation
before and the presentation should have been a revision. You will
do well in this module if you know transcription and translation
inside out. There are numerous websites and textbooks in which
transcription and translation are described.
Do not proceed until you can explain the following terms and
reactions well:
DNA, intron, exon
Transcription factor, transcription factor binding site, RNA
polymerase II, mRNA
Translation, peptide, protein
Introduction
Part 3
Exercise changes transcription and
translation
Exercise regulates transcription and translation
Exercise stimulates acute and chronic adaptations probably in every
organ of the body. Many of these adaptations are mediated by socalled signal transduction pathways that regulate the transcription
and translation of genes. The sequence of events is:
Exercise
signals

activation/inhibition
of
signal
transduction pathway  change in gene transcription,
translation or other cell function (= adaptation).
Today, Molecular Exercise Physiology researchers aim to trace this
sequence of events for many adaptations to exercise. Here, I will
briefly introduce how trancription and translation may be regulated
by exercise.
How exercise may regulate transcription
Exercise signal
(1)
P
Transcription
(2)
P
P
mRNA
Nucleus
(1) In this model, an exercise signal (such as calcium, stretch,
energy stress) leads to the activation of a signalling protein (green)
by phosphorylation. (2) The signalling protein then phosphorylates a
transcription factor (blue) which promotes the translocation of the
transcription factor into the nucleus. (3) The transcription factor
binds to the DNA which increases the transcription of a gene (i.e.
increase in mRNA).
Exercise regulates transcription
Example: Zambon et al. (2003) used so-called DNA microarrays
(gene chips) in order to identify genes whose expression (i.e. whose
mRNA content) was changed in muscle 6 h after resistance training.
DNA microarrays allowed the researchers to measure the gene
expression of 20,000 genes in one two-day experiment. The table
below contains some results (the real results table consists of 20,000
rows!). Column 1 in the table below gives a gene description, column
2 the fold change (exercised versus non-exercised leg) and the pvalue indicates the significance of the change. The data indicate that
the mRNA of the first three genes was significantly decreased
(negative change ) and of that the mRNA of the fourth gene was
significantly increased 6 h after resistance exercise.
GeneCard
Description
Fold change
P value
KIAA0157
KIAA0157 protein
-1.1923
5.03E-05
Cstf1
cleavage stimulation factor,
-1.0481
0.000156
Fgfr2
fibroblast growth factor receptor 2
1.032399
0.000273
Exercise regulates translation
Exercise signal
P
mRNA
(2)
P
(1)
Protein
Translation
Nucleus
(1) In this model, an exercise signal (such as calcium, stretch,
energy stress) leads to the activation of a signalling protein (green)
by phosphorylation. (2) The signalling protein then phosphorylates a
protein (orange) which increases the translation of mRNA into
protein (which is protein synthesis). Muscle protein synthesis and
translation signalling can be increased for 48 h after resistance
exercise.
Exercise regulates translation
Translation is the process in which
mRNA is used as a blueprint to
assemble proteins from amino
acids. Thus, translation is protein
synthesis.
In the recent five years researchers
have shown that strength training
can increase the general rate of
translation. In addition, many of the
regulatory proteins that regulate
translation have been identified.
Example: The figure is taken from
Baar and Esser (1999) and shows a
Western blot of the p70 S6k protein
which is involved in the regulation
of translation/protein synthesis.
The p70 S6k bands are shifted
upwards in response to insulin and
3 h and 6 h after resistance
exercise. The changed banding
indicates that the protein is
activated in these situations.
Task
a) How do the following methods work and how may the be used for
Molecular Exercise Physiology research questions?
- RT-PCR
- Western blots
- DNA microarray
- SNP chips
b) Find two research papers each in which the authors investigate
the effect of exercise on gene expression (i.e. transcription of a
gene) and translation. Use PubMed for your search.
Revise this presentation several times and do a lot of
additional reading. This material is crucial for the rest of the
module.
The End