CIS 595 Bioinformatics

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CIS 595 Bioinformatics

Lecture 2


Based on the book chapter:

Hunter, L., Molecular Biology for Computer Scientists. Artificial
Intelligence for Molecular Biology, Ed. L. Hunter, pp. 1
-
46, AAAI
Press, 1993.


Contains figures taken from


Molecular Biology of the Cell. 4th ed.

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P.,

New York: Garland Publishing; 2002.:

Available from

http://www.ncbi.nlm.nih.gov/books/

DNA


All of the genetic information in any living
creature is stored in deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA)


DNA
-

polymers of four simple nucleic acid units,
called nucleotides.


Four nucleotides (or bases):


Purines: adenine (A) and guanine (G)


Pyrimidines: cytosine (C) and thymine (T) (uracil: U)


Each nucleotide consists of


one of two base molecules (a purine or a pyrimidine)


sugar (ribose in RNA and deoxyribose DNA)


one or more phosphate groups.

Nucleic Acids

Figure 4
-
3. DNA and its building blocks.

DNA is made of four types of nucleotides, which are linked covalently into a polynucleotide chain (a DNA
strand) with a sugar
-
phosphate backbone from which the bases (A, C, G, and T) extend. A DNA molecule is composed of two DNA stra
nds held
together by hydrogen bonds between the paired bases. The
arrowheads

at the ends of the DNA strands indicate the polarities of the two strands,
which run antiparallel to each other in the DNA molecule. In the diagram at the bottom left of the figure, the DNA molecule i
s s
hown straightened out; in
reality, it is twisted into a double helix, as shown on the right. For details, see
Figure 4
-
5


Genes and Genetic Code


Genetic information can be stored in several
DNA molecules (chromosomes)


In diploids, each chromosome contains two similar
DNA molecules, one from each parent


Bacteria have a single circular chromosome


Primary role of DNA: carry the encoding of the
proteins


Each triplet of nucleotides (called codon) codes for a
single AA


Stop codons define the end of a protein


Open reading frame: long enough stretch of codons
without intervening stop codons


There are 6 different ways to start parsing a segment
of DNA


Chromosomes

Figure 4
-
14. Two closely related species of deer with very different chromosome numbers.

In the evolution of the Indian
muntjac, initially separate chromosomes fused, without having a major effect on the animal. These two species have roughly
the same number of genes. (Adapted from M.W. Strickberger, Evolution, 3rd edition, 2000, Sudbury, MA: Jones & Bartlett
Publishers

Genetic Code

Figure 6
-
50. The genetic code.

The standard one
-
letter abbreviation for each amino acid is presented below its three
-
letter
abbreviation (see
Panel 3
-
1
, pp. 132 133, for the full name of each amino acid and its structure). By convention, codons are
always written with the 5
-
terminal nucleotide to the left. Note that most amino acids are represented by more than one codon,
and that there are some regularities in the set of codons that specifies each amino acid. Codons for the same amino acid tend

to

contain the same nucleotides at the first and second positions, and vary at the third position. Three codons do not specify a
ny
amino acid but act as termination sites (stop codons), signaling the end of the protein
-
coding sequence. One codon AUG acts
both as an initiation codon, signaling the start of a protein
-
coding message, and also as the codon that specifies methionine

Organization of Genes

Figure 4
-
15. The organization of genes on a human chromosome.

(A) Chromosome 22, one of the smallest human chromosomes, contains 48
×

106
nucleotide pairs and makes up approximately 1.5% of the entire human genome. Most of the left arm of chromosome 22 consists o
f s
hort repeated
sequences of DNA that are packaged in a particularly compact form of chromatin (heterochromatin), which is discussed later in

th
is chapter. (B) A tenfold
expansion of a portion of chromosome 22, with about 40 genes indicated. Those in
dark brown

are known genes and those in
light brown

are predicted
genes. (C) An expanded portion of (B) shows the entire length of several genes. (D) The intron
-
exon arrangement of a typical gen
e is shown after a
further tenfold expansion. Each exon
(red)

codes for a portion of the protein, while the DNA sequence of the introns
(gray)

is relatively unimportant. The
entire human genome (3.2
×

109 nucleotide pairs) is distributed over 22 autosomes and 2 sex chromosomes (see
Figures 4
-
10

and
4
-
11
). The term
human genome sequence

refers to the complete nucleotide sequence of DNA in these 24 chromosomes. Being diploid, a human somatic cell therefore
contains roughly twice this amount of DNA. Humans differ from one another by an average of one nucleotide in every thousand,
and

a wide variety of
humans contributed DNA for the genome sequencing project. The published human genome sequence is therefore a composite of man
y i
ndividual
sequences. (Adapted from International Human Genome Sequencing Consortium,
Nature

409:860 921, 2001

Reading Frames

Figure 6
-
51. The three possible reading frames in protein synthesis.

In the process of translating a nucleotide sequence
(blue)

into an amino acid sequence
(green),

the sequence of nucleotides in an mRNA molecule is read from the 5 to the 3 end
in sequential sets of three nucleotides. In principle, therefore, the same RNA sequence can specify three completely differen
t
amino acid sequences, depending on the reading frame. In reality, however, only one of these reading frames contains the actu
al
message

Transcription and Translation


Steps in mapping from DNA sequence to
folded protein:


Transcription
: from DNA segment to
messenger RNA (mRNA)


Translation
: from mRNA to protein


Post
-
translational modification
: transformation
of a protein into its functional form (i.e. by
attaching various chemical groups)


From DNA to Protein

Figure 6
-
21. Summary of the steps leading from gene to protein in eucaryotes and bacteria.

The final level of a protein in the cell depends on the
efficiency of each step and on the rates of degradation of the RNA and protein molecules. (A) In eucaryotic cells the RNA mol
ecu
le produced by
transcription alone (sometimes referred to as the primary transcript) would contain both coding (exon) and noncoding (intron)

se
quences. Before it can be
translated into protein, the two ends of the RNA are modified, the introns are removed by an enzymatically catalyzed RNA spli
cin
g reaction, and the
resulting mRNA is transported from the nucleus to the cytoplasm. Although these steps are depicted as occurring one at a time
, i
n a sequence, in reality
they are coupled and different steps can occur simultaneously. For example, the RNA cap is added and splicing typically begin
s b
efore transcription has
been completed. Because of this coupling, complete primary RNA transcripts do not typically exist in the cell. (B) In procary
ote
s the production of mRNA
molecules is much simpler. The 5 end of an mRNA molecule is produced by the initiation of transcription by RNA polymerase, a
nd
the 3 end is produced
by the termination of transcription. Since procaryotic cells lack a nucleus, transcription and translation take place in a co
mmo
n compartment. In fact,
translation of a bacterial mRNA often begins before its synthesis has been completed

E.Coli Example

Figure 7
-
33. The clustered genes in
E. coli

that code for enzymes that manufacture the amino acid tryptophan.

These
five genes are transcribed as a single mRNA molecule, a feature that allows their expression to be controlled coordinately.
Clusters of genes transcribed as a single mRNA molecule are common in bacteria. Each such cluster is called an operon

Transcription


Steps:


Binding of RNA polymerase to promoter region of
DNA


Promoter


DNA region upstream and near a protein coding
region


Creation of a complementary strand of RNA


Contains introns and exons


Ends with 250 or more adenosines (poly
-
A tail)


Splicing our introns and connecting exons using
spliceosome to create mRNA


mRNA is transported to the cytoplasm to bind with a
ribosome

DNA Transcription

Figure 6
-
8. DNA is transcribed by the enzyme RNA polymerase.

The RNA polymerase
(pale blue)

moves stepwise along the DNA, unwinding the
DNA helix at its active site. As it progresses, the polymerase adds nucleotides (here,
small "T" shapes)

one by one to the RNA chain at the
polymerization site using an exposed DNA strand as a template. The RNA transcript is thus a single
-
stranded complementary copy o
f one of the two
DNA strands. The polymerase has a rudder (see
Figure 6
-
11
) that displaces the newly formed RNA, allowing the two strands of DNA behind the
polymerase to rewind. A short region of DNA/RNA helix (approximately nine nucleotides in length) is therefore formed only tra
nsi
ently, and a "window"
of DNA/RNA helix therefore moves along the DNA with the polymerase. The incoming nucleotides are in the form of ribonucleosid
e t
riphosphates (ATP,
UTP, CTP, and GTP), and the energy stored in their phosphate
-
phosphate bonds provides the driving force for the polymerization r
eaction (see
Figure
5
-
4
). (Adapted from a figure kindly supplied by Robert Landick

Transcription Initiation

Figure 6
-
16. Initiation of transcription of a eucaryotic gene by RNA polymerase II.

To begin
transcription, RNA polymerase requires a number of general transcription factors (called TFIIA, TFIIB,
and so on). (A) The promoter contains a DNA sequence called the TATA box, which is located 25
nucleotides away from the site at which transcription is initiated. (B) The TATA box is recognized and
bound by transcription factor TFIID, which then enables the adjacent binding of TFIIB (C). For
simplicity the DNA distortion produced by the binding of TFIID (see
Figure 6
-
18
) is not shown. (D)
The rest of the general transcription factors, as well as the RNA polymerase itself, assemble at the
promoter. (E) TFIIH then uses ATP to pry apart the DNA double helix at the transcription start point,
allowing transcription to begin. TFIIH also phosphorylates RNA polymerase II, changing its
conformation so that the polymerase is released from the general factors and can begin the
elongation phase of transcription. As shown, the site of phosphorylation is a long C
-
terminal
polypeptide tail that extends from the polymerase molecule. The assembly scheme shown in the
figure was deduced from experiments performed
in vitro
, and the exact order in which the general
transcription factors assemble on promoters in cells is not known with certainty. In some cases, the
general factors are thought to first assemble with the polymerase, with the whole assembly
subsequently binding to the DNA in a single step. The general transcription factors have been highly
conserved in evolution; some of those from human cells can be replaced in biochemical experiments
by the corresponding factors from simple yeasts

Transcription
Initiation

Figure 7
-
43. Activation of transcription initiation in eucaryotes by
recruitment of the eucaryotic RNA polymerase II holoenzyme complex.

(A)
An activator protein bound in proximity to a promoter attracts the holoenzyme
complex to the promoter. According to this model, the holoenzyme (which contains
over 100 protein subunits) is brought to the promoter separately from the general
transcription factors TFIID and TFIIA. The "broken" DNA in this and subsequent
figures indicates that this portion of the DNA molecule can be very long and of
variable length. (B) Diagram of an
in vivo

experiment whose outcome supports the
holoenzyme recruitment model for gene activator proteins. The DNA
-
binding
domain of a protein has been fused directly to a protein component of the
mediator, a 20
-
subunit protein complex which is part of the holoenzyme complex,
but which is easily dissociable from the remainder of the holoenzyme. When the
binding site for the hybrid protein is experimentally inserted near a promoter,
transcription initiation is strongly increased. In this experiment, the "activation
domain" of the activator (see
Figure 7
-
42
) has been omitted, suggesting that an
important function of the activation domain is simply to interact with the RNA
polymerase holoenzyme complex and thereby aid in its assembly at the promoter.
The ability of gene activator proteins to recruit the transcription machinery to
promoters has also been demonstrated directly, using chromatin
immunoprecipitation (see
Figure 7
-
32
). DNA
-
bound activator proteins typically
increase the rate of transcription by up to 1000
-
fold, which is consistent with a
relatively weak and nonspecific interaction between the activator and the
holoenzyme (a 1000
-
fold change in affinity corresponds to a change in D
G

of ~4
kcal/mole, which could be accounted for by just a few weak, noncovalent bonds

RNA Splicing

Figure 6
-
26. The RNA splicing reaction.

(A) In the first step, a specific adenine nucleotide in the intron sequence (indicated in
red)

attacks the 5 splice site and cuts the sugar
-
phosphate backbone of the RNA at this point. The cut 5 end of the intron
becomes covalently linked to the adenine nucleotide, as shown in detail in (B), thereby creating a loop in the RNA molecule.
The released free 3
-
OH end of the exon sequence then reacts with the start of the next exon sequence, joining the two exons
together and releasing the intron sequence in the shape of a
lariat.

The two exon sequences thereby become joined into a
continuous coding sequence; the released intron sequence is degraded in due course

Alternative Splicing

Figure 6
-
27. Alternative splicing of the a
-
tropomyosin gene from rat.

a
-
tropomyosin is a coiled
-
coil protein (see
Figure 3
-
11
)
that regulates contraction in muscle cells. The primary transcript can be spliced in different ways, as indicated in the figu
re,

to
produce distinct mRNAs, which then give rise to variant proteins. Some of the splicing patterns are specific for certain type
s o
f
cells. For example, the a
-
tropomyosin made in striated muscle is different from that made from the same gene in smooth
muscle. The arrowheads in the top part of the figure demark the sites where cleavage and poly
-
A addition can occur

Translation


Ribosome


a complex combination of RNAs
and proteins that produces a protein from mRNA


Transfer
-
RNA (tRNA)


critical for translation.
They have


an anti
-
codon (that binds to codon) near one end


The corresponding AA on the other end


After translation and post
-
translation
modification, the protein is transported by


Passive diffusion


And active transport mechanism

mRNA Translation

Figure 6
-
65. Translating an mRNA molecule.

Each amino acid added to the growing end of a polypeptide chain is
selected by complementary base
-
pairing between the anticodon on its attached tRNA molecule and the next codon
on the mRNA chain. Because only one of the many types of tRNA molecules in a cell can base
-
pair with each
codon, the codon determines the specific amino acid to be added to the growing polypeptide chain. The three
-
step
cycle shown is repeated over and over during the synthesis of a protein. An aminoacyl
-
tRNA molecule binds to a
vacant A
-
site on the ribosome in step 1, a new peptide bond is formed in step 2, and the mRNA moves a distance of
three nucleotides through the small
-
subunit chain in step 3, ejecting the spent tRNA molecule and "resetting" the
ribosome so that the next aminoacyl
-
tRNA molecule can bind. Although the figure shows a large movement of the
small ribosome subunit relative to the large subunit, the conformational changes that actually take place in the
ribosome during translation are more subtle. It is likely that they involve a series of small rearrangements within each
subunit as well as several small shifts between the two subunits. As indicated, the mRNA is translated in the 5
-
to
-
3
direction, and the N
-
terminal end of a protein is made first, with each cycle adding one amino acid to the C
-
terminus
of the polypeptide chain. The position at which the growing peptide chain is attached to a tRNA does not change
during the elongation cycle: it is always linked to the tRNA present in the P site of the large subunit

Genetic Regulation


At any particular time, a cell is producing only a
small fraction of the proteins coded for in its
DNA


The amount of each protein must be precisely
regulated in order for the cell to function properly


The cell regulates protein production in
response to the environment or other cues


The production process is controlled by a
complex collection of proteins that influence
which genes are expressed.

Expression Efficiency

Figure 6
-
3. Genes can be expressed with different efficiencies.

Gene A is transcribed and translated much more efficiently
than gene B. This allows the amount of protein A in the cell to be much greater than that of protein B

Gene Expression

Figure 7
-
3. Differences in mRNA expression patterns among
different types of human cancer cells.

This figure summarizes a
very large set of measurements in which the mRNA levels of 1800
selected genes (arranged
top

to
bottom)

were determined for 142
different human tumors (arranged
left

to
right),

each from a different
patient. Each small
red

bar indicates that the given gene in the
given tumor is transcribed at a level significantly higher than the
average across all the cell lines. Each small
green

bar indicates a
less
-
than
-
average expression level, and each
black

bar denotes an
expression level that is close to average across the different
tumors. The procedure used to generate these data mRNA
isolation followed by hybridization to DNA microarrays is described
in
Chapter 8

(see pp. 533 535). The figure shows that the relative
expression levels of each of the 1800 genes analyzed vary among
the different tumors (seen by following a given gene
left

to
right

across the figure). This analysis also shows that each type of tumor
has a characteristic gene expression pattern. This information can
be used to "type" cancer cells of unknown tissue origin by matching
the gene expression profiles to those of known tumors. For
example, the unknown sample in the figure has been identified as a
lung cancer. (Courtesy of Patrick O. Brown, David Botstein, and the
Stanford Expression Collaboration

6 Steps of Expression Control

Figure 7
-
5. Six steps at which eucaryotic gene expression can be controlled.

Controls that operate at steps 1 through
5 are discussed in this chapter. Step 6, the regulation of protein activity, includes reversible activation or inactivation b
y
protein phosphorylation (discussed in
Chapter 3
) as well as irreversible inactivation by proteolytic degradation (discussed in
Chapter 6


Nucleosome Disruption

Figure 4
-
34. A cyclic mechanism for nucleosome disruption and re
-
formation.

According to this model, different chromatin remodeling complexes
disrupt and re
-
form nucleosomes, although, in principle, the same complex might catalyze both reactions. The DNA
-
binding protein
s could function in
gene expression, DNA replication, or DNA repair, and in some cases their binding could lead to the dissociation of the histon
e c
ore to form nucleosome
-
free regions of DNA like those illustrated in
Figure 4
-
30
. (Adapted from A. Travers,
Cell

96:311 314, 1999.)

Control Regions


The control mechanisms are responsive to the level of a
product already in the cell as well as to a tremendous
variety of extracellular signals.


Regulatory proteins recognize and bind to very specific
sequences in the DNA (
control regions
)


Control regions tend to border the protein coding regions
of genes


Control regions just upstream of the coding region that
encourage expression are called
promoters


Similar regions either downstream of the coding region
or relatively far upstream are called
enhancers


Sequences that tend to prevent the production of a
protein are called
repressors

Switching Tryptophan Genes

Figure 7
-
34. Switching the tryptophan genes on and off.

If the level of tryptophan inside the cell is low, RNA polymerase binds to the promoter and
transcribes the five genes of the tryptophan
(trp)

operon. If the level of tryptophan is high, however, the tryptophan repressor is activated to bind to the
operator, where it blocks the binding of RNA polymerase to the promoter. Whenever the level of intracellular tryptophan drops
, t
he repressor releases its
tryptophan and becomes inactive, allowing the polymerase to begin transcribing these genes. The promoter includes two key blo
cks

of DNA sequence
information, the
-
35 and
-
10 regions highlighted in
yellow

(see
Figure 6
-
12


Repressor Proteins

Figure 7
-
49. Five ways in which eucaryotic gene repressor proteins can
operate.

(A) Gene activator proteins and gene repressor proteins compete for
binding to the same regulatory DNA sequence. (B) Both proteins can bind DNA, but
the repressor binds to the activation domain of the activator protein thereby
preventing it from carrying out its activation functions. In a variation of this strategy,
the repressor binds tightly to the activator without having to be bound to DNA
directly. (C) The repressor interacts with an early stage of the assembling complex
of general transcription factors, blocking further assembly. Some repressors also
act at late stages in transcription initiation, for example, by preventing the release of
the RNA polymerase from the general transcription factors. (D) The repressor
recruits a chromatin remodeling complex which returns the nucleosomal state of the
promoter region to its pre
-
transcriptional form. Certain types of remodeling
complexes appear dedicated to restoring the repressed nucleosomal state of a
promoter, whereas others (for example, those recruited by activator proteins) render
DNA packaged in nucleosomes more accessible (see
Figure 4
-
34
). However the
same remodeling complex could in principle be used either to activate or repress
transcription: depending on the concentration of other proteins in the nucleus, either
the remodeled state or the repressed state could be stabilized. According to this
view, the remodeling complex simply allows chromatin structure to change. (E) The
repressor attracts a histone deacetylase to the promoter. Local histone
deacetylation reduces the affinity of TFIID for the promoter (see
Figure 7
-
46
) and
decreases the accessibility of DNA in the affected chromatin. A sixth mechanism of
negative control inactivation of a transcriptional activator by heterodimerization was
illustrated in
Figure 7
-
26
. For simplicity, nucleosomes have been omitted from (A)
-
(C), and the scale of (D) and (E) has been reduced relative to (A)
-
(C

Complex Example

Figure 7
-
50. Eucaryotic gene regulatory proteins often assemble into complexes on DNA.

Seven gene regulatory
proteins are shown in (A). The nature and function of the complex they form depends on the specific DNA sequence that
seeds their assembly. In (B), some assembled complexes activate gene transcription, while another represses transcription.
Note that the
red

protein is shared by both activating and repressing complexes

Computational Challenge:

Consensus Sequences

Figure 6
-
17. Consensus sequences found in the vicinity of eucaryotic RNA polymerase II start points.

The name given to each consensus
sequence
(first column)

and the general transcription factor that recognizes it
(last column)

are indicated. N indicates any nucleotide, and two nucleotides
separated by a slash indicate an equal probability of either nucleotide at the indicated position. In reality, each consensus

se
quence is a shorthand
representation of a histogram similar to that of
Figure 6
-
12
. For most RNA polymerase II transcription start points, only two or three of the four sequences
are present. For example, most polymerase II promoters have a TATA box sequence, and those that do not typically have a "stro
ng"

INR sequence.
Although most of the DNA sequences that influence transcription initiation are located "upstream" of the transcription start
poi
nt, a few, such as the DPE
shown in the figure, are located in the transcribed region

Computational Challenge:

Consensus Sequences

Figure 6
-
28. The consensus nucleotide sequences in an RNA molecule that signal the beginning and the end of most introns in huma
ns.

Only the three blocks of nucleotide sequences shown are required to remove an intron sequence; the rest of the intron can be
occ
upied by any
nucleotide. Here A, G, U, and C are the standard RNA nucleotides; R stands for either A or G; Y stands for either C or U. The

A
highlighted in
red

forms the branch point of the lariat produced by splicing. Only the GU at the start of the intron and the AG at its end are i
nva
riant nucleotides in the
splicing consensus sequences. The remaining positions (even the branch point A) can be occupied by a variety of nucleotides,
alt
hough the indicated
nucleotides are preferred. The distances along the RNA between the three splicing consensus sequences are highly variable; ho
wev
er, the distance
between the branch point and 3 splice junction is typically much shorter than that between the 5 splice junction and the br
anc
h point

Mechanisms of Evolution


The similarities and differences among closely related
molecules provide important information about their
structure and function


Molecules (or their sequences) which are related to one
another are said to be homologous


Sources of variation:


Point mutation (insertion, deletion, change)


Gene duplication (create pseudogenes)


Crossover (sexual reproduction)


Effects of mutations:


Neutral (in introns, third codon position); basis of genetic drift


Lethal (very often)


Advantageous (rarely); get fixed in the population


4 Types of Innovation

Figure 1
-
23. Four modes of genetic innovation and their effects on the DNA sequence of an organism


Effect of Mutation

Figure 1
-
29. A mutant phenotype reflecting the function of a gene.

A normal yeast (of the species
Schizosaccharomyces pombe)

is compared with a mutant in which a change in a single gene has converted the cell from a
cigar shape
(left)

to a T shape
(right).

The mutant gene therefore has a function in the control of cell shape. But how, in
molecular terms, does the gene product perform that function? That is a harder question, and needs biochemical analysis to
answer it. (Courtesy of Kenneth Sawin and Paul Nurse.)

Gene Duplication

Figure 1
-
51. The consequences of gene duplication for mutational analyses of gene function.

In this hypothetical example, an ancestral
multicellular organism has a genome containing a single copy of gene G, which performs its function at several sites in the b
ody
, indicated in
green
. (A)
Through gene duplication, a modern descendant of the ancestral organism has two copies of gene G, called G1 and G2. These hav
e d
iverged somewhat
in their patterns of expression and in their activities at the sites where they are expressed, but they still retain importan
t s
imilarities. At some sites, they
are expressed together, and each independently performs the same old function as the ancestral gene G (alternating
green

and
yellow stripes);

at other
sites, they are expressed alone and serve new purposes. (B) Because of a functional overlap, the loss of one of the two genes

by

mutation
(red cross)

reveals only a part of its role; only the loss of both genes in the double mutant reveals the full range of processes for whi
ch
these genes are responsible.
Analogous principles apply to duplicated genes that operate in the same place (for example, in a single
-
celled organism) but are

called into action
together or individually in response to varying circumstances. Thus, gene duplications complicate genetic analyses in all org
ani
sms

Human vs. Mouse

Figure 1
-
53. Human and mouse: similar genes and similar development.

The human baby and the mouse shown here
have similar white patches on their foreheads because both have mutations in the same gene (called
kit),

required for the
development and maintenance of pigment cells. (From R.A. Fleischman,
Proc. Natl. Acad. Sci. USA

88:10885 10889, 1991.
© National Academy of Sciences

Life as a Biochemical Process


The revolution in biology over the last three
decades resulted from the understanding cells in
terms of their chemistry


The functions of life (e.g. cell division, immune
reaction) are the interactions of complex, self
-
regulating networks of chemical reactions


Biomolecules
-

the substances that carry out and
regulate these activities:


macromolecules (proteins, carbohydrates, lipids)


a variety of small molecules.

Life as a Biochemical Process


The
genetic material

specifies how to
create proteins, when and how much to
create


Proteins
control the flow of energy and
materials through the cell


including the creation and transformation of
carbohydrates, lipids and other molecules,


Proteins ultimately accomplish all of the
functions that the cell carries out.

Life as a Biochemical Process


In even the simplest cell, there are thousands of
kinds of biomolecules interacting with each other


The amount and concentration of molecules in
various cell compartments determines what
influence they have


The concentrations vary over time, on scales of
seconds to decades.


Interactions among biomolecules are highly non
-
linear, as are their interactions with molecules
from outside the cell.

Catalysis and Metabolic Pathways


Intermediary
metabolism
: most essential chemical
processes within a cell


catabolic processes: breaking down proteins, fats and
carbohydrates


anabolic processes: building new materials.


Processes in intermediary metabolism are almost
all
catalyzed reactions


require special compounds (catalysts or enzymes) that
facilitate the reaction


Enzymes

are mostly proteins


materials transformed by catalysts are called substrates


catalysts are not changed by the reactions they
participate in

Catalysis and Metabolic Pathways


Pathways
: combinations of reactions which
accomplish tasks like turning foods into useable
energy or compounds


Even the basic transformations of intermediary
metabolism can involve dozens or hundreds of
catalyzed reactions.


Allosteric regulation:


the presence or absence of substrates can affect the
behavior of the enzymes themselves


Competitive inhibition:


substance other than the usual substrate of the
enzyme binds to the active site of the enzyme


this is preventing it from having an effect on its
substrate.

How Enzymes Work

Figure 2
-
47. How enzymes work.

Each enzyme has an active site to which one or two
substrate

molecules bind, forming an enzyme
-
substrate
complex. A reaction occurs at the active site, producing an enzyme
-
product complex. The
product

is then released, allowing the enzyme to bind
additional substrate molecules

How Enzymes Work

Figure 3
-
52. Some general strategies of enzyme catalysis.

(A) Holding substrates together in a precise alignment. (B) Charge stabilization of
reaction intermediates. (C) Altering bond angles in the substrate to increase the rate of a particular reaction

Positive regulation

Figure 3
-
57. Positive regulation caused by conformational coupling between two distant binding sites.

In this example, both glucose and
molecule X bind best to the
closed

conformation of a protein with two domains. Because both glucose and molecule X drive the protein toward its
closed conformation, each ligand helps the other to bind. Glucose and molecule X are therefore said to bind
cooperatively

to the protein

Negative regulation

Figure 3
-
58. Negative regulation caused by conformational coupling between two distant binding sites.

The scheme here resembles that in the
previous figure, but here molecule X prefers the
open

conformation, while glucose prefers the
closed

conformation. Because glucose and molecule X
drive the protein toward opposite conformations (closed and open, respectively), the presence of either ligand interferes wit
h t
he binding of the other

Metabolic chain

Figure 2
-
34. How a set of enzyme
-
catalyzed reactions generates a metabolic pathway.

Each enzyme catalyzes a particular chemical reaction,
leaving the enzyme unchanged. In this example, a set of enzymes acting in series converts molecule A to molecule F, forming a

me
tabolic pathway

Metabolic
Pathways

Figure 2
-
35. Some of the metabolic pathways and their
interconnections in a typical cell.

About 500 common metabolic
reactions are shown diagrammatically, with each molecule in a
metabolic pathway represented by a filled circle, as in the
yellow

box in
Figure 2
-
34


Model Organisms


Most valuable methods in biological research


are invasive


require organisms to be sacrificed


require many generations of observation


require observations on large populations


Much of this work is impractical or unethical to
carry out on humans


research involving humans can be done ethically


in some areas of biomedical research, such as final
drug testing, it is obligatory


Biologists have selected a variety of model
organisms for experimentation.

Model Organisms


E. coli (the ubiquitous intestinal bacterium Escherichia
coli)


fast reproduction time, safe and easy to work with


common target for genetic engineering, (genes from other
organisms are inserted into its genome and produced in
quantity)


Saccharomyces cervesiae (brewer’s yeast)


safe, easy to grow, short generation time


yeasts are eukaryotes


useful in in sequencing large amounts of DNA


Arabidopsis thaliana (a common weed)


useful in agriculture research


very little repetitive DNA (genome 30 times less DNA than corn)


Model Organisms


Caenorhabditis elegans (the nematode worm)


has exactly 959 cells doing exactly the same thing


one of the simplest creatures with a nervous system (1/3 of its
cells)


useful to gaining insight in gain insight into the mechanisms of
aging and development


Drosophila melanogaster (the fruit fly)


short generation times


many different genetically determined morphological
characteristics


a staple of classical genetics research


Mus musculus (laboratory mouse)


mammals


genetically practically identical to people


Experimental Methods


Important for understanding the strengths and
limitations of various sources of data


Methods:


Imaging/microscopy; new technologies (e.g. Atomic
Force Microscope) offer the potential to create
images of individual molecules.


Gel electrophoresis; a method for separation of a
protein mixture by their charge and size


The resulting individual spots or bands can be removed from
the gel for further processing, in a procedure called blotting


Experimental Methods


Cloning


A group of cells with identical genomes (also called a cell
line)


individual genes could be cloned


by inserting a piece of DNA containing a gene of interest into
the bacterial genome; creating recombinant DNA)


how: put the new gene into vectors
-

a virus that infects
bacteria or a plasmid; add antibiotic resistance genes


Genetic engineering: inserting foreign genes into plants,
animals, even people.


Hybridization / Immunological staining; to identify the
presence or absence of a particular molecule (or even
a region of a molecule) in vanishingly small amounts


Application in microarrays to measure expression of all
genes in a genome

Experimental Methods


Gene Mapping and Sequencing; to identify the
location and size of all of the genes


Polymerase chain reaction (PCR); to rapidly produce
huge amounts of a specific region of DNA, simply by
knowing a little bit of the sequence around the desired
region


Crystallography and NMR; to determine 3D structure
of biomolecules


Mass spectrometry; to identify sequence of protein,
RNA, or DNA fragments


Yeast
-
Two
-
Hybrid systems; to assess protein
-
protein
interactions