Laboratory #9B/10A: Molecular genetics
simulations1
Objectives: This is partially an observational lab and partially an experimental
lab, wherein you will do simulations that help to understand the genetic code.
When you finish this lab, you should:
1. Have an understanding of the experiments that deciphered the genetic
code
2. Understand how changes in DNA sequence produce changes in the
primary structure of a protein
3. Understand how hypotheses regarding genetically-based diseases can be
tested using DNA sequencing and protein analysis.
Background: Even after Watson and Crick had proposed a molecular structure for
DNA, it remained a mystery how 4 nucleotides could be used to code for 20 amino
acids. In the decades prior to the development of modern mechanized molecular
analysis such as amino acid sequencing and DNA sequencing, researchers had to use
other experimental techniques to put together the puzzle.
Even before these experiments, scientists were fairly certain that the genetic
code would involve sequences of three nucleotides for each amino acid. You can
follow their logic by answering these questions:
a) If the code consisted of 2 nucleotides per amino acid, how many different
amino acids could be uniquely identified? Is there any redundancy (more unique
nucleotide sequences than amino acids)? Hint: to calculate the number of unique
“words” in a particular alphabet, use the equation N=lx where N is the number of
words, l is the total number of letters, and x is the number of letters per word.
b) If the code consisted of 4 nucleotides per amino acid, how many different
amino acids could be uniquely identified? Is there any redundancy?
c) If the code consisted of 3 nucleotides per amino acid, how many different
amino acids could be uniquely identified? Is there any redundancy?
Although scientists were fairly certain that the code would consist of 3-nucleotide
codons, the arrangement of those codons was unknown. In particular, would they be
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Modified from BiologyLabsOnline, Translation and Hemoglobin exercises
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overlapping or non-overlapping? An overlapping code would mean that if codon 1
started at position 1 in the DNA, codon 2 would start at position 2 or 3. A nonoverlapping code would mean that codon 2 would start at DNA position 4. And, not
least, they still did not know what 3-nucleotide “words” would correspond to each of
the 20 amino acids. Our first two exercises will address these questions.
In the early 1960s, Nirenberg published his discovery that cell-free extracts of
the bacterium E. coli could translate synthetic RNA into small proteins, or
polypeptides. Initially synthetic RNA could be “composed” only randomly, but later
developments allowed researchers to synthesize RNA with specific nucleotide
sequences. By providing specific RNA nucleotide sequences to the cell-free
extracts, they were able to completely decode the genetic code. We will first
simulate these experiments.
After the code was deciphered, the question remained of whether and how small
changes in a gene, such as single nucleotide substitutions, could affect the protein.
We will explore this question in the second exercise of the lab.
Exercise I: General instructions: Go to the Translation Lab in BiologyLabsOnline,
and click on “Start Experiment”. For each of the four bottles of ribonucleotides,
you can click on an arrow to select a nucleotide. If you then click Make RNA, an
RNA macromolecule will be synthesized using the ribonucleotides in the sequence
you have selected. Clicking on Translation Mix will produce the amino acid
sequence(s) that are synthesized by your RNA sequence. We have found that the
program may crash if you write to the simulation notebook, and recommend that you
make notes in your laboratory notebook for each RNA sequence and the
corresponding protein sequence(s).
Part a: Simple mononucleotide sequences. The simplest codons will be those
involving only one ribonucleotide such as UUU. Synthesize each of these
mononucleotide RNA molecules and determine the corresponding amino acids for
these codons. Can you use these results to determine if the codons are overlapping
or non-overlapping?
Part b: Simple dinucleotide sequences. Synthesize an RNA molecule with two
nucleotides (“XYXYXY”), and translate it to determine the amino acid sequence.
Your nucleotide sequence here_________________
Your protein sequence here____________________
Now reverse the RNA (“YXYXYX”). Do the results change?
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Can you use these results to determine whether the codons are overlapping or nonoverlapping? Hint: what would the codons be if the code was overlapping? What if
the code was non-overlapping?
Part c: Are codons overlapping? A test using trinucleotide sequences. Make an
RNA molecule with three nucleotides (“XYZXYZ”). Before synthesizing the protein,
consider the possible outcomes.
Question
Hypothesis 1
Are codons overlapping?
Overlapping codons in RNA are “read” to produce an amino acid
sequence.
Prediction 1
Your nucleotide sequence_______________________
The successive codons if read in overlapping fashion:
How many different amino acids will be incorporated into a single
protein?
Hypothesis 2
Non-overlapping codons in RNA are “read” to produce an amino acid
sequence.
Prediction 2
Your nucleotide sequence_______________________
The successive codons if read in non-overlapping fashion:
How many different amino acids will be incorporated into a single
protein?
Repeat this experiment with three more combinations of ribonucleotides.
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Results: Fill your results in this table
Experimental RNA sequences
Protein sequence(s) produced
A)
B)
C)
D)
Evaluation and inferences: Use your results to answer the following questions:
1. Do your results cause you to reject either the “overlapping codon” or “nonoverlapping codon” hypothesis? Explain your reasoning.
2. From parts a and b, which codons have you now linked to a specific amino acid?
3. You likely got qualitatively different results in parts b and c, with different
numbers of resulting protein sequences for each RNA sequence. What does this
tell you about how translation is initiated in the cell-free synthesis?
4. Did any of your sequences from part c have a different number of proteins
compared to the others? Why might this be?
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Exercise II: The affect of changes in nucleotide sequence on protein structure
and function. Modern molecular techniques combined with medical science have
greatly enhanced our understanding of the bases for many genetically-linked
diseases. Hemoglobin is the protein that, bound to iron in 4-protein complexes,
binds oxygen and moves it from the lungs to the rest of the body. The complex is
so large that it’s structure largely determines the shape of the red blood cells in
which it is synthesized. In this section, we will explore how changes in nucleotide
sequence (mutations) in the gene for hemoglobin B alter the structure and function
of the protein.
before you start: Because this laboratory alternates with a wet lab, some of you
may not have yet discussed the “Central Dogma” of molecular genetics, that DNA is
transcribed to RNA which is translated to a protein. If this is the case, or if you
are still confused by these concepts, please take some time right now to work
through a transcription and translation simulation at the University of Utah
Genetics web site, http://gslc.genetics.utah.edu/units/basics/index.cfm
part a: Gel electrophoresis Gel electrophoresis is a technique that separates
proteins by size and shape. Longer, more bulky or highly charged proteins move
through the gel more slowly. In a study, different samples are placed into each well
on the gel, an external standard of “normal protein” is included in one well for
comparison. Go to the Hemoglobin lab in BiologyLabsOnline. There are a variety
of “cases” listed. Click on “Gel Electrophoresis” and view several different cases.
Choose two cases for further study.
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part b: Observations Explore the “patient history”, “blood samples”and
“microscope samples” for one case. Briefly note below your observations. Finally,
click on “peptide sequence”. Search for differences in sequence by scanning or by
clicking on “find difference”. Note the difference below, and then repeat for a
second patient.
Case 1 :
Case 2:
family history: do any
relatives have similar
conditions?
gel electrophoresis:
blood color:
cell structure:
Protein sequence:
part c) Testing hypotheses. Based upon your observations and the genetic code,
develop an explicit hypothesis concerning how the gene for each patient’s heme
molecule might differ from normal. In particular, note what changes in the gene
you expect to find and where (which codon) you expect to find them. Note that you
will be working with DNA sequences, not RNA as in exercise I.
Hypothesis, case 1:
Hypothesis, case 2:
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Use the “Edit DNA” function of the lab to test your hypothesis. The general
protocol is as follows:
First, find the beginning of the coding region of the gene by typing in the DNA
sequence that codes for “start” (methionine) and hitting return. Note the location
of “start”, and then click on “bracket codons”. Now the gene is bracketed into 3
base codons.
Calculate which base location(s) will correspond to your predicted differences for
case 1, and scroll or jump to that number (jump by typing in the base number and
hitting return). Make the change(s) that you predicted by clicking on a base, then
clicking on the base you wish to change it to in the “change to” box; repeat until
you have made all of your desired changes. Lastly, click “translate”. The patient’s
protein sequence, the normal sequence, and the protein sequence produced by your
experimental DNA will all appear. Find the appropriate amino acid location, and
note your results.
Repeat for the second patient.
Results: What changes did your alterations of the DNA sequence generate in the
protein sequence?
Evaluation: For each patient and corresponding hypothesis, did you correctly
predict the changes in gene sequence resulting in differences in protein sequence
(in other words, do your results cause you to reject your hypothesis)?
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