2020_SS1_Bis2a_Facciotti_Reading_25 - Biology

Learning Objectives Associated with 2020_SS1_Bis2a_Facciotti_Reading_25

  • Describe how genotype and phenotype are linked. Predict a phenotype if given a genotype and related molecular mechanisms, and vice versa.
  • Describe how environmental information can shape transcriptional and translational output in ways that lead to different phenotypes and cellular specialization.
  • Define and explain the different vocabulary terms used to describe mutations (point, deletion, insertion, nonsense, frameshift, null, loss of function and gain of function) and be able to predict their impact on protein function.
  • Use a codon table and your knowledge of protein structure and function to make predictions of how specific changes at the DNA level might influence protein structure and function.
  • Explain the possible different mechanisms by which mutations can cause changes in phenotype. Include mutations to both protein coding regions and non-protein-coding regions in your discussion.
  • Explain the potential influence of mutations on the specificity and affinity of protein-DNA interactions and the potential impact of these mutations on gene expression.


Errors occurring during DNA replication are not the only way by which mutations can arise in DNA. Induced mutations are those that result from an exposure to chemicals, UV rays, x-rays, or some other environmental agent. Spontaneous mutations occur with no exposure to any environmental agent; they result from spontaneous biochemical reactions taking place within the cell.

Mutations may have a wide range of effects.

Some mutationscreate no observable impact; we call these

silent mutations. Point mutations are those mutations that affect a single base pair. The most common nucleotide mutations are substitutions,

in which one base is replaced by another

. These can be of two types, either transitions or transversions. Transition substitution refers to a purine or pyrimidine being replaced by a base of the same kind; for example,

a purine such as adenine may be replaced by the purine guanine

. Transversion substitution refers to a purine being replaced by a pyrimidine, or vice versa; for example, cytosine, a pyrimidine,

is replaced

by adenine, a purine. Mutations can also

be the result of

the addition of a nucleotide, known as an insertion, or the removal of a base, also known as deletion. Sometimes a piece of DNA from one chromosome may get translocated to another chromosome or to another region of the same chromosome;

this is known

as translocation.

As we will visit later, when a mutation occurs in a protein coding region, it may have several effects. Transition or transversion mutants may lead to no change in the protein sequence (known as silent mutations), change the amino acid sequence (known as missense mutations), or create

what is known as

a stop codon (known as a nonsense mutation). Insertions and deletions in protein coding sequences lead to frameshift mutations. Missense mutations that lead to conservative changes results in the substitution of similar but not identical amino acids. For example, the acidic amino acid glutamate being substituted for the acidic amino acid aspartate would

be considered

conservative. We do not expect these types of missense mutations to be as severe as a non-conservative amino acid change; such as a glutamate substituted for a valine. Drawing from our understanding of functional group chemistry, we can correctly infer that this

type of

substitution may lead to severe functional consequences, depending upon location of the mutation.

Note: Vocabulary Watch

Note that the preceding paragraph had a lot of potentially new vocabulary - it would be a good idea to learn these terms.

Figure 1. Mutations can lead to changes in the protein sequence encoded by the DNA.

Mutations: Some nomenclature and considerations


Etymologically, the term mutation means a change or alteration. In genetics, a mutation is a change in the genetic material - DNA sequence - of an organism. By extension, a mutant is the organism in which a mutation has occurred. But what is the change compared to? The answer to this question is that it depends. We can make the comparison with the direct progenitor (cell or organism) ortopatterns seen in a population of the organism in question. It mostly depends on the specific context of the discussion. Since genetic studies often look at a population (or key subpopulations) of individuals, we begin by describing the term "wild-type".

Wild Type vs Mutant

What do we mean by "wild type"? Since the definition can depend on context, this concept is not entirely straightforward. Here are a few examples of definitions you may run into:

Possible meaningsof "wild-type"

  1. An organism having an appearance characteristic of the species in a natural breeding population (i.e. a cheetah's spots and tear-like dark streaks that extend from the eyes to the mouth).
  2. The form or forms of a gene most commonly occurring in nature in agivenspecies.
  3. A phenotype, genotype, or gene that predominates in a natural population of organisms or strain of organisms in contrast to that of natural or laboratory mutant forms.
  4. The normal, as opposed to the mutant gene or allele.
The common thread to all of the definitions listed above is based

on the "norm" for a set of characteristics

with respect to

a specific trait compared to the overall population. In the "Pre-DNA sequencing Age"

species were classified

based on common phenotypes (what they looked like, where they lived, how they behaved, etc.). A "norm"

was established

for the species in question. For example, Crows display a common set of characteristics, they are large, black birds that live in specific regions, eat certain types of food and behave in a certain characteristic way. If we see one, we know


a crow based on these characteristics. If we saw one with a white head, we would think


either it is a different bird (not a crow) or a mutant, a crow that has some alteration from the norm or wild type.

In this class we take what is common about those varying definitions and adopt the idea that "wild type" is


a reference standard against which we can compare members of a population.

Possible NB Discussion Point

If you were assigning wild type traits to describe a dog, what would they be? What is the difference between a mutant trait and variation of a trait in a population of dogs? Is there a wild type for a dog that we could use as a standard? How would webegin tothink about this conceptwith respect todogs?

Figure 2. Mutations can lead to changes in the protein sequence encoded by the DNA that then impact the outward appearance of the organism.

Mutations aresimplychanges from the "wild type", reference or parental sequence for an organism. While the term "mutation" has colloquially negative connotations, wemustremember that change is neither inherently "bad". Mutations (changes in sequences) should not primarilybe thoughtof as "bad" or "good", butrathersimplyas changes and a source of genetic and phenotypic diversity on which evolution by natural selection can occur. Natural selection ultimately determines the long-termfateof mutations. If the mutation confers a selective advantage to the organism, the mutation willbe selectedand may eventually become very common in the population. Conversely, if the mutation is deleterious, natural selection will ensure thatthe mutation will be lostfrom the population. If the mutation is neutral, that is it neither provides a selective advantage or disadvantage, then it may persist in the population. Differentforms ofa gene, including those associated with "wild type" and respective mutants, in a populationare termedalleles.

Consequences of Mutations

For an individual, the consequence of mutations may mean little or it may mean life or death. Some deleterious mutations are null or knock-outmutationswhich result in a loss of function of the gene product. These mutations can arise by a deletion ofthe eitherthe entire gene, a portion of the gene, or by a point mutation in a critical region of the gene that renders the gene product non-functional. These types of mutations are also referred to as loss-of-function mutations. Alternatively, mutations may lead to a modification of an existing function (i.e. the mutation may change the catalytic efficiency of an enzyme, a change in substrate specificity, or a change in structure). In rare cases a mutation may create a new or enhanced function for a gene product;this is often referredto as a gain-of-function mutation.Lastly, mutations may occur in non-coding regions of DNA. These mutations can have a variety of outcomes including altered regulation of gene expression, changes in replication rates or structural properties of DNA and other non-protein associated factors.

Mutations and cancer

Mutations can affect either somatic cells or germ cells. Sometimes mutations occur in DNA repair genes, in effect compromising the cell's ability to fix other mutations that may arise. If,as a resultof mutations in DNA repair genes, many mutations accumulate in a somatic cell, they may lead to problems such as the uncontrolled cell division observed in cancer. Cancers, including forms of pancreatic cancer, colon cancer, and colorectal cancer havebeen associatedwith mutations like these in DNA repair genes. If, by contrast,a mutation in DNA repair occurs in germ cells (sex cells), the mutation willbe passedon to the next generation, asin the case ofdiseases like hemophilia and xeroderma pigmentosa.Inthe case of xerodermapigmentoasindividuals with compromised DNA repair processes become very sensitive to UV radiation. In severe cases these individuals may get severe sun burns with just minutes of exposure to the sun. Nearly half of all children with this condition develop their first skin cancers by age 10.

Consequences of errors in replication, transcription and translation

Something key to think about:

Cells have evolved a variety of ways to make sure DNA errors are both detected and corrected,romproof reading by the various DNA-dependent DNA polymerases, to more complex repair systems. Why did so many mechanisms evolve to repair errors in DNA?By contrast, similarproof-reading mechanisms did NOT evolve for errors in transcription or translation. Why might this be? What would be the consequences of an error in transcription? Would such an error effect the offspring? Would it be lethal to the cell? What about translation? Ask the same questions about the process of translation. What would happen ifthe wrong amino acid was accidentally putinto the growing polypeptide during the translation of a protein? Contrast this with DNA replication.

Mutations as instruments of change

Mutations are how populations can adapt to changing environmental pressures

Mutationsare randomly createdin the genome of every organism, and thisin turncreates genetic diversity and a plethora of different alleles per gene per organism in every population on the planet. If mutations did not occur, and chromosomeswere replicatedand transmitted with 100% fidelity, how would cells and organisms adapt? Whether evolution keeps mutations in a population depends on whether the mutation provides selective advantage, poses some selective cost orisat the very least, not harmful.Indeed, mutationsthat appear neutral may persist in the population for many generations and only be meaningful when a populationis challengedwith a new environmental challenge.At this point,theapparently previously neutral mutations may provide a selective advantage.

Example: Antibiotic resistance

The bacterium E. coli is sensitive to an antibiotic called streptomycin, which inhibits protein synthesis by binding to the ribosome.The ribosomal proteinL12can be mutatedsuch that streptomycin no longer binds to the ribosome and inhibits protein synthesis. Wild type and L12 mutants grow equallywelland the mutation appears to be neutral in theabsence of the antibiotic. In thepresence of the antibioticwild typecells die and L12 mutants survive. This example shows how genetic diversity is important for the population to survive. If mutations did not randomly occur, when the populationis challengedby an environmental event, such as the exposure to streptomycin, the entire population would die. For most populations this becomes a numbers game. If the mutation rate is 10-6 then a population of 107 cells would have 10 mutants; a population of 108 would have 100 mutants, etc.

Uncorrected errors in DNA replication lead to mutation. In this example,an uncorrected error was passedonto a bacterial daughter cell. This error is in a gene that encodes for part of the ribosome. The mutation results in a different final 3D structure of the ribosome protein. While thewildtyperibosome can bind to streptomycin (an antibiotic that will kill the bacterial cell by inhibiting the ribosome function) the mutant ribosome cannot bind to streptomycin.Thisbacteriaisnow resistant to streptomycin.
Source: Bis2A Team original image

An example: Lactate dehydrogenase

Lactate Dehydrogenase (LDH), the enzyme that catalyzes the reduction of pyruvate into lactic acid in fermentation, while virtually every organism has this activity, the corresponding enzyme and therefore gene differs immensely between humans and bacteria.The proteins are clearlyrelated,they perform the same basic function but have a variety of differences, from substrate binding affinities and reaction rates to optimal salt and pH requirements. Each of these attributes havebeen evolutionarily tunedfor each specific organism through multiple rounds of mutation and selection.

Possible NB Discussion Point

We can use comparative DNA sequence analysis to generate hypotheses about the evolutionary relationships between three or more organisms. One way to accomplish this is to compare the DNA or protein sequences of proteins found in each of the organisms we wish to compare. Let us, for example, imagine that we were to compare the sequences of lactate dehydrogenase (LDH) from three different organisms. The schematic below depicts the primary structures of LDH proteins from Organisms A, B, and C. The letters in the center of the proteins' line diagram represent amino acids at a unique position and the proposed differences in each sequences (Attribution:Marc T. Facciotti [original work]). Question: Is Organism C more closely related to Organism A or B? The simplest explanation is that Organism A is the earliest form, a mutation occurred giving rise to Organism B. Over time a second mutation arose in the B lineage to give rise to the enzyme found in Organism C. This is the simplest explanation, however we cannot rule out other possibilities. Can you think of other ways the differentforms ofthe LDH enzyme arose these three organisms?

Real-life Application:

As we have seen in the "Mutations and Mutants" module, changing even one nucleotide can have major effects on the translated product. Read more about an undergraduate's work on point mutations and GMOs here.


induced mutation:

mutationthat results from exposure to chemicals or environmental agents


variation in the nucleotide sequence of a genome

mismatch repair:

type of repair mechanism in whichmismatched bases are removedafter replication

nucleotide excision repair:

type of DNA repair mechanism in which the wrong base, along with a few nucleotides upstream or downstream,are removed


function of DNApolin which it reads the newly added base before adding the next one

point mutation:

mutationthat affects a single base

silent mutation:

mutationthatis not expressed

spontaneous mutation:

mutationthat takes place in the cellsas a resultof chemical reactions taking place naturally without exposure to any external agent

transition substitution:

when a purineis replacedwith a purine or a pyrimidineis replacedwith another pyrimidine

transversion substitution:

when a purineis replacedby a pyrimidine or a pyrimidineis replacedby a purine

2020_SS1_Bis2a_Facciotti_Reading_25 - Biology

Introduction A series of laws, called the laws of thermodynamics, describe how energy is transferred and dispersed in a reaction. We consider two of these. The first law states that the total amount of energy in the universe is constant. This means that energy can’t be created or destroyed in a reaction or process, only transferred. The second law of thermodynamics states the entropy of the universe is always increasing. We describe the general relevance of these two laws and their application in biology.

Laws of Thermodynamics

Thermodynamics is concerned with describing the changes in systems before and after a change. This usually involves a discussion about the energy transfers and its dispersion within the system. In nearly all practical cases, these analyses require that the system and its surroundings be completely described. For instance, when discussing the heating of a pot of water on the stove, the system may includes the stove, the pot, and the water and the environment or surroundings may include everything else. Biological organisms are what are called open systems energy is transferred between them and their surroundings.

1st Law of Thermodynamics The first law of thermodynamics deals with the total amount of energy in the universe. It states that this total amount of energy is constant. In other words, there has always been, and always will be, exactly the same amount of energy in the universe. According to the first law of thermodynamics, energy may be transferred from place to place (module 4.0), but it cannot be created or destroyed. The transfers of energy take place around us all the time. Light bulbs transfer energy an electrical power station into heat and photons to produce light. Gas stoves transfer energy stored in the bonds of chemical compounds into heat and light. Heat, by the way, is the amount of energy transferred from one system to another because of a temperature difference. Plants perform one of the most biologically useful energy transfers on earth: they transfer energy in the photons of sunlight into the chemical bonds of organic molecules. In every one of these cases energy is neither made or destroyed and we must try to account for all of the energy when we examine some of these reactions.

1st Law and the Energy Story The first law of thermodynamics is deceptively simple. Students often understand that energy cannot be created or destroyed. Yet, when describing an energy story of a process they often make the mistake of saying things such as "energy is produced from the transfer of electrons from atom A to atom B". While most of us will understand the point the student is trying to make, the wrong words are being used. Energy is not made or produced, it is simply transferred. To be consistent with the first law, when telling an energy story, make sure that you try to explicitly track all of the places that ALL of the energy in the system at the start of a process goes by the end of a process.

2nd Law of Thermodynamics An important concept in physical systems is that of entropy . Entropy is related to the with the ways in which energy can be distributed or dispersed within the particles of a system. The 2nd Law of Thermodynamics states that entropy is always increasing in a system AND its surroundings (everything outside the system). This idea helps explain the directionality of natural phenomena. In general the notion is that the directionality comes from the tendency for energy in a system to move towards a state of maximal dispersion. The 2nd law, therefore, means that in any transformation we should look for an overall increase in entropy (or dispersion of energy), somewhere. A idea that is associated with increased dispersion of energy in a system or its surroundings is that as dispersion increases the ability of the energy to be directed towards work decreases.

There will be many examples of where the entropy of a system decreases. To be consistent with the second law, however, we must try to find something else (likely a closely connected system in the surroundings) that must compensate for the "local" decrease in entropy with an equal or greater increase in entropy.

The entropy of a system can increase when:(a) it gains energy(b) a change of state occurs from solid to liquid to gas (c) mixing of substances occurs(d) the number of particles increases during a reaction.

An increase in disorder can happen in different ways. An ice cube melting on a hot sidewalk is one example. Here, ice is displayed as a snowflake, with organized, structured water molecules forming the snowflake. Over time, the snowflake will melt into a pool of disorganized, freely moving water molecules.

If we consider the first and second laws together (the conservation of energy and the need for entropy to increase if a process happens) we come to a useful conclusion. In any process where energy is transferred or redistributed within a system entropy must increase. This increase in entropy is related to how "useful" the energy is to do work (generally becoming less available as entropy increases). So, we can conclude that in any transformation we consider that while all of the energy must be conserved the required change increase in entropy means that some of the energy will become distributed in a way that makes it less useful for work. In many cases, particularly in biology, some of the increase in entropy can be tracked to a transfer of energy to heat in the environment.

Free Energy

If we want to describe transformations, therefore, it is useful to have a measure of (a) how much energy is in a system and (b) the dispersal of that energy within the system and of course how these change between the start and end of a process. The concept of free energy , often referred to as Gibbs free energy or free enthalpy (abbreviated with the letter G), in some sense does just that. Gibbs free energy can be defined in several interconvertible ways, but a useful one in the context of biology is the enthalpy (internal energy) of a system minus the entropy of the system scaled by the temperature. The difference in free energy when a process takes place is often reported in terms of the change (delta) of enthalpy (internal energy) denoted H, minus the temperature scaled change (delta) in entropy, denoted S. See the equation below.

The Gibbs energy is often interpreted as the amount of energy available to do useful work. With a bit of handwaving we can interpret this by invoking the idea presented above that the dispersion of energy (required by the Second Law) associated with a positive change in entropy somehow renders some of the energy that is transferred less useful to do work. One can say that this is reflected in part in the T∆S term of the Gibbs equation.

To provide a basis for fair comparisons of changes in Gibbs free energy amongst different biological transformations or reactions the free energy change of a reaction is measured under a set of common standard experimental conditions. The resulting standard free energy change of a chemical reaction is expressed as an amount of energy per mole of the reaction product (either in kilojoules or kilocalories, kJ/mol or kcal/mol 1 kJ = 0.239 kcal) when measured at a standard pH, temperature, and pressure conditions. Standard pH, temperature, and pressure conditions are generally calculated at pH 7.0, 25 degrees Celsius, and 100 kilopascals (1 atm pressure), respectively. It is important to note that cellular conditions vary considerably from these standard conditions, and so actual ∆G inside a cell will differ considerably from those calculated under standard conditions.

Endergonic and Exergonic Reactions

Reactions that have a ∆G < 0 means that the products of the reaction have less free energy than the reactants. Since ∆G is the difference between the enthalpy and entropy changes in a reaction a net negative ∆G can arise in different ways. The left panel of Figure 2 below shows a common graphical representation an exergonic reaction. Free energy is plotted on the y-axis and the x-axis in arbitrary units shows model for the progress of a reaction. This type of graph is called a reaction coordinate diagram. In the case of an exergonic reaction depicted below the chart indicates two key things: (1) the difference between the free energy of the reactants and products is negative and (2) the progress of the reaction requires some input of free energy (shown as an energy hill). This graph does not tell us how the energy in the system was redistributed, only that the difference between enthalpy and entropy is negative. Reactions that have a negative ∆G and consequently are termed exergonic reactions . These reactions are occur spontaneously. Understanding which chemical reactions are spontaneous is extremely useful for biologists that are trying to understand whether a reaction is likely to "go" or not.

It is important to note that the term spontaneous - in the context of thermodynamics - does NOT imply anything about how fast the reaction proceeds. The change in free energy only describes the difference between beginning and end states NOT how fast that transition takes. This is somewhat contrary to the everyday use of the term which usually carries the implicit understanding that something happens quickly. As an example, the oxidation/rusting of iron is a spontaneous reaction. However, an iron nail exposed to air does not rust instantly - it may take years.

A chemical reaction with a positive ∆G means that the products of the reaction have a higher free energy than the reactants (see the right panel of Figure 2). These chemical reactions are called endergonic reactions , and they are NOT spontaneous. An endergonic reaction will not take place on its own without the transfer of energy into the reaction or increase of entropy somewhere else.

Exergonic and endergonic reactions result in changes in Gibbs free energy. In exergonic reaction the free energy of the products is lower than that of the reactants meanwhile in endergonic the free energy of the products is higher than that of the reactants.

The building of complex molecules, such as sugars, from simpler ones is an anabolic process and is endergonic. On the other hand, the catabolic process, such as the breaking down of sugar into simpler molecules is generally exergonic. Like the example of rust above, while the breakdown of biomolecules is generally spontaneous these reactions don’t necessarily occur instantaneously(quickly). Figure 3 shows some other examples of endergonic and exergonic reactions. But remember, the terms endergonic and exergonic only refer to the difference in free energy between the products and reactants - they don't tell you about the rate of reaction (how fast it happens). The issue of rate will be discussed in later sections.

Shown are some examples of endergonic processes (ones with positive changes in free energy between products and reactants) and exergonic processes (ones with negative changes in free energy between products and reactants). These include (a) a compost pile decomposing, (b) a chick hatching from a fertilized egg, (c) sand art being destroyed, and (d) a ball rolling down a hill. (credit a: modification of work by Natalie Maynor credit b: modification of work by USDA credit c: modification of work by “Athlex”/Flickr credit d: modification of work by Harry Malsch)

An important concept in the study of metabolism and energy is that of chemical equilibrium. Most chemical reactions are reversible. They can proceed in both directions, often transferring energy into their environment in one direction, and transferring energy in from the environment in the other direction. The same is true for the chemical reactions involved in cell metabolism, such as the breaking down and building up of proteins into and from individual amino acids, respectively. Reactants within a closed system will undergo chemical reactions in both directions until a state of equilibrium is reached. This state of equilibrium is one of the lowest possible free energy and a state of maximal entropy. Equilibrium in a chemical reaction, is the state in which both reactants and products are present in concentrations which have no further tendency to change with time. Usually, this state results when the forward reaction proceeds at the same rate as the reverse reaction. NOTE THIS LAST STATEMENT! Equilibrium means that the relative concentrations of reactants and products is not changing in time BUT it does NOT mean that there is no interconversion between substrates and products - it just means that when reactant is converted to product that product is converted to reactant at an equal rate.

Either a rebalancing of substrate of product concentrations (by adding or removing substrate or product) or a positive change in free energy, typically by the transfer of energy from outside the reaction, is required to move a reaction out of a state of equilibrium. In a living cell, most chemical reactions do not reach a state of equilibrium - this would require that they reach their lowest free energy state. Energy is therefore required to keep biological reactions out of their equilibrium state. In this way, living organisms are in a constant energy-requiring, uphill battle against equilibrium and entropy.

At equilibrium, do not think of a static unchanging system. Instead, picture molecules moving , in equal amounts from one area to another. Here, at equilibrium, molecules are still moving from left to right and right to left. The net movement however, is equal. There will still be about 15 molecules in each side of this flask once equilibrium is reached.