Berg J.M., Tymoczko J.L., Stryer L. Biochemistry
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I. The Molecular Design of Life 7. Exploring Evolution 7.3. Examination of Three-Dimensional Structure Enhances Our Understanding of Evolutionary Relationships
Figure 7.16. Sequence Alignment of Internal Repeats. (A) An alignment of the sequences of the two repeats of the
TATA-box-binding protein. The amino-terminal repeat is shown in green and the carboxyl-terminal repeat in blue. (B)
Structure of the TATA-box-binding protein. The amino-terminal domain is shown in green and the carboxyl-terminal
domain in blue.
I. The Molecular Design of Life 7. Exploring Evolution 7.3. Examination of Three-Dimensional Structure Enhances Our Understanding of Evolutionary Relationships
Figure 7.17. Convergent Evolution of Protease Active Sites. The relative positions of the three key residues shown are
nearly identical in the active sites of the serine proteases chymotrypsin and subtilisin.
I. The Molecular Design of Life 7. Exploring Evolution 7.3. Examination of Three-Dimensional Structure Enhances Our Understanding of Evolutionary Relationships
Figure 7.18. Structures of Chymotrypsin and Subtilisin. The β strands are shown in yellow and α helices in blue. The
overall structures are quite dissimilar, in stark contrast with the active sites, shown at the top of each structure.
I. The Molecular Design of Life 7. Exploring Evolution 7.3. Examination of Three-Dimensional Structure Enhances Our Understanding of Evolutionary Relationships
Figure 7.19. Comparison of RNA Sequences. (A) A comparison of sequences in a part of ribosomal RNA taken from a
variety of species. (B) The implied secondary structure. Bars indicate positions at which Watson-Crick base-pairing is
completely conserved in the sequences shown, whereas dots indicate positions at which Watson-Crick base-pairing is
conserved in most cases.
I. The Molecular Design of Life 7. Exploring Evolution 7.3. Examination of Three-Dimensional Structure Enhances Our Understanding of Evolutionary Relationships
Figure 7.20. An Evolutionary Tree for Globins. The branching structure was deduced by sequence comparison,
whereas the results of fossil studies provided the overall time scale showing when divergence occurred.
I. The Molecular Design of Life 7. Exploring Evolution
7.4. Evolutionary Trees Can Be Constructed on the Basis of Sequence Information
Conceptual Insights, Sequence Analysis, offers insights into evolutionary
trees through interactive analysis of simulated evoluntionary histories.
The observation that homology is often manifested as sequence similarity suggests that the evolutionary pathway relating
the members of a family of proteins may be deduced by examination of sequence similarity. This approach is based on
the notion that sequences that are more similar to one another have had less evolutionary time to diverge from one
another than have sequences that are less similar. This method can be illustrated by using the three globin sequences in
Figures 7.10 and 7.12, as well as the sequence for the human hemoglobin β chain. These sequences can be aligned with
the additional constraint that gaps, if present, should be at the same positions in all of the proteins. These aligned
sequences can be used to construct an evolutionary tree in which the length of the branch connecting each pair of
proteins is proportional to the number of amino acid differences between the sequences (Figure 7.20).
Such comparisons reveal only the relative divergence times
for example, that myoglobin diverged from hemoglobin
twice as long ago as the α chain diverged from the β chain. How can we estimate the approximate dates of gene
duplications and other evolutionary events? Evolutionary trees can be calibrated by comparing the deduced branch points
with divergence times determined from the fossil record. For example, the duplication leading to the two chains of
hemoglobin appears to have occurred 350 million years ago. This estimate is supported by the observation that jawless
fish such as the lamprey, which diverged from bony fish approximately 400 million years ago, contain hemoglobins built
from a single type of subunit (Figure 7.21).
These methods can be applied to both relatively modern and very ancient molecules, such as the ribosomal RNAs that
are found in all organisms. Indeed, it was such an RNA sequence analysis that led to the suggestion that Archaea are a
distinct group of organisms that diverged from Bacteria very early in evolutionary history.
I. The Molecular Design of Life 7. Exploring Evolution 7.4. Evolutionary Trees Can Be Constructed on the Basis of Sequence Information
Figure 7.21. The Lamprey. A jawless fish whose ancestors diverged from bony fish approximately 400 million years
ago, the lamprey contains hemoglobin molecules that contain only a single type of polypeptide chain. [Brent P. Kent.]
I. The Molecular Design of Life 7. Exploring Evolution
7.5. Modern Techniques Make the Experimental Exploration of Evolution Possible
Two techniques of biochemistry have made it possible to examine the course of evolution more directly and not simply
by inference. The polymerase chain reaction (Section 6.1.5) allows the direct examination of ancient DNA sequences,
releasing us, at least in some cases, from the constraints of being able to examine existing genomes from living
organisms only. Molecular evolution may be investigated through the use of combinatorial chemistry, the process of
producing large populations of molecules en masse and selecting for a biochemical property. This exciting process
provides a glimpse into the types of molecules that may have existed in the RNA world.
7.5.1. Ancient DNA Can Sometimes Be Amplified and Sequenced
The tremendous chemical stability of DNA (Section 2.2.7) makes the molecule well suited to its role as the storage site
of genetic information. So stable is the molecule that samples of DNA have survived for many thousands of years under
appropriate conditions. With the development of PCR methods, such ancient DNA can sometimes be amplified and
sequenced. This approach has been applied to mitochondrial DNA from a Neanderthal fossil estimated at between
30,000 and 100,000 years of age found near Düsseldorf, Germany, in 1856. Investigators managed to identify a total of
379 bases of sequence. Comparison with a number of the corresponding sequences from Homo sapiens revealed between
22 and 36 substitutions, considerably fewer than the average of 55 differences between human beings and chimpanzees
over the common bases in this region. Further analysis suggested that the common ancestor of modern human beings and
Neanderthals lived approximately 600 million years ago. An evolutionary tree constructed by using these and other data
revealed that the Neanderthal was not an intermediate between chimpanzees and human beings but, instead, was an
evolutionary "dead end" that became extinct (Figure 7.22).
Note that earlier studies describing the sequencing of much more ancient DNA such as that found in insects trapped in
amber appear to have been flawed; contaminating modern DNA was responsible for the sequences determined.
Successful sequencing of ancient DNA requires sufficient DNA for reliable amplification and the rigorous exclusion of
all sources of contamination.
7.5.2. Molecular Evolution Can Be Examined Experimentally
Evolution requires three processes: (1) the generation of a diverse population, (2) the selection of members based on
some criterion of fitness, and (3) reproduction to enrich the population in more fit members (Section 2.2). Nucleic acid
molecules are capable of undergoing all three processes in vitro under appropriate conditions. The results of such studies
enable us to glimpse how evolutionary processes might have generated catalytic activities and specific binding
abilities
important biochemical functions in all living systems.
A diverse population of nucleic acid molecules can be synthesized in the laboratory by the process of combinatorial
chemistry, which rapidly produces large populations of a particular type of molecule such as a nucleic acid. A population
of molecules of a given size can be generated randomly so that many or all possible sequences are present in the mixture.
When an initial population has been generated, it is subjected to a selection process that isolates specific molecules with
desired binding or reactivity properties. Finally, molecules that have survived the selection process are allowed to
reproduce through the use of PCR; primers are directed toward specific sequences included at the ends of each member
of the population.
As an example of this approach, consider an experiment that set a goal of creating an RNA molecule capable of binding
adenosine triphosphate and related nucleotides. Such ATP-bonding molecules are of interest because they might have
been present in the RNA world. An initial population of RNA molecules 169 nucleotides long was created; 120 of the
positions differed randomly, with equimolar mixtures of adenine, cytosine, guanine, and uracil. The initial synthetic pool
that was used contained approximately 10
14
RNA molecules. Note that this number is a very small fraction of the total
possible pool of random 120-base sequences. From this pool, those molecules that bound to ATP, which had been
immobilized on a column, were selected (Figure 7.23).
The collection of molecules that were bound well by the ATP affinity column was allowed to reproduce by reverse
transcription into DNA, amplification by PCR, and transcription back into RNA. This new population was subjected to
additional rounds of selection for ATP-binding activity. After eight generations, members of the selected population
were characterized by sequencing. Seventeen different sequences were obtained, 16 of which could form the structure
shown in Figure 7.24. Each of these molecules bound ATP with high affinity, as indicated by dissociation constants less
than 50 µ M.
The folded structure of the ATP-binding region from one of these RNAs was determined by nuclear magnetic resonance
(Section 4.5.1) methods (Figure 7.25). As expected, this 40-nucleotide molecule is composed of two Watson-Crick base-
paired helical regions separated by an 11-nucleotide loop. This loop folds back on itself in an intricate way to form a
deep pocket into which the adenine ring can fit. Thus, a structure was generated, or evolved, that was capable of a
specific interaction.
I. The Molecular Design of Life 7. Exploring Evolution 7.5. Modern Techniques Make the Experimental Exploration of Evolution Possible
Figure 7.22. Placing Neanderthal on an Evolutionary Tree. Comparison of DNA sequences revealed that Neanderthal
is not on the line of direct descent leading to Homo sapiens but, instead, branched off earlier and then became extinct.
I. The Molecular Design of Life 7. Exploring Evolution 7.5. Modern Techniques Make the Experimental Exploration of Evolution Possible
Figure 7.23. Evolution in the Laboratory. A collection of RNA molecules of random sequences is synthesized by
combinatorial chemistry. This collection is selected for the ability to bind ATP by passing the RNA through an ATP
affinity column (Section 4.1.3). The ATP-binding RNA molecules are released from the column by washing with excess
ATP, and replicated. The process of selection and replication is then repeated several times. The final RNA products
with significant ATP-binding ability are isolated and characterized.
I. The Molecular Design of Life 7. Exploring Evolution 7.5. Modern Techniques Make the Experimental Exploration of Evolution Possible
Figure 7.24. A Conserved Secondary Structure. The secondary structure shown is common to RNA molecules
selected for ATP binding.
I. The Molecular Design of Life 7. Exploring Evolution 7.5. Modern Techniques Make the Experimental Exploration of Evolution Possible
Figure 7.25. RNA Molecule Binds ATP. (A) The Watson-Crick base-pairing pattern, (B) the folding pattern, and (C) a
surface representation of an RNA molecule selected to bind adenosine nucleotides. The bound ATP is shown in part B,
and the binding site is revealed as a deep pocket in part C.
I. The Molecular Design of Life 7. Exploring Evolution
Summary
Homologs Are Descended from a Common Ancestor
Exploring evolution biochemically often means searching for homology, or relatedness, between molecules, because
homologous molecules, or homologs, evolved from a common ancestor. Paralogs are homologous molecules that are
found in one species and have acquired different functions through evolutionary time. Orthologs are homologous
molecules that are found in different species and have similar or identical functions.
Statistical Analysis of Sequence Alignments Can Detect Homology
Protein and nucleic acid sequences are two of the primary languages of biochemistry. Sequence-alignment methods are
the most powerful tools of the evolutionary detective. Sequences can be aligned to maximize their similarity, and the
significance of these alignments can be judged by statistical tests. The detection of a statistically significant alignment
between two sequences strongly suggests that two sequences are related by divergent evolution from a common ancestor.
The use of substitution matrices makes the detection of more distant evolutionary relationships possible. Any sequence
can be used to probe sequence databases to identify related sequences present in the same organism or in other
organisms.
Examination of Three-Dimensional Structure Enhances Our Understanding of
Evolutionary Relationships
The evolutionary kinship between proteins may be even more profoundly evident in the conserved three-dimensional
structures. The analysis of three-dimensional structure in combination with analysis of especially conserved sequences
has made it possible to determine evolutionary relationships that are not possible to detect by other means. Sequence-
comparison methods can also be used to detect imperfectly repeated sequences within a protein, indicative of linked
similar domains.
Evolutionary Trees Can Be Constructed on the Basis of Sequence Information
Construction of an evolutionary tree based on sequence comparisons revealed approximate times for the gene duplication
events separating myoglobin and hemoglobin as well as the α and β subunits of hemoglobin. Evolutionary trees based on
sequences can be compared to those based on fossil records.
Modern Techniques Make the Experimental Exploration of Evolution Possible
The exploration of evolution can also be a laboratory science. In favorable cases, PCR amplification of well-preserved
samples allows the determination of the nucleotide sequences from extinct organisms. Sequences so determined can help
authenticate aspects of an evolutionary tree constructed by other means. Molecular evolutionary experiments performed
in the test tube can examine how molecules such as ligand-binding RNA molecules might have been generated.
Key Terms
homolog
paralog
ortholog
sequence alignment
conservative substitution
substitution matrix
sequence template
self-diagonal plot
divergent evolution
convergent evolution
evolutionary tree
combinatorial chemistry
I. The Molecular Design of Life 7. Exploring Evolution
Problems
1.
What's the score? Using the identity-based scoring system (Section 7.2), calculate the score for the following
alignment. Do you think the score is statistically significant?
See answer
2.
Sequence and structure. A comparison of the aligned amino acid sequences of two proteins each consisting of 150
amino acids reveals them to be only 8% identical. However, their three-dimensional structures are very similar. Are
these two proteins related evolutionarily? Explain.
See answer
3.
It depends on how you count. Consider the following two sequence alignments:
Which alignment has a higher score if the identity-based scoring system (Section 7.2) is used? Which alignment has
a higher score if the Blosum-62 substitution matrix (Figure 7.9) is used?
See answer
4.
Discovering a new base pair. Examine the ribosomal RNA sequences in Figure 7.19. In sequences that do not
contain Watson-Crick base pairs, what base tends to be paired with G? Propose a structure for your new base pair.
See answer
5.
Overwhelmed by numbers. Suppose that you wish to synthesize a pool of RNA molecules that contain all four bases
at each of 40 positions. How much RNA must you have in grams if the pool is to have at least a single molecule of
each sequence? The average molecular weight of a nucleotide is 330 g mol
-1
.
See answer
6.
Form follows function.The three-dimensional structure of biomolecules is more conserved evolutionarily than is
sequence. Why is this the case?
See answer
7.
Shuffling. Using the identity-based scoring system (Section 7.2), calculate the alignment score for the alignment of
the following two short sequences:
Generate a shuffled version of sequence 2 by randomly reordering these 10 amino acids. Align your shuffled
sequence with sequence 1 without allowing gaps, and calculate the alignment score between sequence 1 and your
shuffled sequence.
See answer
8.
Interpreting the score. Suppose that the sequences of two proteins each with 200 amino acids are aligned and that
the percentage of identical residues has been calculated. How would you interpret each of the following results in
regard to the possible divergence of the two proteins from a common ancestor?
(a) 80%, (b) 50%, (c) 20%, (d) 10%
See answer
9.
A set of three. The sequences of three proteins (A, B, and C) are compared with one another, yielding the following
levels of identity:
Assume that the sequence matches are distributed relatively uniformly along each aligned sequence pair. Would you
expect protein A and protein C to have similar three-dimensional structures? Explain.
See answer
10.
RNA alignment. Sequences of an RNA fragment from five species have been determined and aligned. Propose a
likely secondary structure for these fragments.
See answer
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