1. What four non-covalent interactions stabilize the tertiary structure of polypeptides?
2. Draw a tripeptide. The R groups of the three amino acids can be designated as R1, R2, and R3. On your diagram, draw a rectangle around the planar regions.
3. Describe the "charge-relay" that activates the serine 195 residue of chymotrypsin.
4. Illustrate the secondary structure of B-DNA, as defined by Watson and Crick.
5. What is meant by "wobble"? What aspect of the genetic code protects against it?
6. Describe the steps that occur during the elongation phase of protein synthesis in eukaryotes starting at the interaction between eEF1, GTP and tRNA and continuing to the formation fo the peptide bond.
7. Draw a replication fork and indicate on it the
following: leading strand, lagging strand, location of DNA helicase, location
of topoisomerase.
REVIEW SHEET
I. Molecular Organization
A. Monosaccharide structure
1. D form;
alpha versus beta form of glucose
2. glycogen
structure versus cellulose structure; functional implications
B. Phospholipid structure
1. cell
membrane
C. Four weak non-covalent interactions
involved in stabilizing macromolecule structure and interaction
1. Hydrogen bond
2. Ionic interaction
3. Hydrophobic interaction
4. Van der waal's contacts
II. Amino Acid and Protein Structure
A. Amino Acid Structure
1. classification
of amino acids
B. Primary Structure of Polypeptides
1. The peptide bond
a. resonance and planarity
b. Amino terminus; Carboxy terminus
C. Secondary Structure
1. alpha-helix
2. beta-pleated
sheet
3. motifs
D. Tertiary Structure
1. domains,
structural and functional
E. Quarternary Structure
1. interacting
subunits
2. hemoglobin
example
III. Protein Function
A. Free energy changes during
chemical reactions
B. Entropy changes
C. Activation energy; Transition
state
D. Catalysts
E. Enzymes as catalysts
F. Mechanism of chymotrypsin catalysis
of the breaking of a peptide bond.
1. induced
fit
2. charge-relay
3. two
step reaction (two tetrahedral intermediates)
IV. DNA Structure
A. The nucleotide
1. pyrimidines
and purines in DNA and RNA
2. nucleosides.
Ribose in RNA; deoxyribose in DNA
3. nucleoside
monophosphates, diphosphates, triphosphates.
B. The DNA polymer
1. The
phosphodiester bond
2. 5'end
defined
3. 3'end
defined
C. B-DNA
1. double-stranded
2. two
antiparallel alpha helices
3. sugar-phosphates
to the outside
4. bases
to the inside
5. bases
hydrogen bond: A and T; C and G
6. bases
of a strand also interact by hydrophobic interactions
7. about
10 base pairs per turn of the helix
8. major
and minor grooves
D. Evidence for B-DNA structure
1. X-ray
crystallography patterns
2. Chargaff's
experiments: conc. of A equals T; conc. of C equals G in all DNAs
3. Linus
Pauling's description of protein alpha-helix stabilized by hydrogen bonds
4. DNA
loses viscosity when heated (hydrogen bonds involved)
E. Chromatin structure
1. Nucleosomes
a. Histone octamer (2 each of H2A, H2B, H3, H4) with
DNA wound around twice
2. Linkers
of DNA connecting nucleosomes, average of 200 base pairs, variable.
3. HI
bound to linker regions
a. helps fold the chromatin into a 30nm fiber or solenoid
4. Chromatin
structure correlated to gene activity. (Covered later in the course.)
IV. Genetic Code
A. Triplet code
B. Reading Frame
C. Degenerate Code
D. Start and Stop Codons
E. Second base U=hydrophobic amino
acid; C=hydrophobic or polar; A or G=polar or charged.
V. Major Players in Translation
A. messenger RNA (eukaryotes)
1. Cap
at 5' end
2. 5'
untranslated region
3. AUG
start codon
4. open
reading frame (coding region)
5. 3'
untranslated region
6. poly
A tail
B. transfer RNA
1. stem-loop
structures
2. anticodon-loop
a. codon-anticodon base pairing
b. wobble position
c. conncection between wobble and the degeneracy of the
genetic code.
3. amino
acid attachment to 3'end
C. amino-acyl-tRNA synthetase
1. catalyzes
attachment of amino acid to either the 2' or 3' carbon of the ribose of
the adenine at the 3' end of tRNA molecule.
2. two
step reaction
a. Pyrophosphate removed from ATP; AMP attached to amino
acid through COOH group.
b. AMP removed and amino acid attached to the tRNA
3. specificity
of the enzyme assures the correct attachment
D. Ribosomes
1. Two
subunits, large and small
2. Consist
of ribosomal RNAs and ribosomal proteins
3. Contain
the enzymatic activity needed during protein synthesis
4. Function
in the cytoplasm
VI. The Translation Mechanism
A. Initiation
1. sets
the reading frame
2. small
subunit, initiator tRNA, initiation factors and GTP interact
3. Other
initiation factors bind to the Cap on mRNA and to the 5' untranslated region
4. complex
(step 2) interacts with factors at the Cap
5. factors
at the cap and at the 5' untranslated region melt any stem-loop in the
5'untranslated region
6. small
subunit complex scans along until it finds the first AUG
7. initiator
tRNA anticodon binds to AUG codon
8. large
ribosomal subunit joins creating the functional ribosome with a P and A
site.
B. Elongation
1. eEF1
bound to GTP binds to tRNA.
2. This
complex moves to the A site of the ribosome.
3. If
the anticodon on the tRNA is correct for the codon, it is held there long
enough for
4. GTP
to be hydrolyzed to GDP causing the eEF1 to disconnect from the tRNA and
leave
5. The
energy released in step 4 stably attaches the tRNA to the A site.
6. Peptidyl
transferase activity (probably rRNA in the ribosome) catalyzes the breaking
of the bond holding methionine to the initiator tRNA and using the energy
released to attach the methionine to the amino acid attached to the tRNA
at the A site by a peptide bond.
7. With
the help of eEF2 and GTP, the ribosome moves one codon along the mRNA so
that the tRNA is now at the P site and the A site awaits another tRNA complex.
Called translocation.
8. The
process repeats itself over and over until a stop codon is at the A site.
9. The
eEF1 bound to GDP (see step 1) is regenerated to the GTP bound form by
eEF1b binding to the GDP bound form, causing its displacement and allowing
the GTP to bind. It can now interact with another tRNA.
10. This mechanism provides
a kinetic proofreading that insures that only the correct tRNA at the A
site will remain there long enough to allow the above events to happen
before a peptide bond is made.
C. Termination
1. When a stop codon is
at the A site of the ribosome, termination factors bind there.
2. Peptidyl
transferase breaks the attachment between the polypeptide and the tRNA
at the P site.
3. The
polypeptide is released with an intact COOH terminus; the tRNA at the P
site exits; and with the help of GTP, the entire ribosomal complex comes
apart.
D. Additional Considerations
1. The
ribosome also has an E site where the exiting tRNA moves during translocation.
2. The
tRNA orientation with the ribosome during translation begins with P/P.
3. Then
a tRNA enters the A site first with its anticodon (A/T orientation)
4. Then
the amino acid side binds (A/A orientation)
5. After
peptide bond formation, both tRNAs lean over such that the P site tRNA
has its 3' end at the E site (P/E orientation) and the A site tRNA has
its amino acid end at the P site (A/P orientation).
6. Following translocation,
the previous P site tRNA is now attached to the E site only by its 3' end
and will soon leave completely. (E orientation). And at the P site now
is the tRNA that had been at the A site, completely attached (the P/P orientation).
7. Consult
page 137 of the text for pictures.
E. Prokaryotic Differences
1. Initiation
and setting of the reading frame can occur in more than one place (polycistronic)
a. uses Shine-Delgarno interactions between mRNA codons
before the AUG interacting with ribosomal RNA sequences.
2. prokaryotes
can couple transcription and translation
3. the
initiating methionine is formylated in prokaryotes
VII. DNA Replication
A. Overall
1. Semi-conservative
2. DNA
polymerase is the major enzyme involved.
B. Properties of DNA polymerases
1. Require
a pre-existing 3'OH to which to attach the incoming dNTP.
2. Need
a template strand
a. base-pairs the incoming nucleotide to the template
strand to select the correct one.
3. Has
three domains
a domain where the 5'-3' polymerizing reaction is catalyzed
b. domain where the 3'-5' exonuclease activity is
- involved in the proofreading mechanism that removes incorrectly added
nucleotides
c. domain where the 5'-3' exonuclease activity is
- involved in removing the RNA primers and in DNA repair mechanisms.
C. Types of DNA polymerases
1. Prokaryotic
a. Pol I: involved in DNA repair
b. Pol III: the major DNA polymerase that works during
DNA replication
2. Eukaryotic
a. DNA polymerase alpha: synthesizes the Okazaki fragments.Short
processivity.
b. DNA polymerase delta: synthesizes the leading strand
c. DNA polymerase beta: works during DNA repair
d. DNA polymerase gamma: works in the mitochondria
e. DNA polymerase epsilon: also works during DNA repair
D. Replication Fork
1. New
DNA polymers grow 5'-3'
2. Leading
strand grows continuously
3. Lagging
strand grows discontinuously using Okazaki fragments
4. DNA
helicase melts the hydrogen bonds between the bases to open the fork
5. Primase
makes a short RNA primer to begin each new strand.
6. DNA
ligase connects the Okazaki fragments by making the last, untemplated,
phosphodiester bond.
7. Ligase
reaction is two-step reaction.
a. Pyrophosphate removed from ATP and AMP attached to
the 5'P..
b. AMP removed and phosphodiester bond made between the
5'P and the 3'OH of the other nucleotide.
8. Topoisomerase
I works ahead of the replication fork, relieving torsional strain
a. Covalently links to the phosphate of one of the phosphodiester
bonds, thus breaking that DNA polymer temporarily
b. Helix unwinds through the nick, relieving the strain.
c. Topoisomerase I then reforms the original phosphodiester
bond.
9. Single-stranded
DNA binding proteins attach to the lagging strand as DNA helicase melts
the hydrogen bonds, keeping the region single stranded until the replication
apparatus moves through.
10. PCNA
works with DNA polymerase delta on the leading strand to increase its processivity,
allowing the synthesis to be continuous.
11. It
is believed that DNA polymerase delta actually proofreads both strands
during DNA replication.
12. In
three dimensions, the lagging strand winds around the DNA polymerase to
facilitate the priming of new Okazaki fragments.
13. We
believe that the primase function associates with DNA polymerase alpha.
E. Initiation of DNA replication
1. occurs
at origins of replication
a. palindromic sequences that interact with initiator
protein
2. initiator
protein melts the double helix at the origin of replication allowing the
helicase- primase-DNA polymerase alpha complex to load on
3. A replication
fork begins, moving in one direction from the origin
4. Shortly,
a second replication fork begins in the opposite direction.
5. DNA
polymerase delta quickly replaces alpha on the leading strands of both
replication forks once the priming and initial synthesis is done.
6.
This is called bidirectional DNA synthesis and creates replication bubbles
7. Replication
continues until two oppositely moving bubbles meet each other.
8. The
ends of eukaryotic DNA chromosomes must be copied by an unusual reaction
involving DNA telomerase.
Note: The above review is simply an organized list of topics that we have covered in class. It does not necessarily include every detail and aspect of what we discussed but should be a guide for you to look further at the notes and book to be able to apply and discuss the topics on the exam.
1. The hydrophobic interaction occurs primarily due to entropy. When a hydrophobic molecule or portion of a molecule are in a water environment, water molecules must form an ordered cage around the hydrophobic groups in order to be able to form hydrogen bonds with one another. This decreases the entropy of the system as is undesirable, energetically. By having hydrophobic groups pull together in a hydrophobic interaction, this undesirable increase in entropy can be minimized. An example could include: phospholipid bylayers or micelles; hydrophobic side chains of amino acids within proteins interacting and contributing to tertiary structure; many others.
2. The alpha-helical structure is a secondary structure that can form along the polypeptide backbone if the amino acid sequence is appropriate. It is a right-handed helix that is stabilized by hydrogen bonds between the C=O of one peptide linkage and the H-N of another peptide linkage located four linkages away. This occurs for every peptide bond along the helix. The side chains of the amino acids face to the outside of the helix. Consult the textbook for an illustration.
3.
A The active site of trypsin contains
a binding pocket lined with polar or charged amino acids that can interact
through hydrogen bonds and ionic interactions with lysine and arginine
side chains. Trypsin catalyzes the breaking of a peptide bond following
lysine and arginine amino acids in a polypeptide. Chymotrypsin breaks bonds
following bulky hydrophobic side chains like phenylalanine. Its binding
pocket is lined with nonpolar amino acids that form hydrophobic interactions
with the bulky aromatic side chain.
B. When the substrate binds an induced fit brings the amino acids aspartic acid, histidine, and serine, located at the active site, closer together. The negative charge on the aspartic acid side chain pulls a proton towards aspartic acid from histidine causing a negative charg on the histidine. This negative charge pulls a proton from the serine towards histidine. This activates the oxygen of the serine side chain which attacks the C=O of the peptide bond, causing the C to bond covalently to the serine, creating the first tetrahedral intermediate.
4. Chromatin describes the interaction of double-stranded DNA with histone proteins. It consists of a series of nucleosomes connected by linker regions. The nucleosomes are approximately 146 base pairs of DNA wound twice around a histone octamer that consists of two molecules each of histones 2A, 2B, 3, and 4. The DNA that is not wound into the nucleosome structure serves as the linker between these nucleosomes. Linker regions are of variable length, on average 200 base-pairs. Histone H1 binds to the linkers near the junction with the nucleosomes and packs the nucleosomes into a more densely folded structure called the 30nm fiber or solenoid.
5. See the diagram in your textbook. p.104
6. Wobble is nonconventional, stable base-pairing
between nucleotide bases at the 1 position of an anticodon with the 3 position
of a codon. For example: codon 5'XXC3'
anticodon 3'XXI5'
The genetic code is degenerate because of this such that the 3 position
of all codons that can bind in such wobble relationships is irrelevant
to coding, therefore all tRNAs with the appropriate bases at anticodon
positions 2 and 3 carry the same amino acids.
7. Consult the diagram in your textbook. p. 137
8.
A. Amino acyl tRNA synthetase
attaches the correct amino acid to the correct tRNA in a two-step reaction.
First, the amino acid and ATP bind to the active site. Pyrophosphate is
removed from ATP and AMP attached to the carboxyl terminus of the amino
acid, transiently. This first binding requires that the enzyme have the
correct shape at the active site to interact with a particular amino acid.
The successful binding of step one causes the enzyme to alter the conformation
of its active site such that it can only now bond to the appropriate tRNA
molecule. Then AMP is removed from the amino acid and the amino acid is
linked to the 3' end of the tRNA at either the 2' or 3' OH of the ribose.
Since the enzyme has to have two specific conformations, the second dependent
upon the first being correct, it helps insure the accuracy of the amino
acid-tRNA attachment.
B. A complex between eEF1-GTP and a charged tRNA forms in the cytoplasm. This commplex randomly floats into the A site of the ribosome. The anticodon of tRNA contacts the codon. If three interactions can occur between them, the tRNA will be held there long enough for GTP to be hydrolyzed to GDP. This alters the structure of the eEF1 which no longer can remain bonded to the tRNA. It leaves, relieving the steric hindrance on the tRNA molecule and allowing it to stably attach to the A site. The energy that was released by the GTP hydrolysis assists this attachment. Now peptidyl transferase is activated. If the codon-anticodon interaction was not stable (less than 3 interactions), kinetic energy will knock the complex out of the A site before the GTP hydrolysis can occur. This prevents stable attachment of an incorrect tRNA to the A site, helping insure that the correct amino acid be on the tRNA there before peptidyl transferase can act.
9.
A. 2
B. primer 1 can bind to the DNA
sequence at the right end of the molecule but then leaves only a 5'phosphate
end available to extend, which cannot be done.
primer 3 has two problems. There are T's in it so it is not a typical RNA
molecule. Also, it cannot bind in an antiparallel and complementary way
to the DNA sequence at all.
C. DNA polymerase requires a preexisting 3'OH before it can make a phosphodiester bond during DNA polymerization. The primer provides this group.
10. Consult the key in theglass case outside 021 McKinly or your textbook p.375.
11.
A. DNA helicase melts the hydrogen
bonds of the double-stranded DNA molecule as the replication fork opens
up.
B. Topoisomerase I nicks a single strand of the DNA polymer ahead of the replication fork, part of the double-stranded DNA that is not yet melted. The nick allows the other strand to swivel through it, relieving the torsional strain and supercoiling induced by the melting of the double-stranded DNA as the replication fork opens. The enzyme then reseals the nick.
C. PCNA binds to DNA polymerase delta and increases its processivity. This means it helps it remain associated with DNA template strand for long periods as it synthesizes the leading strand.
D. Primase synthesizes a short RNA polymer called the primer that provides the 3'OH for DNA polymerase alpha to begin synthesizing a DNA Okazaki fragment. This also happens during initiation.
E. DNA ligase catalyzes formation of the phosphodiester linkages between Okazaki fragments.
Group Work Question:
Several possible answers here. The simplest is to synthesize a polymer XXXXXXXXXXXX and feed it into an in-vitro translation system to make a polypeptide from it. Then amino acid sequence the polypeptide. Should get only one amino acid, the one coded by XXX.
Can also synthesize XXX and mix it with ribosomes and all other factors needed to carry out translation including tRNAs. Then add all 20 amino acids with one of them labelled. Filter this and see if the radioactive label remains on the filter paper, indicating that the labelled amino acid is carried on a tRNA that has an anticodon for the XXX codon. This will be the amino acid coded by XXX. If the label flows through, the labelled amino acid was not associated with the correct tRNA. Try another with a different labelled amino acid until one remains on the filter. If none do, it may be a stop codon.
Graders:
Questions 1 and 2: Joy He
3 and 4 Sheba Aragawal
5 and 6 Ya Chen
7 and 9 Rania Al-Shami
10 and 11 Wei Chen
8 and Group Dr. Schmieg
Procedure:
1. Check math. Errors to Dr. Schmieg
2. Check key.
3. Grade-related questions taken to the grader for
that question.
4. Initialed grade changes shown to Dr. Schmieg.
5. Must be done by April 12.
6. Must do steps 2 and 3 first!!
1. The hydrophobic interaction is a major determinant of molecular behaviors. Explain why this interaction happens. Your answer should be made in terms of energy considerations. (5 pts) Also give one example of a hydrophobic interaction (5 pts)
2. Describe the alpha helical secondary structure of a polypeptide.
In your answer, include a description of what stabilizes this
structure. (7 pts)
3. Trypsin and chymotrypsin both catalyze the breaking of a peptide bond by a similar mechanism.
A. What is different about the active site of chymotrypsin and
trypsin that causes the specificity of these two similar enzymes? (5 pts)
B. Describe the initial steps in the reaction that they catalyze
that creates the first tetrahedral intermediate. (8 pts)
4. Eukaryotic DNA is found as chromatin in the nucleus. Describe this chromatin structure. (7 pts)
5. Draw one phosphodiester bond in a DNA polymer. Do not draw
in the bases, simply indicate them by the letter B. (7 pts)
6. Illustrate, using an example, what is meant by "wobble" and
what influence did this have on the evolution of the genetic code?
(8 pts)
7. Illustrate the orientation of the tRNA molecules at the ribosome
during the sequential steps of elongation during translation.
Remember that three ribosomal locations need to be considered, E, P, and
A. (8 pts)
8. To avoid having incorrect amino acids put into a polypeptide, the cell has evolved a number of ways to guarantee that no mistakes will be made. Choose one of the following events that occur during or previous to translation and explain how it helps insure the accuracy of the process. Note that to get full credit for this answer you cannot simply describe the process or reaction but must also indicate how this helps guarantee accuracy. (10 pts)
A. the charging reaction
B. the way that tRNAs stably associate with the A site of the
ribosome
9. Consider this situation: A new graduate student is preparing to synthesize a DNA polymer using a reaction mixture that contains DNA polymerase and all other necessary components. All he needs to add is the RNA primer to get things going. His DNA template has the sequence 5’CTTGCAAT……..ATTCGGCT3’. There are three RNA primers in the refrigerator that he could use. They have the following sequences:
primer 1: 5’GAACGUUA3’
primer 2: 5’AGCCGAAU3’
primer 3: 5’UTTGCAAG3’
He chooses a primer and is pleased to see that his experiment has worked quite well.
A. Which primer did he use? (2 pts)
B. Why would the other primers not work? (3 pts)
C. Why did he need to use a primer in the first place? (3 pts)
10. Draw a replication fork, indicating the polarity of the strands
and show which is the leading strand. (7 pts)
11. What role do the following molecules play in the process of DNA replication? (15 pts)
A. DNA helicase
B. topoisomerase I
C. PCNA
D. primase
E. DNA ligase
Group Work Question
How would you determine what amino acid was coded for by the XXX sequence
in the alien mRNA? Be sure to include all components necessary and be clear
as to how the experiment could be interpreted.