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[I] MCB 3201 Gene Expression
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Instructor: Dr. Thomas T. Chen
Office: TLS Rm 413A; Te: 860-486-5481; E-mail:
[email protected]; Office hour: Tue 11:00 a.m.
- 1:00 p.m. or by appointment
Class Meeting Time: Tue and Thu 9:30 – 10:45 a.m. in
TLS 263
Text Book:
 Molecular Cell Biology (7th edition) by Lodish et al.
 Course Website: www.sp.uconn.edu/~ttc02001/MCB3201/
A recommended extra-reading:
 RNA, Life’s indispensable molecule (by James Darnell), published by
Cold Spring Harbor Laboratory Press (can be purchased through
amazon.com)
Course Grade:
 Average of two in-class exams
MCB 201 Gene Expression (II)
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Two in-class Exams:
 Exam I: Thu, 03/03 (Tue)
 Exam II: Tue, to be announced
I will lecture 75 minutes in each lecture slot
Exam questions will consist of definitions, short and long
answers and problem solving questions. Exam materials will be
taken from lecture slides, assigned pages in the textbook and
assigned papers on the MCB 3201 website
The course grade will be determined by averaging the scores of
two exams
Some Facts About Gene Expression in
Eukayotes
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The central dogma of molecular biology is that DNA
produces RNA through transcription which in tern
produces proteins through translation
While the content of DNA of different tissues and cell
types in a specific species of organism is the same, the
presence and the relative abundance of mRNAs and
proteins are different
It implies that control of gene expression must operate to
produce different mRNA population in different cell types
from the same DNA through regulation at:
 Transcriptional level
 Post-transcriptional level
 Translational level
[I] Principle of Supramacromolar
Assembly in the Biological System
An important principle in the biological system
Chemical Composition of Living Cells
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Hydrogen, oxygen, nitrogen, carbon, sulfur, and
phosphorus normally makeup more than 99% of the
mass of living cells
About 70% percent by mass of the molecules inside
living cells are water molecules
Cells normally contain more proteins than nucleic
acids (DNA & RNA)
Cells also contain carbohydrates, saturated and
unsaturated fatty acids, steroids, cholesterol, lipids,
amino acids and inorganic elements
An important question: How are these compounds
associate together to form cells with specific
structures and functions? How is regulation of gene
expression achieved?
Types of Biochemical Bondings
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Covalent bonding: -50 to -100 Kcal/mol
Ionic bonding: -1 to -80 Kcal/mol
Hydrogen bonding: -3 to -6 Kcal/mol
Van der Wallas attraction: -0.5 to -1 Kcal/mol
Hydrophobic interaction: -0.5 to -3 Kcal/mol
Weak chemical interactions: ionic bonding,
hydrogen bonding, Van der Walls interaction
and hydrophobic interaction
Amino Acids
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Different protein
molecules are
made up of the
same 20 natural
occurring amino
acids but with
specific sequence
Each amino acid
contains two
functional groups:
amino group and
carboxyl group
Unique Property
of Amino Acids
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Zwitterion
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Isoelectric point
The pH of an amino acid
solution at Zwitterion form
is called isoelectric point of
the amino acid
Why amino acids or
proteins can serve as a
good buffer?
Nonpolar Amino Acids
Nonpolar amino acids contain R groups that are nonpolar in nature. Of 20 amino acids, 9 amino acids are
non-polar
Polar or Charged Amino Acids
Types of Chemical Bonds in Biologically
Important Molecules (I)
• Covalent bond: bond strength -50 to 100 Kcal/mol
Types of Chemical Bonds in Biologically
Important Molecules (II)
•Ionic bond: bond strength -1 to -80 Kcal/mol
Bonds formed between
the charged amino
group of basic amino
acids (lys, arg, and his)
and the charged
carboxyl group of acidic
amino acids (asp and
glu)
Types of Chemical Bonds in Biologically
Important Molecules (III)
•Hydrogen bond: -3 to -6 Kcal/mol
d+
d-
Types of Chemical Bonds in Biologically
Important Molecules (IV)
•Van der Waals attraction: -0.5 to -1 Kcal/mol
The electron cloud around any nonpolar atom will
fluctuate, producing a flicking dipole. Such dipoles
will transiently induce an oppositely polarized
flickering dipole in a near-by atom. This interaction
generates an attraction between atoms that is very
weak. However, since many atoms can be
simultaneously in contact when two surfaces fit
closely, the net result is often significant
Types of Chemical Bonds in Biologically
Important Molecules (V)
•Hydrophobic interaction: -0.5 to -3 Kcal/mol
Nonpolar amino acids:
gly. Leu. Ilu, val, ala, trp,
met, phe, pro
Levels of Structures of Proteins
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Primary structure: Peptide bond formation (covalent
bonds)
Secondary structure: Hydrogen bonding form within
one polypeptide chain(a-helical and b-sheet
structure)
Tertiary structure: Ionic interaction, hydrophobic
interaction, hydrogen bonding and Van der Waals
attraction formed among moieties within one
polypeptide chain
Quaternary structure: Weak chemical interactions
among different polypeptide chains
Supramolecular assembly of macromolecules: Weak
chemical interactions of different macromolecules
Making a
Peptide Chain
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When the carboxyl group
of one amino acid is
brought adjacent to
amino group of another
amino acid, an enzyme
(peptide synthetase) can
catalyze an dehydration
reaction to form a peptide
bond
When this reaction is
repeated over and over, a
polypeptide will be
formed
The a-Helical Structure
of a Polypeptide
Hydrogen bond is formed
between the N-H of every
peptide bond and the C=O of a
neighboring peptide bond
located four peptide bonds
away in the same chain
The b-Sheet Structure of a Polypeptide
Individual peptide
chains run in opposite
directions and
hydrogen bonds are
formed between
peptide bonds in
different strands
Structure of a b-turn
Tertiary Structure of a Polypeptide
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Chemical properties of the side chains (i.e., the R groups) of amino
acids help define the tertiary structure of a peptide
 Disulfide bonds between the side chains of cysteine residues in some
proteins covalently link regions of proteins, thus help to stablize the
tertiary structure of a protein
 Amino acid with charged hydrophilic polar side chains tend to in the
outside surface of proteins, by interacting with water molecules, can
help proteins to be soluble in aqueous solutions and form noncovalent interactions with other water-soluble molecules
 Amino acids with hydrophobic nonpolar side chains are usually
sequestered away from the water-facing surfaces of a protein, forming
a water-insoluble central core
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Proteins usually fall into one of the
three broad categories based on
their tertiary structure: fibrous
proteins, globular proteins and
integral membrane proteins
Tertiary Structure of a Polypeptide
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but undergoes continual, minute fluctuations
Tertiary structure refers to
the overall conformation of
a polypeptide chain – that
is the three dimensional
arrangement of all its
amino acid residues
Tertiary structure is
stabilized by hydrophobic
interaction between nonpolar side chains, and
hydrogen bonding of polar
side chains and peptide
bonds
Since the stabilizing
interactions are weak, the
tertiary structure of a
protein is not rigidly fixed,
Motifs of Protein Secondary Structure
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Structural motifs are regular combinations of secondary and tertiary
structures of proteins
Any particular structural motif often performs a common function in
different proteins
The primary sequences responsible for a given structural motif may
be very similar to one another. However, it is possible for seemingly
unrelated primary sequences to result in folding into a common
structure motif
Structural and Functional Domains
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Domains: Distinct regions of protein tertiary structure are often referred
as domains
Three main classes of protein domains: structural domain, functional
domain and topological domain
Functional domain: a region of a protein that exhibits a particular
activity characteristic of the protein even when it is isolated from the
rest of the protein
A structural domain is a region ~40 or more amino acids in length,
arranged in a stable, distinct secondary or tertiary structure, that often
fold into its characteristic structure independently of the rest of
the protein
Topological domain: Distinctive special relationships with the
rest of protein
Denaturation and Renaturation of
Ribonuclease A
1. Ribonuclease A is a single
chain polypeptide.
2. Dr. Chris Anfinsen showed
that denatuation of RNase A
resulted in loosing the
activity of the enzyme and
re-naturation of the
polypeptide regained the
enzyme activity.
3. This discovery resulted in
receiving a Nobel Prizes in
1973 (Assigned reading [I])
Hypothetical ProteinFolding Pathway
(a). Primary structure
(b)–(d). Secondary structure
(e). Tertiary structure
Chaperonin-Mediated Protein Folding in E. coli
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Prokartyotic GroEL in E. coli is a hollow barrel-shaped complex of 14
identical 60,000 MW submits arranged in two stacked rings
In the absence of ATP or presence of ADP, GeoEL esxist in a tight
conformation state that binds partially folded or mis-folded proteins
Binding of ATP shifts GroEL to a more open relaxed state, which releases
the folded protein
GroES, a co-chaperonin of 10,000 MW, helps the folding process
Hsp70-Like Proteins Mediate Protein Folding in
Eukaryotic Cells
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Hsp70 family
proteins are
molecular
chaperones
DnaJ/Hsp40 and
GrpE/BAG1 are two
co-chaperone
accessory proteins
involved in helping
Hsp70 to promote
the assembly of
proteins
Hsp70 in cytosol and mitochondrial matrix, BiP in endoplasmic reticulum,
and DnaK in bacteria are molecular chaperones. Hsp70 and its homologs
are major chaperones in all organisms
Hsp90 Protein Mediates Protein Folding in
Eukaryotic Cells
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In addition to Hsp70
family proteins, Hsp90
family proteins are
another group of
molecular chaperones
Hsp90s are critical in cells
to cope with denatured
proteins generated by
stress
Hsp90s help to stabilize
transcription factors and
kinases
Hsp90s function as a
dimer in cycle in which
ATP binding, hydrolysis
and ADP release resulting
in protein folding
Quaternary Structure
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Individual protein subunits interact between or among one
another to form a complex entity
Hydrophobic or hydrophilic interaction between the side
chains of amino acids in one submit with the side chains of
amino acids in the other submit is responsible for formation
of quaternary structure of a protein
The submits in the quaternary of proteins can be either
identical submits or un-identical submits
With the formation of quaternary structure, proteins frequently
quire additional functions
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3o and 4o structure of
hemagglutinin (HA), a
surface protein of
influenza virus
This multimeric molecule
is made up of three
identical submits, each
composed of two
polypeptide chains (HA1
and HA2)
The 4o structure of HA
composed of 3 submits
and the distal globular
domain of each submit
binds sialic acid on the
surface of the target cells
Aspartate
Transcarbamoylase
(ATCase) in E. coli
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ATCase catalyzes the formation of
N-carbamoyl aspartate from
carbamoyl phosphate
This enzyme is a multimeric
enzyme consists of catalytic
submit (c=33 kD) and regulatory
submit (r=17 kD)
The intact ATCase is 300 kD
consists of c6r6
C submit has catalytic activity
alone. By combining with r submit,
it assume allosteric effect
CTP has inhibit effect on ATCase
and ATP has activation effect
Formation of 4o structure resulted
in assuming allosteric effect
Myoglobin and Hemoglobin
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Myoglobin is a single chain polypeptide which can bind oxygen.
Hemoglobin consists of 2 a-globin chains and 2 b-globin chains. By
forming the complete hemoglobin molecule, it assumes an allosteric
effect
Assembly of Transcription Initiation Complex
•By binding
transcription factors
and RNA polymerase II
to the promoter (TATA
box ) region of a gene,
transcription can take
place precisely.
•This is another of how
transcription can be
initiated through
macromolecular
assembly
Assembly of Tobacco Mosaic Virus
Assembly of T4 Phage
T4 Phage
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Another example
of this is the in
vitro packaging
of lambda phage
by mixing the
coat proteins of
lambda phage
with its genomic
DNA in a testube
Overview of
Supramolecular
Assembly of
Macromolecules and
the Biological
Activities
Assigned Reading [I]
1. Nobel lecture by Chris Anfensen
2. Chaperonin-associated protein folding
3. Protein folding in the cell