Transcript Lecture 5-8 Bio200 Summer2011s

Peer Instruction
5 end
of mRNA
3
2
2
Start
of gene
1
(3 end of
template strand)
1
Protein
1
Ribosome
RNA polymerase
1) What is happening?
2) Why is this useful for the prokaryote?
3) Why can’t eukaryotes do this?
End of gene
(5 end of
template strand)
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
Ribosome
Protein
Translation
in cytoplasm
(not covered in your reading…) The following
structure is sometimes found in the
cytoplasm of a eukaryotic cell.
What is going on here, and why?
Peer Instruction
A ‘guide’ protein that binds
to both ends of the RNA
5’ cap
A ribosome
PolyA tail
An RNA molecule
Hint: Only one
molecule is
really moving…
A new protein
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
SECOND BASE
Codon tables
Phenylalanine (Phe)
Serine (Ser)
Cysteine (Cys)
Stop codon
Stop codon
Stop codon
Tryptophan (Trp)
Histidine (His)
Leucine (Leu)
Arginine (Arg)
Proline (Pro)
Glutamine (Glu)
Isoleucine (Ile)
Asparagine (Asn)
Serine (Ser)
Lysine (Lys)
Arginine (Arg)
Threonine (Thr)
Methionine (Met)
(Start codon)
Aspartic acid (Asp)
Valine
(Val)
Alanine
(Ala)
Glycine (Gly)
Glutamic acid (Glu)
Notice the redundancies
(mostly, but not always, in the 3rd position)
THIRD BASE
FIRST BASE
Leucine (Leu)
Tyrosine (Tyr)
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
-35
-10 +1
TAC
AUG
UAG
Do you think biologists
are consistent in how
they represent genes?
N
5
C
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
Phenylalanine (Phe)
Serine (Ser)
Cysteine (Cys)
Stop codon
Stop codon
Stop codon
Tryptophan (Trp)
Histidine (His)
Leucine (Leu)
Arginine (Arg)
Proline (Pro)
Glutamine (Glu)
Isoleucine (Ile)
Asparagine (Asn)
Serine (Ser)
Lysine (Lys)
Arginine (Arg)
Threonine (Thr)
Methionine (Met)
(Start codon)
Aspartic acid (Asp)
Valine
(Val)
Alanine
(Ala)
Glutamic acid (Glu)
Glycine
(Gly)
THIRD BASE
FIRST BASE
Leucine (Leu)
Tyrosine (Tyr)
Peer Instruction
Here is a piece of DNA.
There is only one start codon. Translate!
5’–ATCTTAGCGGGAATTCATAGTC-3’
5’–TAGAATCGCCCTTAAGTATCAG-3’
SECOND BASE
Phenylalanine (Phe)
Serine (Ser)
Cysteine (Cys)
Stop codon
Stop codon
Stop codon
Tryptophan (Trp)
Histidine (His)
Leucine (Leu)
Arginine (Arg)
Proline (Pro)
Glutamine (Glu)
Isoleucine (Ile)
Asparagine (Asn)
Serine (Ser)
Lysine (Lys)
Arginine (Arg)
Threonine (Thr)
Methionine (Met)
(Start codon)
Aspartic acid (Asp)
Valine
(Val)
Alanine
(Ala)
Glutamic acid (Glu)
Glycine
(Gly)
THIRD BASE
FIRST BASE
Leucine (Leu)
Tyrosine (Tyr)
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”.
5’–AATGAAGCGGGAATTCTAAGTC-3’
What is the incorrect assumption in this request?
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’
Where does sigma bind?
Peer Instruction
In which direction does the RNA polymerase enzyme move?
Termination signal
for mRNA H
-10
Promoter H
-35
Where does the new RNA start and end?
Termination signal
for mRNA H
This gene encodes the
mRNA sequence shown.
Peer Instruction
-10
Promoter H
-35
1: 5’-NNNNNNNNNNNNNCUUCAGC
:20
21:
GCCUGGUUUGCGCAUGUCCU
:40
41:
AUUGAGGGGACUUCUUAAAG
:60
61:
UGGNNNNNNN-3’
Where is the Start Codon?
How long is the encoded protein?
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.
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
What is a point mutation?
Peer Instruction
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?
Is this a normal human karyotype?
For a difference that you noticed:
How many genes did this change impact?
Peer Instruction
Peer Instruction
Match the following mutation types to
the genes from the information found in this example:
Genes:
Regulatory enzyme
Structural protein
Mutation Type:
Deletion
Frameshift
Ribosome factor
Insertion
ATP producer
Missense
Synonymous
16
Peer Instruction
It would be helpful to define a gene.
What would we need to include in a robust definition?
17
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
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’
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
1.
2.
3.
4.
5.
6.
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
Match the following mutation types to
the mutations found in this example:
Genes:
Regulatory enzyme
Structural protein
Peer Instruction
Mutation Type:
Deletion
Frameshift
Ribosome factor
Insertion
ATP producer
Missense
Synonymous
21
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
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
3
5
5
3
What is an ‘origin’?
5
5
3
Old DNA
New DNA
Replication
fork
Why do eukaryotic chromosomes have multiple origins?
Why is replication able to go in both directions?
Primase synthesizes
RNA primer
(supplying a 3’ OH)
5
Peer Instruction
Topoisomerase
relieves twisting
forces
3
5
Single-strand DNAbinding proteins (SSBP)
Helicase opens
double helix
Why can’t DNA replication start without a primer?
Why is ssBP important?
What would be the phenotype of a mutation in:
• Topoisomerase
• Helicase
Leading
strand
3
Peer Instruction
Sliding clamp holds
DNA polymerase III in place
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?
In which direction is:
• the replication fork moving?
• the polymerase on this strand moving?
Peer Instruction
What is the engineering problem faced by
the enzymes on the lagging strand?
RNA
primer
5
3
5
Topoisomerase
SSBPs
Primase
Helicase
Describe the mechanisms shown here.
5
3
Okazaki
fragment
Sliding
clamp
3
5
5
DNA polymerase
III
5
3
3
5
Okazaki
fragment
Okazaki
fragment
5
Peer Instruction
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?
Homework: Replication Enzymes Chart
Enzyme
ssDBP
Topoisomerase
Helicase
DNA
Polymerase I
DNA
Polymerase III
Sliding Clamp
Primase
Ligase
Function Location Mutation
Effects?
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?