[I] MCB 3201 Gene Expression

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I MCB 3201 Gene Expression

• Instructor Dr. Thomas T. Chen
• Office TLS Rm 413A Te 860-486-5481 E-mail
  Thomas.Chen_at_uconn.edu Office hour Tue 1100
  a.m. - 100 p.m. or by appointment
• Class Meeting Time Tue and Thu 930 1045 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, Lifes 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

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MCB 201 Gene Expression (II)

• 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

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Some Facts About Gene Expression in Eukayotes

• 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

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I Principle of Supramacromolar Assembly in the
Biological System
An important principle in the biological system
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Chemical Composition of Living Cells

• 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?

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Types of Biochemical Bondings

• 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

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Amino Acids

• 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

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Unique Property of Amino Acids

• 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?

Zwitterion
Isoelectric point
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Nonpolar Amino Acids
Nonpolar amino acids contain R groups that are
non-polar in nature. Of 20 amino acids, 9 amino
acids are non-polar
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Polar or Charged Amino Acids
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Types of Chemical Bonds in Biologically Important
Molecules (I)

• Covalent bond bond strength -50 to -100 Kcal/mol

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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)
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Types of Chemical Bonds in Biologically Important
Molecules (III)

• Hydrogen bond -3 to -6 Kcal/mol

d
d-
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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
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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
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Levels of Structures of Proteins

• 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

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Making a Peptide Chain

• 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

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The a-Helical Structure of a Polypeptide
Hydrogen bond is formed between the N-H of every
peptide bond and the CO of a neighboring peptide
bond located four peptide bonds away in the same
chain
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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
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Tertiary Structure of a Polypeptide

• 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 non-covalent 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

• Proteins usually fall into one of the three broad
  categories based on their tertiary structure
  fibrous proteins, globular proteins and integral
  membrane proteins

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Tertiary Structure of a Polypeptide

• 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 non-polar 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,

but undergoes continual, minute fluctuations
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Motifs of Protein Secondary Structure

• 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

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Structural and Functional Domains

• 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

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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)

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Hypothetical Protein-Folding Pathway
(a). Primary structure (b)(d). Secondary
structure (e). Tertiary structure
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Chaperonin-Mediated Protein Folding in E. coli

• 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

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Hsp70-Like Proteins Mediate Protein Folding in
Eukaryotic Cells

• 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
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Hsp90 Protein Mediates Protein Folding in
Eukaryotic Cells

• 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

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Quaternary Structure

• 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

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Aspartate Transcarbamoylase (ATCase) in E. coli

• ATCase catalyzes the formation of N-carbamoyl
  aspartate from carbamoyl phosphate
• This enzyme is a multimeric enzyme consists of
  catalytic submit (c33 kD) and regulatory submit
  (r17 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

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Myoglobin and Hemoglobin

• 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

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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

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Assembly of Tobacco Mosaic Virus
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Assembly of T4 Phage
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T4 Phage

• 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

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Overview of Supramolecular Assembly of
Macromolecules and the Biological Activities
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• Assigned Reading I
• Nobel lecture by Chris Anfensen
• Chaperonin-associated protein folding
• Protein folding in the cell