Tutorial 5: Gene Expression

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Tutorial 5 Gene Expression

• Purpose The Central Dogma of
• DNA RNA Protein
• has been well established.
• Put yourself in the role of the researcher and
  interpret the results from the following
  experiments which have helped us to elucidate the
  mechanisms of gene expression.

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Experiment 1

• In response to a change in the bacteria
  environment (lactose addition), new proteins are
  synthesized and ß-galactosidase (an enzyme that
  breaks down lactose) activity appears.

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Experiment 1 Interpretation

• In present day we know that there is an
  intermediate step between DNA Protein
  synthesis. Looking at the previous slide, what
  is the evidence that an intermediate exists?
• What does this experiment show with regards to
  the speed in which this intermediate is
  synthesized?

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Experiment 1 Answer

• In present day we know that there is an
  intermediate step between DNA Protein
  synthesis. Looking at the previous slide, what
  is the evidence that an intermediate exists?
• There is a slight lag before protein production
  occurs.
• Protein production stops when lactose is removed.
• What does this experiment show with regards to
  the speed in which this intermediate is
  synthesized?
• Rapid synthesis

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Experiment 1 Answer
Kinetics of induction not compatible with a
stable DNA template
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Experiment 1 Answer
E1 -Rapid synthesis
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Experiment 2

• Now that you know an intermediate exists, you
  want to determine the nature of the molecule
  (i.e. is it a polynucleotide?) and show that it
  associates with ribosomes (the protein synthesis
  machinery).
• You grow bacteria in the presence of 15N-13C in
  order to radioactively label the ribosomes.

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Experiment 2 (contd)

• You then infect the bacteria in the presence of
  14N-12C which will label newly synthesized
  ribosomes, Methionine35S which will label newly
  synthesized proteins and Uracil32P which will
  label the intermediate IF it is RNA.
• You then collect different fractions of the cell
  lysate by density centrifugation and analyze the
  fractions for the presence of 15N-13C or 14N-12C
  (old/new ribosomes), Methionine35S (proteins) and
  Uracil32P (RNA).

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14N-12C
15N-13C
15N-13C
14N-12C Uracil32P-Methionine35S
High density
Low density
32P-35S ?
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High density
Low density
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Experiment 2 Interpretation

• Looking at the previous 2 slides, what is the
  evidence that the intermediate is RNA?
• What does this experiment show with regards to
  RNAs and proteins association with ribosomes?
• What does this experiment tell you about the
  specificity of ribosomes (i.e. does an individual
  ribosome only have the ability to produce 1 types
  of protein)? What is the evidence?

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Experiment 2 Answer

• Looking at the previous 2 slides, what is the
  evidence that the intermediate is RNA?
• Polynucleotide is produced with Uracil32P
• What does this experiment show with regards to
  RNAs and proteins association with ribosomes?
• RNA associated with pre-existing ribosomes
• New proteins synthesized by pre-existent
  ribosomes
• What does this experiment tell you about the
  specificity of ribosomes (i.e. does an individual
  ribosome only have the ability to produce 1 types
  of protein)?
• Ribosomes are not specialized, synthesize
  proteins according to the RNA associated

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Experiment 2 Answer
E2 -Intermediate should be a polynucleotide E2
-Should associate with ribosomes E1 -Rapid
synthesis
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Experiment 3

• You are gathering more evidence that RNA is the
  intermediate in gene expression.
• You infect a bacterial culture with phage in the
  presence of Uracil32P. You then replace the
  culture medium with media containing
  Methionine35S.
• Finally you assay the cell lysate over time for
  the presence of Uracil32P and Methionine35S (N.B.
  Uracil32P and Methionine35S that is not
  incorporated into RNA or protein, respectively,
  is not counted)

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Uracil32P
Methionine 35S
Radioactivity
time
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Experiment 3 Interpretation

• Looking at the previous slide, what does this
  experiment tell you about the stability of RNA
  and protein?

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Experiment 3 Answer

• Looking at the previous slide, what does this
  experiment tell you about the stability of RNA?
• RNA is degraded quickly.
• Because protein still accumulates, the
  degradation of RNA is not due to cell death.

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Experiment 3 Answer
E3 -Unstable E2 -Intermediate should be a
polynucleotide E2 -Should associate with
ribosomes E1 -Rapid synthesis
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Experiment 4

• You are still gathering more evidence that RNA is
  the intermediate in gene expression.
• You infect a bacterial culture with phage in the
  presence of Uracil32P and collect the RNA.
• You electrophorese the RNA on an agarose gel to
  separate the RNA based on size and expose the gel
  to an X-ray film (N.B. anywhere there is
  radioactivity the film will turn black).
• Before exposing the gel to X-ray film you stain
  it with Ethidium Bromide (which allows you to
  visualize nucleic acids- in this case Total RNA).

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RNA
Uracil32P
X-ray
Total
3000
2000
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Experiment 4 Interpretation

• Looking at the X-ray, what does this experiment
  tell you about the size of RNA produced?
• Why does the Ethidium Bromide stained gel (Total)
  look different? What are the 3 bands?

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Experiment 4 Answer

• Looking at the X-ray, what does this experiment
  tell you about the size of RNA produced?
• RNA produced is heterogeneous in size.
• Why does the Ethidium Bromide stained gel (Total)
  look different? What are the 3 bands?
• Ethidium Bromide (EtBr) stains ALL RNA
• EtBr only allows you to visualize as low as 100ng
  of RNA vs pg amounts for detection of
  radioactivity.
• 3 band are the 3 rRNAs which represent appox. 70
  of all RNA in a cell!

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Experiment 4 Answer
E4 Heterogeneous size E3 -Unstable E2
-Intermediate should be a polynucleotide E2
-Should associate with ribosomes E1 -Rapid
synthesis
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Experiment 5

• You are still gathering more evidence that RNA is
  the intermediate in gene expression.
• You grow 3 different bacterial cultures
• 1) Culture grown in the presence of Uracil32P
  and collect the bacterial DNA radioactive
  bacterial RNA.
• 2) You infect the bacterial culture with phage
  in the presence of Uracil32P collect the
  radioactive phage RNA
• 3) You infect the bacterial culture with phage
  and collect the phage DNA

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Uracil 32P
Uracil 32P
hour
minutes
RNA DNA
RNA
DNA
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Experiment 5 (contd)

• Now that you have bacterial DNA, radioactive
  bacterial RNA, radioactive phage RNA phage DNA,
  you now perform 8 density centrifugation
  experiments on various DNA/RNA mixtures
• 1) mix bacterial DNA radioactive bacterial RNA
• 2) mix bacterial DNA radioactive bacterial RNA,
  however before density centrifugation you heat
  denature and allow renaturation.
• 3) mix radioactive bacterial RNA phage DNA
• 4) mix radioactive bacterial RNA phage DNA,
  however before density centrifugation you heat
  denature and allow renaturation.
• 5) mix radioactive phage RNA bacterial DNA
• 6) mix radioactive phage RNA bacterial DNA,
  however before density centrifugation you heat
  denature and allow renaturation.
• 7) mix radioactive phage RNA phage DNA
• 8) mix radioactive phage RNA phage DNA, however
  before density centrifugation you heat denature
  and allow renaturation.

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RNA DNA
RNA DNA
RNA DNA
RNA DNA
RNA DNA
RNA DNA
RNA DNA
RNA DNA
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Experiment 5 Interpretation

• Looking at the previous 2 slides, what do the
  results tell you about the base composition of
  the RNA? Explain the evidence.

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Experiment 5 Answer

• Looking at the previous 2 slides, what do the
  results tell you about the base composition of
  the RNA? Explain the evidence.
• Compare experiment 12 shows that bacterial RNA
  hybridizes with bacterial DNA.
• Experiment 4 shows that bacterial RNA does not
  hybridize with phage DNA therefore hybridization
  in experiment 2 is because hybridization is
  sequence specific
• Similar to previous two points experiment 6 shows
  that phage RNA does not hybridize with bacterial
  DNA but experiment 8 shows it does hybridize with
  phage DNA therefore sequence specific.

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Experiment 5 Answer
E5 Base composition should reflect DNA E4
Heterogeneous size E3 -Unstable E2 -Intermediate
should be a polynucleotide E2 -Should associate
with ribosomes E1 -Rapid synthesis
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Gene Expression

• From the previous 5 experiments you have
  identified 6 characteristics of RNA which make it
  the ideal candidate as an intermediate in gene
  expression. What are they and why is it
  important that the RNA intermediate have these
  six characteristics?

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Gene ExpressionAnswer

• From the previous 5 experiments you have
  identified 6 characteristics of RNA which make it
  the ideal candidate as an intermediate in gene
  expression. What are they and why is it
  important that the RNA intermediate have these
  six characteristics?
• E5 Base composition should reflect DNA
• Important in order to get specific protein
  production encoded by DNA
• E4 Heterogeneous size
• Able to encode various sizes of proteins
• E3 Unstable
• Need to be able to turn off expression quickly
• E2 -Intermediate should be a polynucleotide
• To base pair with DNA
• E2 -Should associate with ribosomes
• To be translated into proteins.
• E1 -Rapid synthesis
• To quickly react to cellular signals and.

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Experiment 6

• You have identified RNA as the intermediate in
  gene expression. Now you want to show what is
  necessary for the production of RNA.
• You take a bacterial cellular extract and assay
  for the production of RNA in vitro in the
  presence of Uracil32P. If RNA is produced it
  will bind to a filter which will make the filter
  radioactive. If no RNA is produced the Uracil32P
  will flow through the filter.

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RNA Polymerase activity
Filter
Filter 32P
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Experiment 6 (contd)

• Now that you have isolated the cell extract you
  perform the following 7 in vitro experiments
  followed by filter hybridization to assay for RNA
  production
• 1) Extract Uracil32P
• 2) Heat denature or Protease treat Extract then
  add Uracil32P.
• 3) DNase digest Extract, inactivate DNase, then
  add Uracil32P.
• 4) RNase digest Extract, inactivate RNase, then
  add Uracil32P.
• 5) DNase digest Extract, inactivate DNase, then
  add double-stranded DNA Uracil32P.
• 6) DNase digest Extract, inactivate DNase, then
  add single-stranded DNA Uracil32P.
• 7) DNase digest Extract, inactivate RNase, then
  add RNA Uracil32P.

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Uracil 32P
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Experiment 6 Answer

• Looking at the previous 2 slides, what do the
  results tell you about what is required for RNA
  synthesis? Explain the evidence.
• Experiment 2 Proteins are required
• Experiment 3 DNA is required
• Experiment 4 RNA is not required
• Experiment 5 Double-stranded DNA
  can serve as the template
• Experiment 6 Single-stranded DNA
  can serve as the template
• Experiment 7 RNA does not rescue
  DNase treatment (RNA not the
  template)