Transcript Protein Synthesis in a Eukaryotic Cell.

Introduction to
Studying Proteins
Chapter 5
Learning Outcomes
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Describe the structure of proteins, including the significance of amino
acid R-groups and their impact on the three-dimensional structure of
proteins.
Explain the steps of transcription and translation in protein synthesis.
Discuss the role of naturally occurring proteins and recombinant
proteins in biotechnology.
Differentiate proteins that function as part of structure, as antibodies,
and as enzymes.
Describe the structure of antibodies and explain the relationship
between antibodies and antigens.
Discriminate among the classes of enzymes and discuss the effect of
reaction conditions on enzyme activity.
Summarize polyacrylamide gel electrophoresis and identify its
usefulness for studying proteins.
5.1 The Structure and Function of Proteins
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Virtually all biotechnology products have
something to do with proteins.
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Protein has a three-dimensional structure.
Protein Molecule Structure
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Polymers composed of amino acids
Twenty different kinds of amino acids are found in protein
Most proteins contain tens of hundreds of amino acids
Function of Structural Proteins
For an HIV particle to recognize, attach, and infect a T-helper cell, the gp210 structure
must be a precise shape and must exactly match its human cell membrane receptors
Function of Antibody Proteins
Recognize and bind foreign proteins or other molecules (antigens) for removal from
the body
Vocabulary
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X-ray crystallography – a technique used to determine the three-dimensional structure of a protein
Polar – the chemical characteristic of containing both a positive and negative charge on opposite sides
of a molecule
Primary structure – the order and type of amino acids found in a polypeptide chain
Secondary structure – the structure of a protein (alpha helix and beta sheets) that results from
hydrogen bonding
Tertiary structure – the structure of a protein that results from several interactions, the presence of
charged or uncharged “R” groups, and hydrogen bonding
Quaternary structure – the structure of a protein resulting from the association of two or more
polypeptide chains
Glycosylated – descriptive of molecules to which sugar groups have been added
CD4 cells – referring to human white blood cells, which contain the cell surface recognition protein
CD4
Reverse transcriptase – an enzyme that transcribes a complementary strand of DNA from a strand
of RNA
Antigens – foreign proteins or molecules that are the target of binding by antibodies
Epitope – the specific region on a molecule that an antibody binds to
ELISA – short for enzyme-linked immunospecific assay, a technique that measures the amount of
protein or antibody in a solution
Monoclonal antibody – a type of antibody that is directed against a single epitope
Hybridoma – a hybrid cell used to generate monoclonal antibodies that results from the fusion of
immortal tumor cells with specific antibody-producing white blood cells (B-cells)
5.1 Review Questions
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How many different kinds of amino acids are found in proteins?
What distinguishes one amino acid from another?
What causes polypeptide chains to fold into functional proteins?
How many polypeptide chains are found in an antibody, and how
are they held together in a protein?
What is the value of monoclonal antibody technology?
5.2 The Production of Protein
Until recently, proteins could only be made in cells.
Now small polypeptide chains can be synthesized in the
laboratory.
Overview of Protein Synthesis
Protein synthesis occurs continuously throughout a cell’s life
Eukaryote and prokaryote – protein synthesis is similar
Protein Synthesis in a Eukaryotic Cell. In a eukaryotic cell, DNA is located
within chromosomes in the nucleus. The mRNA transcripts carry the DNA code out
to the ribosomes, which translate the code into a strand of amino acids.
Transcription and Translation
Protein synthesis is a twostep process:
First Step:
Genetic code must be
rewritten onto a
messenger molecule
Second Step:
mRNA nucleotide
code is rendered
into a sequence of
amino acids
The Importance of Proteins in Biotech R&D
The ability to synthesize and modify peptides or proteins is
crucial to the production of virtually every biotechnology
product.
Vocabulary
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Protein synthesis – the generation of new proteins from amino acid subunits; in the
cell, it includes transcription and translation
Transcription – the process of deciphering a DNA nucleotide code and converting it into
RNA nucleotide code; the RNA carries the genetic message to a ribosome for translation
into a protein code
Codon – a set of three nucleotides on a strand of mRNA that codes for a particular amino
acid in a protein chain
Translation – the process of reading an mRNA nucleotide code and converting it into a
sequence of amino acids
tRNA – a type of ribonucleic acid (RNA) that shuttles amino acids into the ribosome for
protein synthesis
Peptidyl transferase – an enzyme found in the ribosome that builds polypeptide chains
by connecting amino acids into long chains through peptide bonds
Phosphorylation – adding phosphate groups
Cleavage – process of splitting the polypeptide into two or more strands
Taq polymerase – a DNA synthesis enzyme that can withstand the high temperatures
used in PCR
5.2 Review Questions
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Distinguish between transcription and translation.
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If a structural gene’s code is “TAC GGC ATG CCC TTA CGC ATC,” what
will the mRNA transcript be?
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If the mRNA transcript from question No. 2 were translated into a
peptide, what would the amino-acid sequence of the peptide be?
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What is the name of the machine that can make small sections of
amino-acid chains?
5.3 Enzymes: Protein Catalysts
Enzymes are proteins that act as catalysts
Enzymes are involved in virtually every reaction in a cell
Many companies have focused on producing enzymes for sale
Enzymes and Their Substrates
The molecules upon which enzymes act are called substrates
Factors That Affect Enzyme Activity
Amount of substrate in a solution
Temperature of a reaction
Acidity or alkalinity
Vocabulary
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Substrate – the molecule that an enzyme acts on
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Cofactors – an atom or molecule that an enzyme requires to function
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Lock and key model – a model used to describe how enzymes function, in which the
enzyme and substrate make an extract molecular fit at the active site, triggering catalysis
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Induced fit model – a model used to describe how enzymes function, in which a
substrate squeezes into an active site and induces the enzyme’s activity
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Optimum temperature – the temperature at which an enzyme achieves maximum
activity
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Denaturation – the process in which proteins lose their conformation or threedimensional shape
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Optimum pH – the pH at which an enzyme achieves maximum activity
5.3 Review Questions
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Name three examples of enzymes and their substrates.
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What happens if an enzyme is at a temperature significantly above
its optimum temperature?
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What happens if an enzyme is at a pH significantly above or below
its optimum level?
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What would an enzyme be called if it moved methyl groups (-CH3)
between molecules?
5.4 Studying Proteins
A technician loads protein samples on a vertical gel. Vertical
gel boxes operate in a fashion similar to horizontal gel boxes.
Vertical Gel Electrophoresis. Although vertical gel boxes vary from one manufacturer to another, all are basically
of the same design. The gel cassettes are snapped or screwed in place (right). Running buffer is added behind the
gel, covering the wells. Buffer is poured in the front of the gel cassette to cover the front opening. When the top is
placed on the box (left) and the power is turned on, electricity flows from the top (negative charge) to bottom
(positive charge). Negatively charged samples move down the gel toward the positive electrode.
Silver stain is much more sensitive than Coomassie® Blue. When samples have
low concentrations of protein or DNA, silver-staining is the method of choice.
Vocabulary
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PAGE – short for polyacrylamide gel electrophoresis, a process in which proteins and
small DNA molecules are separated by electrophoresis on vertical gels made of the
synthetic polymer, polyacrylamide
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Coomassie® Blue – a dye that stains proteins blue and allows them to be visualized
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Silver stain – a stain used for visualizing proteins
5.4 Review Questions
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What does “PAGE” stand for, and what samples are studied using
PAGE?
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What separates molecules on a PAGE gel?
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PAGE gels are usually run at what amount of current?
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A technician has a stock protein solution with a concentration of 1
mg/mL. He prepares a 1:4 serial dilution of the stock and runs
the samples on a PAGE gel. What is the preferred method of
staining and why?
5.5 Applications of Protein Analysis
Protein profile of cells and tissues
A protein’s structure can help explain its function
Chemical processes in cells
Evolution and taxonomic relationships
Vocabulary
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Taxonomic relationships – how species are related to one another in
terms of evolution
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Biomanufacturing – industry focusing on the production of proteins and
other products created by biotechnology
5.5 Review Questions
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What causes the difference between normal and sickled cells in
sickle cell disease?
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Give an example of proteins studied to understand evolutionary
relationships.
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What is NCBI, how can you access it, and what important
information is found there?
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Do all protein scientists work at biotechnology companies? Explain.
Questions and Comments?