1. Why is this useful for the prokaryote? Why can’t eukaryotes do this?
Peer Instruction
5 end
of mRNA 1 2 3
Protein
Ribosome
RNA polymerase
(3 end of
template strand)
Start
of gene
End of gene
(5 end of
What is happening?
Why is this useful for the prokaryote?
Why can’t eukaryotes do this?

2. What is a disadvantage of the eukaryotic system?
Peer Instruction
What is a disadvantage of the eukaryotic system?
What is an advantage of the eukaryotic system?
mRNA
Transcription and
RNA processing
in nucleus
DNA
Mature
mRNA
Mature
mRNA
Translation
in cytoplasm
Ribosome
Protein

3. What is going on here, and why?
(not covered in your reading…) The following structure is sometimes found in the cytoplasm of a eukaryotic cell.
Peer Instruction
A ‘guide’ protein that binds
to both ends of the RNA
What is going on here, and why?
5’ cap
A ribosome
PolyA tail
An RNA molecule
Hint: Only one molecule is really moving…
A new protein

4. After this class, you should be able to:
Monday January 23rd, 2017
Class 13 Learning Goals
Genetic Codes
After this class, you should be able to:
Read and use a codon table for nearly every organism on Earth
Decode an open reading frame of DNA:
When given the frame
When you know that there is a start codon somewhere
On double-stranded DNA
Describe the advantages and disadvantages of coupling between transcription and translation in prokaryotes

5. Codon tables Notice the redundancies
SECOND BASE
FIRST BASE
Phenylalanine (Phe)
Leucine (Leu)
Isoleucine (Ile)
Methionine (Met)
(Start codon)
Valine (Val)
Alanine (Ala)
Threonine (Thr)
Proline (Pro)
Serine (Ser)
Tyrosine (Tyr)
Stop codon
Histidine (His)
Glutamine (Glu)
Asparagine (Asn)
Lysine (Lys)
Aspartic acid (Asp)
Glutamic acid (Glu)
Glycine (Gly)
Arginine (Arg)
Tryptophan (Trp)
Cysteine (Cys)
THIRD BASE
Notice the redundancies
(mostly, but not always, in the 3rd position)

6. In the diagram shown, what represents DNA?
Peer Instruction
What competitive disadvantage would you expect for a species that used a 2-base code? A 4-base code?
In the diagram shown, what represents DNA? 3 5
TAC 5 3
AUG
UAG
Do you think biologists are consistent in how they represent genes? N C

7. 5’ – AUG-UAC-GGC-CCU-UAA-3’
Peer Instruction
This RNA sequence starts from the start codon Translate this sequence using the codon table.
5’ – AUG-UAC-GGC-CCU-UAA-3’
SECOND BASE
FIRST BASE
Phenylalanine (Phe)
Leucine (Leu)
Isoleucine (Ile)
Methionine (Met)
(Start codon)
Valine (Val)
Alanine (Ala)
Threonine (Thr)
Proline (Pro)
Serine (Ser)
Tyrosine (Tyr)
Stop codon
Histidine (His)
Glutamine (Glu)
Asparagine (Asn)
Lysine (Lys)
Aspartic acid (Asp)
Glutamic acid (Glu)
Glycine (Gly)
Arginine (Arg)
Tryptophan (Trp)
Cysteine (Cys)
THIRD BASE
N-Met-Tyr-Gly-Pro-STOP-C
N-Met-Met-Pro-Gly-C
N-Tyr-Gly-Pro-C
N-Met-Tyr-Gly-Pro-C

8. Here is a piece of DNA. There is only one start codon. Translate!
Peer Instruction
Here is a piece of DNA. There is only one start codon. Translate!
5’–ATCTTAGCGGGAATTCATAGTC-3’
5’–TAGAATCGCCCTTAAGTATCAG-3’
SECOND BASE
FIRST BASE
Phenylalanine (Phe)
Leucine (Leu)
Isoleucine (Ile)
Methionine (Met)
(Start codon)
Valine (Val)
Alanine (Ala)
Threonine (Thr)
Proline (Pro)
Serine (Ser)
Tyrosine (Tyr)
Stop codon
Histidine (His)
Glutamine (Glu)
Asparagine (Asn)
Lysine (Lys)
Aspartic acid (Asp)
Glutamic acid (Glu)
Glycine (Gly)
Arginine (Arg)
Tryptophan (Trp)
Cysteine (Cys)
THIRD BASE
Answer = 1

9. 5’–AATGAAGCGGGAATTCTAAGTC-3’
Peer Instruction
What are the pieces of information that you need to be able to translate from any piece of DNA?
Often, molecular biologists will say something like:
“Please translate the sequence shown”.
What is the incorrect assumption in this request?
5’–AATGAAGCGGGAATTCTAAGTC-3’
Answer = 1
Imagine that this DNA is in an open reading frame. How many
possible protein sequences could be encoded here?
5’–AACGAAGCG-3’
3’–TTGCTTCGC-5’

10. In which direction does the RNA polymerase enzyme move?
Peer Instruction
Where does sigma bind?
In which direction does the RNA polymerase enzyme move?
Termination signal
for mRNA H ‘5 ‘3
-35
-10 +1
Promoter H
CCACTTTAAGAAGTCCCCTCAATGGGACATGCGCAAACCAGGCGCTGAAG
GGTGAAATTCTTCAGGGGAGTTATCCTGTACGCGTTTGGTCCGCGACTTC
Where does the new RNA start and end?

11. 1: 5’-NNNNNNNNNNNNNCUUCAGC :20 21: GCCUGGUUUGCGCAUGUCCU :40
CCACTTTAAGAAGTCCCCTCAATGGGACATGCGCAAACCAGGCGCTGAAG
GGTGAAATTCTTCAGGGGAGTTATCCTGTACGCGTTTGGTCCGCGACTTC
Termination signal
for mRNA H ‘5 ‘3
-35
-10 +1
Promoter H
Peer Instruction
This gene encodes the
mRNA sequence shown.
1: 5’-NNNNNNNNNNNNNCUUCAGC :20
21: GCCUGGUUUGCGCAUGUCCU :40
41: AUUGAGGGGACUUCUUAAAG :60
61: UGGNNNNNNN-3’
Where is the Start Codon?
How long is the encoded protein?

12. Concept Questions
A single-celled eukaryote produces proteins more slowly than bacteria of similar size.
Why is this lack of coupling an advantage in highly mutagenic environments?
Why does the bacteria have an advantage in dark ocean-bottom environments with sporadic food surpluses?
Create a random stretch of DNA of 40 bases long.
Translate in each direction as if the AUG was oriented to start the open reading frame at the 5’ end.
Then, retranslate by finding any start codons.
Do you have any unnecessary STOP codons in this DNA?
From your DNA, change the sequence to make it encode a 3-amino-acid protein. Do this with the least changes possible.

13. After this class, you should be able to:
Tuesday January 24th, 2017
Class 13 Learning Goals
Mutations
After this class, you should be able to:
Assess whether or not a molecule is a likely mutagen based on specific chemical effects
Classify point mutations based on changes to a protein product
Predict the outcome of two different point mutations and infer which would likely have more influence on organism fitness
Predict relative effects of point mutations and chromosomal-scale mutations for organisms or cells
Define a gene

14. What is a point mutation?
Peer Instruction
What is a point mutation?
Examine the following changes. Does each change many proteins, change one protein, or have no impact?
Δ in the DNA of a skin cell
Δ in the DNA of a sperm cell
Δ in a protein in a sperm cell
Δ in an mRNA in a skin cell
Which of these changes can impact fitness?
Which of these changes is heritable?

15. Is this a normal human karyotype?
Peer Instruction
Is this a normal human karyotype?
Duesberg et al. paper 2005; breast cancer cell line
Non-normal = lots of aneuploidy (e.g. 3, 5, 6, 9, 10 …)
MANY translocations
Missing chromosomes (e.g. 2, 15, X or Y)
For a difference that you noticed:
How many genes did this change impact?

16. Peer Instruction
Match the following mutation types to the genes from the information found in this example:
Genes:
Regulatory enzyme
Structural protein
Ribosome factor
ATP producer
Mutation Type:
Deletion
Frameshift
Insertion
Missense
Synonymous

17. Peer Instruction
It would be helpful to define a gene. What would we need to include in a robust definition?

18. A working definition of the “gene”.
A gene is a unit of genetic material that
encodes the information necessary to
produce one protein.
Usually DNA
Not necessarily continuous
Often guided by a promoter region

19. Concept Questions
Create a random stretch of protein-coding DNA. Flip a coin, and if heads imagine that the promoter is on the left (and add the DNA needed to encode a start codon there as well).
Pick any single base, and predict the mutation class:
If you remove the base
If you replace the base with two As
Change the base to a different base
Which of these changes for your DNA is most likely to destroy function of the protein?
Why are prenatal doctors much more likely to test for small chromosomal breakages than for point mutations of 5-20 bases?
Which is more likely to be mutagenic:
A cosmic ray that only changes A’s to U’s in DNA
A radioactive isotope that changes tyrosines to phenyalanines in proteins
An enzyme that can replace the DNA sequence ‘AGCGAGGTT’ with ‘AGTTAGGTT’

20. Here is section of double-stranded DNA.
Assuming all possible mRNAs are made,
which protein sequence or sequences will be created?
5-ACTAATGAGACCAGTATCATGTTAACG-3
3-TGAATACTCTGGTCATAGTACAATTGC-5
A 6-amino-acid protein
A 5-amino-acid protein
A 4-amino-acid protein
A 6-amino-acid protein and a 5-amino-acid protein
A 5-amino-acid protein and a 4-amino-acid protein
A 6-amino-acid protein and a 4-amino-acid protein
Answer is 4

21. Peer Instruction
Match the following mutation types to the mutations found in this example:
Genes:
Regulatory enzyme
Structural protein
Ribosome factor
ATP producer
Mutation Type:
Deletion
Frameshift
Insertion
Missense
Synonymous

22. After this class, you should be able to:
Wednesday, January 25th, 2017
Class 14 Learning Goals
DNA Replication
After this class, you should be able to:
Describe the genome-wide process of initiation of DNA replication
Define the role and predict a loss-of-function mutation result for each of the enzymes involved in replication
Given a diagram of replicating DNA, locate likely sites of action for each enzyme involved in replication
Assign descriptive terms appropriately to replication on the leading or lagging strands of a particular replication fork
By the end of this first half of the class, you’ll have an understanding of what these images are trying to describe.

23. A chromosome being replicated
Peer Instruction
A chromosome being replicated
Circular chromosomes (like in prokaryotes)
Old DNA
New DNA
Origin of
replication
Replication
bubble
Linear chromosomes (like most eukaryotes) 3 5 3 5 3 5 5
Old DNA 3
New DNA
Replication
fork
What is an ‘origin’?
Why do eukaryotic chromosomes have multiple origins?
Why is replication able to go in both directions?

24. Why can’t DNA replication start without a primer?
Peer Instruction
Primase synthesizes RNA primer (supplying a 3’ OH)
Topoisomerase relieves twisting forces 5 3 5
Helicase opens double helix
Single-strand DNA-binding proteins (SSBP)
Why can’t DNA replication start without a primer?
Why is ssBP important?
What would be the phenotype of a mutation in:
Topoisomerase
Helicase

25. The sliding clamp has no effect on DNA pol’s ability to
Peer Instruction
Leading strand
Sliding clamp holds
DNA polymerase III in place 3 5
RNA primer 5
The sliding clamp has no effect on DNA pol’s ability to
catalyze one reaction. What does the sliding clamp do?
Why is this called the ‘leading’ strand?

26. the replication fork moving? the polymerase on this strand moving?
Peer Instruction
In which direction is:
the replication fork moving?
the polymerase on this strand moving?
What is the engineering problem faced by the enzymes on the lagging strand?
RNA
primer 5 3 5
Topoisomerase
SSBPs
Primase
Helicase

27. Describe the mechanisms shown here.
Peer Instruction 5 3
Sliding clamp
Okazaki fragment 3 5 5
DNA polymerase III 5 3
Okazaki fragment
Okazaki fragment 5 3 5

28. What is DNA polymerase I doing?
Peer Instruction 5 3
DNA polymerase I 3 5 5
What is DNA polymerase I doing? 5 3
DNA ligase 5 3 5
What would be the phenotype of a deleterious mutation in the ligase-encoding gene?

29. Homework: Replication Enzymes Chart
Function
Location
Mutation
Effects?
ssDBP
Topoisomerase
Helicase
DNA Polymerase I
DNA Polymerase III
Sliding Clamp
Primase
Ligase

30. Concept Questions
How does replication begin on a single small linear chromosome? What proteins are used?
How would this be different for an extremely large circular chromosome?
Complete the given chart for the enzymes involved in replication. For each enzyme, be able to justify the evolutionary advantage and protein cost of the enzyme.
Draw an upside down ‘Y’. Assume that the tail of the ‘Y’ is double stranded DNA. Fill in the locations of all of the enzymes from the chart based on where they are likely to act. You can assume that each arm of the ‘Y’ is 500 bases long and that an Okazaki fragment is 150 bases long on average.
When finished, complete the replication bubble with the other fork.
Which strand (leading or lagging) is best characterized as:
Simple?
Likely to have mutations?
More complicated in terms of enzymes
Likely to have a very long new strand
Bonus: Slower?