Final Review Session

Bio 99-Luo/Glabe
Nathan Farrokhian
Mai-Anh Vuong-Dac

EVALUATE YOUR TUTORS ONLINE!

Quiz 1 Material

Centra dogma and its exceptions
DNA and RNA similarities and differences
What characteristics make DNA a good carrier of our genetic information?
Chargaffs rules
DNA denaturation
DNA structure
Nucleic acid hybridization
How did scientists prove DNA was the genetic material? Know the purpose and findings of each experiment:
Griffiths bacterial transformation
Avery, MacLeod, McCarty
Hershey and Chase
Beadle and Tatum* (Professor Glabes material ties in with this)
Define gene and genome.
Define introns, transposons, simple sequence repeats.
Supercoiling Problems! (linking number, twisting, writhing)
Histone Proteins and Nucleosomes

Quiz 2 Material

DNA replication (semi-conservative, conservative, and dispersive)
Meselson and Stahl experiment
DNA polymerase reaction
Know important proteins and their functions at initiation, elongation, and termination stages of replication
Know the difference between lagging and leading strands. Be able to identify.
Telomerase reaction
Lesion vs. mutation
Review the different mutations
Proofreading and repair mechanisms

Quiz 3 Material
You were all just tested on this! The review packet for this quiz (and for the other two review sessions) are posted!

Complementation Testing
Answers two questions:
Are two independent mutations in the same
gene?
How many genes are involved in a biosynthetic
pathway?

Example!
0 = No growth
+ = Growth
Only based on the given
information:

Which mutations are on the same
gene?




How many genes are involved in
this biosynthetic pathway?

m1, m5, m7
m2, m4
m3
m6
With the information given, we
could estimate four
Colinearity between DNA and Proteins
Linear relationship between codons (DNA) and AAs
(proteins)

General Experiment Set Up:
1. Allow gene to translate
2. Cleave the protein into smaller
peptides
3. Separate the peptides by mapping
them using electrochromatography
i. Chromatography
ii. Electrophoresis
Perform Experiment:
1. Use a wild type gene and take an identical gene with a point mutation
2. Perform above experiment
3. Observe differences in the electrochromatography results
Overlapping vs Nonoverlapping
Point Mutations:

In the nonoverlapping
model: only a single
amino acid would change
in the final protein
product

In the overlapping model:
three amino acids would
change in the final
protein product






NO READING FRAMES!!
Clicker Question
How will the final protein product change if an
insertion occurs in a gene in the overlapping
model?

A. One amino acid is added
B. Two amino acids are changed
C. All amino acids downstream of the mutation site will be
effected
D. Three amino acids are changed
E. Two of the above
Clicker Question
How will the final protein product change if an
insertion occurs in a gene in the overlapping
model?

A. One amino acid is added
B. Two amino acids are changed
C. All amino acids downstream of the mutation site will be
effected
D. Three amino acids are changed
E. Two of the above
Clicker Question
Do you guys want a doc cam presentation to
explain why the previous question is true?

A. Yes
B. No
Discussion Question
In reality, an insertion or deletion near the
beginning of a wild type gene will change the
__________ to a(n) ________.

A. Closed reading frame; Open reading frame
B. Open reading frame; Closed reading frame
C. Non random sequence; Random sequence
D. Number of Amino Acids in the protein; lower
amount
E. Three of the above


Discussion Question
In reality, an insertion or deletion near the
beginning of a wild type gene will change the
__________ to a(n) ________.

A. Closed reading frame; Open reading frame
B. Open reading frame; Closed reading frame
C. Non random sequence; Random sequence
D. Number of Amino Acids in the protein; lower
amount
E. Three of the above


Open vs Closed Reading Frame
Open reading frame:
purposeful, nonrandom
organization of codons
1 stop per ~300 codons
statistically not by
chance
Closed reading frame:
Random organization of
codons
Statistically there
should be 1 stop codon
per ~20 codons (3
stop/64codons)
Analyze the stop codons!

A frame shift error changes an open reading frame to a closed reading frame
downstream of the mutation. Therefore, you are very likely to end up with a
truncated, useless protein.
Clicker Question
How many reading frames are present in any
given gene?
A. 1
B. 2
C. 3
D. 4
E. 5
Clicker Question
How many reading frames are present in any
given gene?
A. 1
B. 2
C. 3
D. 4
E. 5
Clicker Question
How many of the frames are open and how
many are closed?

A. 3 open 2 closed
B. 1 open 2 closed
C. 3 open zero closed
D. 2 open 1 closed
E. None of the above

Clicker Question
How many of the frames are open and how
many are closed?

A. 3 open 2 closed
B. 1 open 2 closed
C. 3 open zero closed
D. 2 open 1 closed
E. None of the above

Identifying the Codon Protein
Relationship
Breaking the code!
SET UP FOR THE EXPERIMENT:
1.Break open the cells (lyse) extract cell
constituents
2.Destroy DNA with Dnase
i. Removes background
3.Separate cell constituents into 20 test tubes
4.Add a single different radioactively labeled AA
to each tube
Breaking the Code!
PREFORM THE EXPERIMENT!
1. Add synthetic mRNA allow for translation
2. Add trichloro acetic acid (TCA) precipitates
radioactively labeled polypeptides (the AAs
must be bound together for a precipitate to
form!)
3. Determine which test tubes contain a
precipitate.

Clicker Question
What is the maximum number of amino acids
that a polymerized di-nucleotide (NN)
n
can code
for?

A. 1
B. 2
C. 3
D. 4
E. 5
Clicker Question
What is the maximum number of amino acids
that a polymerized di-nucleotide (NN)
n
can code
for?

A. 1
B. 2
C. 3
D. 4
E. 5
Clicker Question
How many reading frames does a polymerized
tri-nucleotide (NNN)
n
contain?

A. 1
B. 2
C. 3
D. 4
E. 5


Clicker Question
How many reading frames does a polymerized
tri-nucleotide (NNN)
n
contain?

A. 1
B. 2
C. 3
D. 4
E. 5


Clicker Question
When using a polymerized tri-nucleotide (NNN)
n,
what is the maximum number of different amino
acids present in a single protein once translation
occurs?

A. 1
B. 2
C. 3
D. 4
E. 5
Clicker Question
When using a polymerized tri-nucleotide (NNN)
n,
what is the maximum number of different amino
acids present in a single protein once translation
occurs?

A. 1
B. 2
C. 3
D. 4
E. 5
Breaking the Code: Doc Cam!
For more complex codons
FILTER BINDING ASSAY!
1.Produce trinucleotides
2.Incubated ribosomes with a mix of cold aminoacyl-
tRNAs and one hot one
3.Add specific trinucleotide codons
4.If the codon in the trinucleotide corresponds to a
specific anti-codon of tRNA, both will stick to ribosome
5.This complex is big; therefore, when filtered everything
else will flow through except for the complex
6.Analyze what amino acid did not make it through the
filter




Filter binding assay
Amplification of DNA
Polymerase Chain Reaction (PCR)
Denaturation - the DNA is
heated to ~95
o
C to break
hydrogen bonds.
Annealing Cool to 45-
72C. This allows the
primers to anneal to their
complementary sequence
Extension Heat to 72
o
C
DNA polymerase extends
the primer
REPEAT!

Clicker Question
What primers should be used for the following
DNA segment to run an optimal PCR reaction?
5 ATCGCAGGCAATGCA 3
3 TAGCGTCCGTTACGT 5
A.3 GCT 5 ; 3 TAG 5
B.3 TCG 5 ; 3 TAG 5
C.3 ATC 5 ; 3 GCA 5
D.3 CGT 5 ; 3 CTA 5

Clicker Question
What primers should be used for the following
DNA segment to run an optimal PCR reaction?
5 ATCGCAGGCAATGCA 3
3 CGT 5
5 ATC 3
3 TAGCGTCCGTTACGT 5

3 CGT 5 ; 3 CTA 5

Sequencing Experiments
Sequencing: Sanger Method
Uses specific dideoxynucleoside triphosphates
(ddNTP).
Importance: No 3 OH. Therefore, you cannot
add an additional nucleotide elongation
halts
How to utilize
ddNTPs
1. Small amounts of a specific
ddNTP into four test tubes
2. Add template + primer +
DNA Pol + normal dNTPs
3. Elongation randomly ceases
at the specific ddNTP in
each tube
4. Mix contents and run an
electrophoresis
5. Analyze using dye/length
Illumina Sequencing
1. Fragment DNA into ~300bp segments
2. Prepare the DNA (steps not important)
3. Add DNA polymerase, primers, and
fluorescent terminator nucleotides
4. Each nucleotide addition will block further
elongation
Illumina Sequencing
4. Excite florescent
probe in order to
identify the specific
nucleotide added
5. Cleave probe and
3 OH blocking
group
6. REPEAT
Determines the sequence
one base at a time
Would you guys like to see a doc cam example
of:

A. Sanger sequencing
B. Illumina sequencing
C. Both
D. Neither
Quick Break
The genetic code is considered
degenerate because_______.
A. RNA is unstable
B. More than one trinucleotide codes for the
same amino acid
C. Constantly changing
D. Lacking some property or order
The genetic code is considered
degenerate because_______.
A. RNA is unstable
B. More than one trinucleotide codes for the
same amino acid
C. Constantly changing
D. Lacking some property or order
The wobble hypothesis
involves_________.
A. tRNA
B. mRNA
C. rRNA
D. Both mRNA and rRNA
E. Both mRNA and tRNA
The wobble hypothesis
involves_________.
A. tRNA
B. mRNA
C. rRNA
D. Both mRNA and rRNA
E. Both mRNA and tRNA
DIFFERENCE BETWEEN WOBBLE
HYPOTHESIS AND DEGENERACY
LETS EXPLORE!
The Wobble Hypothesis
Crick: wobble occurs at the 3
rd
position of the
______ and 1
st
base of ______




Reason: physical flexibility in ______ anticodon
loop
The Wobble Hypothesis
wobble occurs at the 3
rd
position of the codon
and 1
st
base of tRNA anti-codon




Reason: physical flexibility in ______ anticodon
loop
The Wobble Hypothesis
wobble occurs at the 3
rd
position of the codon
and 1
st
base of tRNA anti-codon




Reason: physical flexibility in tRNA anticodon
loop
The Wobble Hypothesis
Example: adenine turns into ________ post-txn.
Deamination

Inosines in anticodon is wobbly and binds to
___, ____, or ____

The Wobble Hypothesis
Example: adenine turns into inosine post-txn.
Deamination
Inosines in anticodon is wobbly and binds to A,
C, or U

1
st
position of tRNA anti-codon is usually an
abnormal base!
Decoding the Genetic code by tRNAs
Wobble Hypothesis allows for third base
_________
-3
rd
base of codon has more/less influence on
AA identity?

Nirenberg: Poly U binds _____ Phe- tRNAs
UUC codon codes for Phe
Implication: ?
Decoding the Genetic code by tRNAs
Wobble Hypothesis allows for third base
degeneracy.
-3
rd
base of codon has more/less influence on
AA identity?

Nirenberg: Poly U binds ____Phe- tRNAs
UUC codon codes for Phe
Implication: ?
Decoding the Genetic code by tRNAs
Wobble Hypothesis allows for third base
degeneracy.
-3
rd
base of codon has more/less influence on
AA identity?

Nirenberg: Poly U binds all Phe- tRNAs
UUC codon codes for Phe
Implication: ?
Decoding the Genetic code by tRNAs
Wobble Hypothesis allows for third base
degeneracy.
-3
rd
base of codon has more/less influence on
AA identity?

Nirenberg: Poly U binds all Phe- tRNAs
UUC codon codes for Phe
Implication: only ONE tRNA for Phe
Decoding the Genetic Code by
tRNAs continued
Robert Holley: single yeast Ala-tRNA binds to
Single/multiple codons

OVERALL CONCLUSION: ?
Decoding the Genetic Code by tRNAs
continued
Robert Holley: single yeast Ala-tRNA binds to
Single/multiple codons

OVERALL CONCLUSION: tRNA can read more
than 1 codon
Wobble Rules
1. It ONLY occurs between 3
rd
codon position (3
end) and 1
st
anti-codon (5 end)

2. In addition to W-C base pairing, G and U can pair
with one another

3. If A is deaminated to I in anti-codon and can bind
to A, U, or C from codon

*REMEMBER: Inosine (I) ONLY occurs in tRNA

Advantages of Wobble
1. Less tRNA genes needed
a. 32 tRNAs needed for the 61 SENSE codons and 3
STOP codons

2. non-canonical (non-W-C) pairing provides
weaker interaction btw. tRNA and mRNA
a. Less stable means higher rate of protein synthesis
Dangers of Wobble
tRNA activation
Which enzyme catalyzes activation?

1. Adenylylation: AA attacks adenosine
triphosphate and releases _____________.
2. creates _______-AMP
3. tRNA __________: 3 end of tRNA attacks
aminoacyl
* This step is called _____________________
tRNA activation
Which enzyme catalyzes activation?

1. Adenylylation: AA attacks adenosine
triphosphate and releases pyrophosphate.
2. creates _______-AMP
3. tRNA __________: 3 end of tRNA attacks
aminoacyl
* This step is called _____________________
tRNA activation
Which enzyme catalyzes activation?

1. Adenylylation: AA attacks adenosine
triphosphate and releases pyrophosphate.
2. creates amino-acyl-AMP
3. tRNA __________: 3 end of tRNA attacks
aminoacyl
* This step is called _____________________
tRNA activation
Which enzyme catalyzes activation?

1. Adenylylation: AA attacks adenosine
triphosphate and releases pyrophosphate.
2. creates amino-acyl-AMP
3. tRNA charging : 3 end of tRNA attacks amino-
acyl AMP
*This step is also called tRNA loading
Ribosome does not check if..
After tRNA is charged, what does ribosome fail
to do?

How do we know this?


Ribosome does not check if..
After tRNA is charged, what does ribosome fail
to do?
Ribosome does NOT check if the correct A.A. is
correctly loaded on a tRNA

How do we know this?


When Cys on a Cys-tRNA is chemically converted
to Ala (alanine) via reductive desulfurization

Alanine will be inserted at Cys codons.
This proves that ribosomes do not proofread to
check that the correct AA is attached to tRNA.
Amino-acyl tRNA synthetases have
their own code: second genetic
code
-correct combo. Of A.A. and tRNA
-aka properly charging tRNA
-its called second genetic code because
without this proper pairing, genetic code
cant be translated properly
Double sieve mechanism
If Tyr is able to pass through the first sieve, and
the second sieve, then what can we say about
Tyr?

a. Too big
b. Too small
c. Just right
Double sieve mechanism
If Tyr is able to pass through the first sieve, and
the second sieve, then what can we say about
Tyr?

a. Too big
b. Too small
c. Just right
Double Sieve Mechanism
If Gly passes through only the first sieve, then
what can we say about Gly?

a. Too big
b. Too small
c. Just right
Double Sieve Mechanism
If Gly passes through only the first sieve, then
what can we say about Gly?

a. Too big
b. Too small
c. Just right
The first sieve acts as the
activation site (By ATP)

The second sieve acts as
the editing site

An A.A. small enough to
fit in the activation and
editing site will be
hydrolyzed
Aminoacyl-tRNA synthetases can
distinguish between tRNAs
Specific tRNAs have identity nucleotides
Synthetase makes contact with these
nucleotides

These are also called identity
Elements
-acceptor, anticodon, and
D-arms

Lecture 19: Translation Steps
Prokaryotic ribosome complex = 70 S
Larger subunit = 50 S
Smaller subunit = 30 S

At the center of the 30 S subunit, where does
catalysis, or peptidy transfer, occur?
a. At a protein
b. at a complex of rRNA and protein
c. At a ribozyme
Lecture 19: Translation Steps
Prokaryotic ribosome complex = 70 S
Larger subunit = 50 S
Smaller subunit = 30 S

At the center of the 30 S subunit, where does
catalysis, or peptidy transfer, occur?
a. At a protein
b. at a complex of rRNA and protein
c. At a ribozyme (no protein at the center)
Ribosome
Three tRNA binding sites
Half of the site is in the 30 S and half is in the
50S subunit

Where does the peptidyl transferase reaction
occur?
Between the P and A site of the 50S

Translation is an Enzymatic
Processive Reaction
Initiation:
Which initiation factor is a GTP binding protein?
eIF2
What is the first A.A. in every polypeptide
chain? And which codon codes for it?
Met, AUG
The ________ complex is comprised of GTP,
eIF2, and Met
Translation is an Enzymatic
Processive Reaction
Initiation:
Which initiation factor is a GTP binding protein?
eIF2
What is the first A.A. in every polypeptide
chain? And which codon codes for it?
Met, AUG
The ternary complex is comprised of GTP, eIF2,
and Met
Initiation
on the mRNA strand, initiation factors screen
for the 5 cap = 5 triphosphate 7-
methylguanylate cap
It serves to connect mRNA to initiation factors

Which consensus sequence establishes the
reading frame? How do we know which AUG
is the start codon?
Because of the Kozak sequence!

What is special about the first
tRNA?
Initiatior Met-tRNA is structurally distinct
Different than the tRNA that codes for
methionine during elongation
Only the initiator tRNA can bind to eIF2
The first tRNA-A.A. binds to the P site which is
the decoding site during initiation
During elongation, the decoding site is the
A site

Initiation
GTP hydrolysis at the eIF2
Initiation factors released
Large subunit binds to small subunit and
makes a 70S complex
Elongation
Polysome formation is directed by 5 cap and
poly A tail (mRNA is looped into circle)

Multiple ribosomes act on one Mrna

Incoming tRNA situate at the A site
mediated by elongation factors that are bound to
GTP
Released later by GTP hydrolysis
Elongation: Peptide Bond
formation
growing POLYPEPTIDE
chain is attached at the
P site (peptidyl site)

Incoming AMINO ACID
attached to the tRNA in
the A site (aminoacyl
site)


Elongation
Next step is for acceptor stem ends of tRNA
move over one site (P E, A P) of 50S
Anticodon does NOT move

Translocation (GTP hydrolysis)
-GTP hydrolyzed on EF-G
-NOW anticodon and tRNA molecule itself
moves over by one codon on 30S
Ribosome proofreading
Ribosome does NOT proofread the A.A. added
onto the tRNA

What does ribosome proofread?
Ribosome proofreads codon-anti-codon base
pairing

If base pairing is incorrect, rejection and
removal
Translation Termination
Energy Cost of Prokaryotic Protein
Synthesis

2N ATPs to charge tRNA (ATP -> AMP + PP -> AMP + 2Pi)
1 GTP for initiation (IF2)
N-1 GTPs to position tRNA for N-1 peptide bonds (EF-Tu)
N-1 GTPs for N-1 translocation steps (EF-G)
1 GTP for termination (RF-3)
====
4N

A total of 4 high energy phosphate bonds is cleaved per AA. Each ATP or GTP
generates ~40 kJ/mol. Each peptide bond costs ~160 kJ/mol in the cell, yet an
uncatalyzed chemical reaction to form a peptide bond costs only ~20 kJ/mol.
Why is it so costly to make a peptide bond on a ribosome? The excess energy is
used for generating an accurate, defined polypeptide sequence, not a random
one or a combination of multiple possibilities.
Puromycin
What did they discover when adding the
molecular mimic of the 3 end of the AA-
tRNA?

Causes premature termination and release of
the growing chain

So we know that only a termination factor is
needed and not a specific tRNA
Antibiotic inhibition of protein
synthesis

Streptomycin: Inhibits initiation by blocking the binding of
fMet-IF2. (Also causes misreading of codons.)

Chloramphenicol: Inhibits peptidyl transferase.

Tetracycline: Inhibits elongation by blocking delivery of
AA-tRNA to A site.

Erythromycin: Inhibits elongation by blocking
translocation.
* inhibitors of cell growth (cytostatic) or cell survival
(cytotoxic).