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Chapter 7

The document describes molecular events that occur at the lac operon in E. coli cells when shifted from glucose to lactose medium. When no lactose is present, the lac repressor binds to the lac operator blocking transcription. When lactose is present, it binds to the lac repressor causing a conformational change that dissociates it from the operator, allowing transcription. When glucose is also present, transcription is very low. Once glucose depletes, cAMP levels rise binding to CAP, stimulating high transcription rates. The document also describes how low phosphate concentrations activate the PhoR sensor, which phosphorylates the PhoB response regulator inducing phosphate response genes.

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Leonita Swandjaja
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326 views

Chapter 7

The document describes molecular events that occur at the lac operon in E. coli cells when shifted from glucose to lactose medium. When no lactose is present, the lac repressor binds to the lac operator blocking transcription. When lactose is present, it binds to the lac repressor causing a conformational change that dissociates it from the operator, allowing transcription. When glucose is also present, transcription is very low. Once glucose depletes, cAMP levels rise binding to CAP, stimulating high transcription rates. The document also describes how low phosphate concentrations activate the PhoR sensor, which phosphorylates the PhoB response regulator inducing phosphate response genes.

Uploaded by

Leonita Swandjaja
Copyright
© © All Rights Reserved
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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Chapter 7

1. Describe the molecular events that occur at the lac operon when E. coli cells are
shifted from a glucose-containing medium to a lactose-containing medium.
Answer:
When no lactose is present, binding of the lac repressor to a sequence called the
lac operator, which overlaps the transcription start site, blocks transcription
initiation by the polymerase. When lactose is present, it binds to specific binding
sites in each subunit of the tetrameric lac repressor, causing a conformational
change in the protein that makes it dissociate from the lac operator. As a result,
the polymerase can initiate transcription of the lac operon. However, when
glucose also is present, the rate of transcription initiation (i.e., the number of
times per minute different polymerase molecules initiate transcription) is very
low, resulting in synthesis of only low levels of lac mRNA and the proteins
encoded in the lac operon.
Once glucose is depleted from the media and the intracellular glucose
concentration falls, E. coli cells respond by synthesizing cyclic AMP, cAMP. As the
concentration of cAMP increases, it binds to a site in each subunit of the dimeric
CAP protein, causing a conformational change that allows the protein to bind to
the CAP site in the lac transcription-control region. The bound CAPcAMP complex
interacts with the polymerase bound to the promoter, greatly stimulating the rate
of transcription initiation. This activation leads to synthesis of high levels of lac
mRNA and subsequently of the enzymes encoded by the lac operon.

2. The concentration of free phosphate affects transcription of some E. coli genes.


Describe the mechanism for this.
Answer:
When the phosphate concentration in the environment falls, it also falls in the
periplasmic space, causing phosphate to dissociate from the PhoR periplasmic
domain, as depicted in Figure 4-18. This causes a conformational change in the
PhoR cytoplasmic domain that activates its protein kinase activity. The activated
PhoR initially transfers a -phosphate from ATP to a histidine side chain in the
PhoR kinase domain itself. The same phosphate is then transferred to a specific
aspartic acid side chain in PhoB, converting PhoB from an inactive to an active
transcriptional activator. Phosphorylated, active PhoB then induces transcription
from several genes that help the cell cope with low phosphate conditions.

In response to low phosphate concentrations in the environment and periplasmic


space, a phosphate ion dissociates from the periplasmic domain of the inactive
sensor protein PhoR. This causes a conformational change that activates a protein
kinase transmitter domain in the cytosolic region of PhoR. The activated
transmitter domain transfers an ATP -phosphate to a conserved histidine in the
transmitter domain. This phosphate is then transferred to an aspartic acid in the
receiver domain of the response regulator PhoB. Several PhoB proteins can be
phosphorylated by one activated PhoR. Phosphorylated PhoB proteins then
activate transcription from genes encoding proteins that help the cell to respond
to low phosphate, including phoA, phoS, phoE, and ugpB.
3. What is the evidence that transcriptional initiation is the primary mechanism of
gene control in complex organisms?
Answer:
Control of transcription initiation is the most important mechanism for
determining whether most genes are expressed and how much of the encoded
mRNAs and, consequently, proteins are produced. RNA processing and various
post-transcriptional mechanism for controlling eukaryotic gene expression that
resulting in gene control.
4. What types of genes are transcribed by RNA polymerases I, II, and III? Design an
experiment to determine whether a specific gene is transcribed by RNA
polymerase II.
Answer:
- RNA polymerase I, located near nucleolus, transcribes genes encoding
precursor rRNA (pre-rRNA), which is processed into 28S, 5.8S and 18S rRNAs.
- RNA polymerase II, transcibes all protein-coding genes; that is, it production
of mRNAs, also produce four of the five small nuclear RNAs that take part in
RNA splicing.
- RNA polymerase III, transcribe genes encoding tRNAs, 5S rRNA, and an array
of small stable RNAs, including one involved in RNA splicing (U6) and the RNA
component of the signal-recognition particle (SRP) involved in directing
nascent proteins to the endoplasmic reticulum.
Experiment to determine whether a specific gene is transcribes by RNA
polymerase II:

Classes of RNA transcribe by the three eukaryotic nuclear RNA polymerases and ther
functions
Polymerases
RNA transcribe
RNA function
RNA polymerase I
Pre-rRNA (28S, 5.8S and Ribosome
components,
18S rRNAs)
protein synthesis
RNA polymerase II

mRNA
snRNAs
miRNAs

RNA polymerase III

tRNAs
5S rRNA
snRNA U6
7S RNA

Other stable short RNAs

Encodes protein
RNA splicing
Post
trnscriptional
gene
control
Protein synthesis
Ribosome
copmponent,
protein synthesis
RNA splicing
Signal-recognition particle
for insertion of polypeptides
into the ER
Various function, unknown
for many

5. The CTD of the largest subunit of RNA polymerase II can be phosphorylated and
hyperphosphorylated at various serine and tyrosine residues. What are the
conditions that lead to phosphorylation versus hyperphosphorylation?
Answer:
CTD (carboxyl terminal domain)
- Phosporylation of CTD occurs once the polymerase initiates transcription and
begins to move away from the promoter
- Remains phosphorylated as the enzyme transcribe the template
hyperphosphorylated, preventing termination and permitting the polymerase
to contionue chain elongation
6. What do TATA boxes, initiators, and CpG islands have in common? Which was the
first of these to be identified? Why?
Answer:
- The three act as promoters in sukaryoic DNA
- CpG
- May contain transcription initiation region in its DNA
7. Describe the methods used to identify the location of DNA control elements in
regulatory regions of genes.
Answer:
DNA recombination
8. What is the difference between a promoter-proximal element and a distal
enhancer?
Answer:
- Promoter-proximal element: control regions lying within 100200 base pairs upstream of
the start site. In some cases, promoter-proximal elements are cell-type-specific; that is, they
function only in specific differentiated cell types.

Distal enhancer: control elements located thousands of base pairs away from the start site.

9. Describe the methods used to identify the location of DNA-binding proteins in the
regulatory regions of genes.
Answer:
Footprinting and Gel-Shift Assays
10. Describe the structural features of transcriptional activator and repressor proteins.
Answer:
11. What happens to transcription of the EGR-1 gene in patients with Wilms tumor?
Why?
Answer:
12. Using CREB and nuclear receptors as examples, compare and contrast the
structural changes that take place when these transcription factors bind to their
co-activators.
Answer:
13. What structural change takes place on polymerase II promoters during
preinitiation complex formation?
Answer:
14. Expression of recombinant proteins in yeast is an important tool for biotechnology
companies that produce new drugs for human use. In an attempt to get a new
gene X expressed in yeast, a researcher has integrated gene X into the yeast
genome near a telomere. Will this strategy result in good expression of gene X?
Why or why not? Would the outcome of this experiment differ if the experiment
had been performed in a yeast line containing mutations in the H3 or H4 histone
tails?
Answer:
15. You have isolated a new protein called STICKY. You can predict from comparisons
with other known proteins that STICKY contains a bHLH domain and a Sin3interacting domain. Predict the function of STICKY and rationale for the
importance of these domains in STICKY function.
Answer:
16. Describe at least one gene you would expect to be able to clone using the
following genes as bait in a yeast two hybrid experiment: alpha-globin; the
catalytic subunit of protein kinase A; and the catalytic subunit of aspartate
transcarbamylase.
Answer:

ANALYZE THE DATA


An electrophoretic mobility shift assay (EMSA) was performed using a radiolabeled DNA
fragment from the sequence upstream of gene X. This DNA probe was incubated with (+)
or without (-) nuclear extract isolated from tissues A (bone); B (lung); C (brain); and D
(skin). The DNA: protein complexes were then fractionated on nondenaturing
polyacrylamide gels. The gels were exposed to autoradiographic film; the results are
presented in the figure.

a. Which tissues contain a binding activity that recognizes the sequence upstream of
gene X? Is the transcription factor the same in each tissue?
b. If the binding activity was purified, what test could be done to verify that this factor is
in fact a transcription factor?
c. What type of assay would be performed to determine the specific DNA sequence(s) to
which the transcription factor binds?
d. If gene X is transcribed in lung and brain tissue but not in bone and skin tissue, what
type of transcription factor is the binding activity? Speculate as to the identity of other
factors that might be complexed at the gene X promoter in bone and skin tissue.
Answer:

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