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Molecular Evolution I
Carol Eunmi Lee
University of Wisconsin
Evolution 410
Molecular Evolution:
pertains to evolution at the levels of
DNA, RNA, and proteins
Outline
(1) Types of Mutations that could affect
function
Structural Changes
Regulatory Changes
(2) Which predominate?
(3) Case study with temperature
adaptation of the enzyme LDH
Previously, we had delved
into some aspects of
molecular evolution when
we discussed
Mutations
Mutations: any change in the genetic code,
including those that arise from errors in DNA
replication or errors in DNA repair
Most Mutations have no Effect
(most mutations are neutral)
• 3.12 billion nucleotides in the human genome
• Most of the genome is non-coding sequence
and has no function (up to 95%):
– Mutations here are “Neutral”
• Mutations that affect function are what matter
(within genes, or within regulatory sequences that
affect the expression of genes)
Mutations
• So, where in the genome do the mutations
matter???
• What exactly are these mutations doing?
• So, let’s go into more detail on what
exactly these mutations are doing…
• And how they affect expression and
function of genes and proteins
Diagram of eukaryotic gene
Each gene is composed of
regulatory and coding region
Eukaryotic
gene (DNA)
Futuyma D.J. (2009)
So… different types of mutations could
include:
• STRUCTURAL: Affects the Property of the gene
product (changes to the allele itself)
– A mutation could occur within the coding sequence of a gene,
and change the amino acid composition of a protein
(structural)
• REGULATORY: Typically affects the Amount of the
gene product
– A mutation could occur within a regulatory element, like a
promoter or enhancer near the gene (cis-regulatory)
– A mutation could occur within a regulatory element, like a
transcription factor that is encoded far away from the gene
(trans-regulatory)
Hierarchical processes that are affected by Mutations
STRUCTURAL
• Primary: Amino Acid composition (Amino
Acid substitutions)
• Secondary, Tertiary, Quaternary structure
REGULATORY
• Protein expression (transcription, RNA
processing, translation, etc)
• Protein activity (allosteric control,
conformational changes, receptors)
Once these mutations have occurred,
creating genetic variation, selection
could then act on genes, gene
expression, and on genetic architecture
(allelic and gene interactions)
STRUCTURAL evolutionary changes
• Mutations in DNA or mRNA that result in
changes in the amino acid composition of a
protein
• Some amino acid changes alter the activity
and/or function of proteins (and enzymes)
“The Central Dogma” of Molecular Biology
Francis Crick (1958)
Codon Bias
 In the case of amino
acids
 Mutations in Position 1,
2 lead to Amino Acid
change
 Mutations in Position 3
often don’t matter
REGULATORY CHANGES
• The protein structure itself does not
change
• Changes in Gene Expression
– Change in amount of expression (amount
of protein made - transcribed or translated)
– Changes in location, timing of expression
REGULATORY
Protein Expression
• Transcription: Mutations at promoters, enhancers
(CIS), transcription factors (TRANS), etc
• RNA Processing: Mutations at splice sites, sites
of polyadenylation, sites controlling RNA export
• Translation: Mutations in ribosomes, regulatory
regions, etc
Protein activity (allosteric control, conformational
changes, receptors)
Gene Expression
• Focusing on Transcription alone:
Cis-regulation (at or near the gene)
Examples: Promoters
Enhancers
Local repressor
Trans-regulation (somewhere else in the genome)
Examples: Gene regulatory proteins (trans-acting factors,
like transcription factors)
• Reading by Emerson et al. -- Focus reading mainly
on Part 1 (Intro) and Part 2
Gene Expression
• Focusing on Trans-Acting Factors:
• Transcription Factors: proteins that bind to
specific DNA sequences, thereby controlling
transcription and gene expression.
– Usually regulates many genes and therefore
often has large pleiotropic effects
Gene Regulation (expression)
trans-acting factor
(e.g. transcription factor)
gene is expressed
ACAGTGA
(promoter or enhancer)
Which types of mutations
predominately contribute to
adaptive evolution?
Structural or Regulatory Changes?
Which types of mutations predominately
contribute to adaptive in evolution?
• Structural Evolution: Hopi Hoekstra & Jerry Coyne (2007):
– Adaptation and speciation probably proceed substantially
through selection on structural mutations
– “There is no evidence at present that cis-regulatory changes
play a major role—much less a pre-eminent one—in adaptive
evolution.”
• Regulatory Evolution: David Stern (2000), Sean Carroll (2000)
– Cis-regulatory elements are the most likely target for the
evolution of gene regulation
– Most mutations causing morphological variation are expected
to reside in the cis-regulatory, rather than the coding, regions
of developmental genes
So who is right?
A Classic Example: temperature
adaptation in Fundulus heteroclitus
LDH
LDH is a glycolytic enzyme which catalyzes the reaction
between Pyruvate and Lactate
Protein function
STRUCTURE
• Amino acid composition (AA substitutions)
• Secondary, Tertiary, Quaternary structure
REGULATORY
• Protein expression (transcription, translation, etc)
• Protein activity (allosteric control, conformational
changes, receptors)
Fundulus heteroclitus
Populations in Maine and Georgia have different
proportions of alleles (isozymes) at LDH-B
Temperature Adaptation and
Enzyme Function
• In cold temperature generally activity of an enzyme
generally slows down
• So, in cold temperature, enzymes generally
compensate, to make up for the slower function.
• How?
• In hot temperature, enzymes have higher activity, but
can denature more readily.
• Enzymes lower the activation energy (Ea) of a
chemical reaction (“catalyzes the reaction”)
• Different isozymes with different properties would
lower the activation energy to differing degrees
• That is, enzymes with different Km or kcat will lower
Ea to differing degrees
Enzyme Reaction
k1
E + S
ES
k2
E + P
k-1
where
E
S
P
ES
= enzyme
= substrate
= product
= enzyme-substrate complex
k1 , k-1 , k2 = enzyme reaction rates
k2 is also called kcat, the catalytic constant
Michaelis-Menten Equation
Velocity (rate of reaction) =
Vmax [S]
Km + [S]
Km = substrate affinity, where Vmax/2
Also called “Michaelis-Menten constant”
[S] = substrate concentration
Vmax = maximum velocity
Michaelis-Menten Equation
Velocity (rate of reaction) =
Vmax [S]
Km + [S]
• Small Km: enzyme requires only a small amount of
substrate to become saturated. Hence, the maximum
velocity is reached at relatively low substrate
concentrations. (greater substrate binding specificity)
• Large Km: Need high substrate concentrations to
achieve maximum reaction velocity.
Enzyme Reaction
k1
E + S
ES
kcat
E + P
k-1
• There could be evolutionary differences in Km
• And kcat among species could evolve
• kcat depends on the G (activation free energy) of the
chemical reaction
Catalytic Efficiency
• Catalytic constant, kcat :
kcat =
Vmax
[E]t
• kcat = turnover number = the rate at which substrate is
converted to product, normalized per active enzyme site; Et is
the concentration of enzyme sites you've added to the assay
• High kcat  greater rate of reaction
• The ratio of kcat / Km is a measure of the enzyme’s catalytic
efficiency
Temperature Adaptation and
Enzyme Function
• In cold temperature generally activity of an enzyme
generally slows down
• So, in cold temperature, enzymes generally
compensate, to make up for the slower function.
• How? –increase in kcat increase in rate of reaction
• but, Km will increase (lower structure integrity)
• In hot temperature, enzymes have higher activity, but
can denature more readily.—want to increase stability
(lower kcat, lower Km)
1° latitude change
= 1°C change in
mean water
temperature
Place and Powers, PNAS 1979
Different alleles
(isozymes) predominate
in North vs South
North: LDH-B b allele
(cold-adapted)
South: LDH-B a allele
(warm-adapted)
The two alleles (proteins)
differ at 2 amino acids
Place and Powers, PNAS 1979
Catalytic efficiency (kcat/km) is higher for the b allele at low
temperature, and higher for the a allele at higher temperature
a allele homozygote
b allele homozygote
Place and Powers, 1979
Catalytic efficiency (kcat/km) is higher for the b allele at low
temperature, and higher for the a allele at higher temperature
• The two allele products (the
enzymes) show genetic
differences in catalytic efficiency
(adaptive differences) across
temperatures
• They also show Genotype x
Environment interactions and
evolutionary tradeoffs in function
across different temperatures,
with the bb homozygote doing
better in the cold, and the aa
homozygote doing better at
higher temperature
Place and Powers, 1979
There are many possible limitations (costs
or constraints) preventing complete
adaptation to an environment due to
evolutionary tradeoffs
For enzyme function, there is often a
tradeoff between functional capacity
and enzyme stability
Evolutionary Tradeoff in enzyme
function at cold vs high temperatures
Tradeoff between flexible vs stable enzyme structure
• Cold Temperature:
•
•
Flexible, can have higher activity to compensate
for cold temperature (higher kcat)
But hard to maintain structural integrity at high
temperature
• Warm Temperature:
•
•
Stable, to maintain structural integrity at high
temperature
But, lower enzyme activity (ok, because
temperature is high)
In damsel fish LDH, a
tradeoff between
functional capacity and
enzyme stability has been
found
More cold-adapted
enzymes are labile (flexible,
higher kcat) and less stable
at higher temperatures
More warm-adapted
enzymes have been found
to be more stable, but less
flexible
In damsel fish LDH, a
tradeoff between
functional capacity and
enzyme stability has been
found
More cold-adapted enzymes
are labile (flexible, higher
kcat) and less stable at higher
temperatures
If too unstable, lose
geometry for ligand
recognition and binding
(higher Km)
Protein could become
inactivated
Tradeoff between
functional capacity
and enzyme stability
Dark areas experience
conformational changes
during ligand binding, such
that amino acid changes
here could affect enzyme
function (kcat or Km)
This Thr -> Ala amino acid
substitution corresponds to
temperate -> tropical shift
A4LDH
This Thr -> Ala amino acid
substitution, at position 219
in the J-1G loop of
A4LDH, corresponds to
temperate -> tropical shift in
Damselfish
Threonine is more
hydrophilic and thought
to make the loop more
flexible (higher Km, kcat)
Threonine -> Alanine amino
acid substitution at a catalytic
loop corresponds to temperate
-> tropical shift in Damselfish
Km and kcat are higher in the
temperate (colder) ortholog
The Alanine amino acid
substitution causes Km and
kcat to be reduced in the
tropical orthologs
Km
Lower stability in colder fish
Chromis punctipinnis
(temperate, colder)
Chromis caudilis
(tropical, warmer)
kcat
Higher reaction rate in colder fish
Threonine is more hydrophilic
and thought to make the loop
more flexible
Johns and Somero 2004
Chromis xanthochira
(tropical, warmer)
Km
Tradeoffs:
Colder (white circles): more
flexible (high kcat), but loss of
binding ability (high Km)
Warmer (black square,
triangle): Less flexible (low
kcat), but higher binding ability
(low Km)
Lower stability in colder fish
Chromis punctipinnis
(temperate, colder)
When cold, you need to
Chromis caudilis
compensate for lower
(tropical, warmer)
rates of reaction activity by
making the enzyme more
flexible  high kcat sacrifice
Km (high Km)
or, fast &sloppy enzymes;
the cold will keep enzyme
more stable
Chromis xanthochira
(tropical, warmer)
kcat
Higher reaction rate in colder fish
Johns and Somero 2004
Protein function
STRUCTURAL
• Amino acid composition (AA substitutions)
• Secondary, Tertiary, Quaternary structure
REGULATORY
• Protein expression (transcription, translation, etc)
• Protein activity (allosteric control, conformational
changes, receptors)
Gene (or protein) expression
• Transcription
– RNA polymerase and promoter
– Enhancers
– Gene regulatory proteins (transcription factors)
•
•
•
•
•
RNA Processing
RNA Transport Control (nucleus to cytoplasm)
Translation
mRNA Degradation Control
Protein Activity Control (allosteric, degradation, etc.)
activity
protein
mRNA
Crawford and Powers, 1989
Common Garden Experiment:
Rear both populations at a
common temperature
The Northern isozyme has BOTH
higher activity and higher level of
expression in fish at constant lab
conditions (20°C temperature)
Higher Gene Expression of
LDH-B in the Northern
Maine population
Maine Florida Georgia New Jersey
Schulte et al. 2000
Transcriptional control
• What controls differences in gene expression of LDH in F.
heteroclitus?
• Mutations within Promoter or Enhancer?
Quick Time™ and a
Photo - JPEG decompressor
are needed to s ee this pic ture.
Doug Crawford: Promoter
Patricia Schulte: Enhancer
Gene expression
• Transcription
Cis-regulation (at or near the gene)
Examples:
– Promoter
– Enhancers
Trans-regulation (somewhere else in the genome)
Examples:
– Gene regulatory proteins (transcription factors)
Schulte et al. 2000
GRE present
control (GRE absent)
Transgenic Fish
GRE
present
Regulatory sequence (an
enhancer) was injected into
Northern and Southern Fish
control
(GRE absent) An enhancer, located
within a 500 base pair
sequence, significantly
increased gene expression
of LDH
Schulte et al. 2000
GRE (gene regulatory element) present
control (GRE absent)
GRE
present
Transgenic Fish
The regulatory sequence
control
from the Northern fish
(GRE absent)
(colder) increased
expression of LDH when
injected into both the
Northern and Southern fish
Which types of mutations contribute to
adaptive in evolution?
• Structural Evolution: Hopi Hoekstra & Jerry Coyne (2007)
– Adaptation and speciation probably proceed substantially
through selection on structural mutations
– “There is no evidence at present that cis-regulatory changes
play a major role—much less a pre-eminent one—in adaptive
evolution.”
• Regulatory Evolution: David Stern (2000), Sean Carroll (2000)
– Cis-regulatory elements are the most likely target for the
evolution of gene regulation
– Most mutations causing morphological variation are expected
to reside in the cis-regulatory, rather than the coding, regions
of developmental genes
So what’s the answer?
• This is a not a good question (binary thinking)
• Obviously, structural and regulatory changes
both contribute to adaptive evolution…
• But, there does appear to be general trends on
which type of mutations predominate
depending on the level of divergence among
taxa (refer to optional slides if you are
interested)
Patterns of Molecular Evolution
• What are mutations? How would structural vs regulatory
mutations affect function?
• Would you expect structural or regulatory evolutionary
differences to predominate?
• What are cis- vs. trans-regulatory mutations?
• When would you expect cis-regulatory evolutionary
differences to predominate? And Why?
• What about trans-regulatory differences?
• What are the possible targets of selection for LDH in
response to temperature?
• How does temperature affect Enzyme Kinetics?
• What changes in enzyme function might enhance a
response to an environmental variable (such as
temperature)? (Vmax, Km, Kcat, Kcat/Km, etc??)
• Why are there tradeoffs between enzyme function and
stability?
• Why are there tradeoffs between cold and warm adaptation
in enzyme function?
• How might organisms evolve in response to global
warming? What about global cooling?
1. When comparing DNA sequences that encode a
protein between two species, the number of
substitutions at nonsynonymous was found to be
much higher than those at synonymous sites. This
result suggests evidence for:
(a) Non-adaptive evolution
(b) Adaptive evolution
(c) Negative selection
(d) Evolutionary constraint
(e) Preferential fixation of synonymous sites
The graph shows the catalytic
efficiency (kcat/Km) for three genotypes
of the LDH-B enzyme across
temperatures for populations of the
fish Fundulus heteroclitus.
2. Which of the following is FALSE regarding the functional differences among
the enzymes above?
(a) The different genotypes appear to show tradeoffs between functioning well
(higher catalytic efficiency) at cold vs warmer temperatures
(b) The performance of the three genotypes shows no evidence for heterozygote
advantage
(c) Adaptation to temperature in these enzymes is likely due to differences in
amino acid composition between the proteins encoded by the a versus b
alleles
(d) kcat/Km is higher for the aa genotype than for the bb genotype at warmer
environments
(e) Differences in allelic function above reflect structural evolutionary changes
and prove that regulatory changes have not occurred
3. A fragment of DNA from an LDH-B allele shows
higher number of nonsynonymous relative to
synonymous substitutions than expected. When this
fragment is injected into a fish, it shows elevated
pyruvate metabolism relative to the equivalent
fragment from another allele.
(a) Evolutionary Adaptation (structural change)
(b) Evolutionary Adaptation (regulatory change)
(c) Linkage
(d) Physical/functional constraint
(e) Insufficient information to determine
• 1B
• 2E
• 3A
• Optional slides (Not required)
So what’s the answer?
• The “debate” between the importance of
structural vs regulatory evolution arose in
part because of lack of communication
between population geneticists and
developmental biologists
• Developmental Biology and the importance
of evolution of gene regulation was in fact
left out of the evolutionary synthesis of the
1930s and 1940s.
So what’s the answer?
• This is a not a good question (binary
thinking)
• Obviously, structural and regulatory
changes both contribute to adaptive
evolution…
• But, there does appear to be general trends
on which type of mutations predominate
depending on the level of divergence among
taxa
Patterns of Evolution
• It appears that patterns of structural,
cis-, trans-regulatory changes varies
depending on levels of divergence
among taxa
• Evolution in different kinds of
populations and over different
evolutionary time scales may result in
selection of different kinds of mutations.
Structural or Regulatory?
• David Stern 2008 performed the first comprehensive
review:
• As of 2008, Cis-regulatory mutations represented ~22%
of 331 identified genetic changes; although, the number
of cis-regulatory changes published annually is rapidly
increasing (there is a bias in the literature as more
studies have examined amino acid changes)
• Above the species level, cis-regulatory mutations
altering morphology are more common than protein
coding changes (supporting the argument of Sean
Carroll, at least at greater divergences)
Cis- vs Trans-Regulatory?
Cis-regulatory changes account for a greater
proportion of the expression differences observed
between rather than within species. Specifically,
cis-regulatory changes seem to accumulate
preferentially over time (Wittkopp et al. 2008).
Cis- vs Trans-Regulatory?
Gene Network and Pleiotropy
• The position of a gene in a regulatory network
is an important parameter to consider when
determining whether cis-regulatory or transregulatory are more likely to contribute to
phenotypic evolution.
Pleiotropy:
when a gene
affects many traits or functions
• Selection might not be able to
act on trait if the gene that
codes the trait is Pleiotropic, and
also affects other traits.
So, changing the gene could
negatively affect the other traits
Gene Network
• Conversely, a seemingly unbeneficial trait might get
selected for because the gene that codes for it also
enhances fitness
• Pleiotropy could sometimes lead to evolutionary tradeoffs
(you can have evolutionary tradeoffs that are not
pleiotropic– between traits encoded by different genes)
• Trans-acting factors, such as
transcription factors, often affect many
other genes… and are highly pleiotropic
Cis- vs Trans-Regulatory?
• Cis-regulation contributes disproportionately to
gene expression divergence between species,
relative to its contribution within species
(Wittkopp et al. 2008)
• This is in agreement with expectations that cisregulatory alleles may be preferentially fixed
because of their weaker pleiotropic effects.
Cis- vs Trans-Regulatory?
• Both contribute to evolutionary change
• There are also other issues (see
optional slides if interested)
Cis- vs Trans-Regulatory?
Dominance between alleles
• Cis-regulation contributes disproportionately
to gene expression divergence between
species, relative to its contribution within
species (Wittkopp et al. 2008)
• Cis-regulatory variants alter allele-specific
expression, whereas trans-regulatory factors
influence expression of both alleles in a
diploid cell.
(1) Cis- vs Trans-Regulatory?
• A promoter or enhancer (cis-) is only going to
regulate the gene that is downstream to it, and
not the other allele (allele-specific expression).
• However, a transcription factor (trans-) will
regulate both alleles (not allele-specific)
• Thus, a fundamental difference in coefficients of
dominance between cis- and trans-regulatory
variation might play significant roles
• Lemos et al. 2008. PNAS.
http://www.pnas.org/content/105/38/14471.full
Cis- vs Trans-Regulatory?
• Cis-regulated alleles tend to have additive effects
(each regulated independently by its own cisregulatory elements – promoters, etc.)
• Cis variation is additive and therefore accessible
to positive selection
• A Trans-regulatory protein could have a
deleterious mutation that is masked from selection
by the other functional allele (due to dominance)
• When Trans variation is masked in the recessive
state it is not purged by negative selection
Selection on cis- vs trans- regulatory elements
Deleterious cisregulatory element
reduced or no expression
expressed
Allele 1
Allele 2
• If a cis-regulatory element has a deleterious effect, it will be
exposed to selection, they will be expressed independently
Allele 1 Allele 2
functional trans-acting
factor takes over (dominant)
Deleterious transacting factor
expressed
expressed
• If a trans-acting factor has a deleterious mutation, the trans-factor
might be masked from selection, as the functional allele might
compensate for reduced function in the other (dominance)
(2) Cis- vs Trans-Regulatory?
Also… Differences in Mutational Targets
between cis- and trans-regulatory elements:
• The mutation variance for trans-variation is
substantially larger than the mutation
variance for cis-variation.
• More mutational targets in a trans-acting
factor (of a protein) than for a promoter or
enhancer
Cis- vs Trans-Regulatory?
• So, what this means is that deleterious
mutations could accumulate more in transalleles  greater mutational target + masking in
the recessive state…
• Such deleterious mutations could accumulate
over time… such that over greater evolutionary
distances the mutational load is higher in the
trans- alleles…
• So, over greater evolutionary distances, cisalleles might be favored over trans-alleles