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Blueprint Series

Complete Solution
Chapter 9

9
TRANSCRIPTIONAL CONTROL
OF GENE EXPRESSION

REVIEW THE CONCEPTS
1
...
After shifting to lactose media,
lactose is transported into the cell where it binds the lac repressor
...
In addition, the cell synthesizes cAMP because
of the low concentration of glucose
...
The lac promoter is a weak promoter in the absence of CAP binding; CAP bound at the CAP site
stimulates RNA polymerase binding and transcription initiation at the lac promoter,
resulting in a high rate of transcription from the lac promoter and maximal induction
of the lac operon
...
In two-component regulatory systems, one protein acts as a sensor and the other
protein is a response regulator
...
coli the proteins NtrB (the sensor) and NtrC
(the response regulator) regulate transcription of glutamine synthetase in response
to the free glutamine concentration
...
Under low glutamine conditions, NtrB undergoes a conformational change that activates the protein
kinase activity and results in the transfer of a phosphate group to NtrC
...


33

34

CHAPTER 9: Transcriptional Control of Gene Expression

3
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b
...

d
...
Similarities:
Both employ protein-binding regulatory DNA sequences/control elements
and specific proteins that bind to these regulatory sequences to control gene transcription
...

Regulatory proteins are able to influence gene transcription by binding to
regulatory sequences proximal or distal to the transcription start site in eukaryotes
...

5
...
RNA
polymerase II is responsible for mRNA transcription
...
Since RNA polymerase
II is uniquely inhibited by a low concentration of a-amanitin, one can determine
if any gene requires this polymerase by measuring gene transcription in the
presence and absence of this compound
...

6
...
The CTD is then further phosphorylated by cyclin T-CDK9
...

7
...
The TATA
box was the first to be identified because it is found in the promoters of most
genes expressed at high level
...
Since the TATA box occurs at a fixed location relative to the
transcription start site (~30 bases upstream), it was relatively easy to recognize
in the DNA sequence
...
To identify DNA-control elements within promoter regions, investigators utilize
5’ deletion mutants and linker scanning mutants
...
The wild-type DNA sequence and mutated
DNA are separately introduced into cultured cells, or transgenic animals are generated with the wild-type and mutant DNA
...
When an activator-binding
site is deleted, expression is reduced
...

9
...
Enhancers are located at greater distances, either upstream
or downstream of the transcription start site
...


CHAPTER 9: Transcriptional Control of Gene Expression

10
...
This assay depends on the ability of protein factors to “protect” DNA
from DNaseI digestion
...
In this assay, an extract is incubated with a
labeled fragment of DNA
...
This technique is often used as an assay
to purify DNA-binding proteins
...
Transcriptional activators and repressors contain a modular structure in which
one or more transcriptional activation or repression domains are connected to a
sequence-specific DNA-binding domain, usually through a flexible domain
...
The location of the sequence on the chromosome may influence whether it is
expressed; for example, if the sequence is adjacent to a telomere, it will be in a
silent (heterochromatin) locus and will therefore not be transcribed due to
condensation of the chromatin at that site
...
Second, DNA/histone methylation or deacetylation can lead to
chromatin condensation and prevent transcription from that site
...
CREB binding to its co-activator (CBP) is regulated by cAMP, which stimulates
phosphorylation of CREB
...
However, in the presence of CBP
the phosphorylated activation domain undergoes a conformational change to
form two a helices that wrap around a larger globular domain of the co-activator
...
When ligand binds to these domains,
they undergo a conformational change that generates a groove in the globular
activation domain that binds a short a helix in a coactivator
...

14
...
This protein folds into a saddle-like
structure that binds to the minor groove of DNA near the TATA box and bends
the DNA
...
TFIIF and Pol II bind and Pol II is positioned over the start
site
...

15
...
Telomeres are usually contained in heterochromatin, which is tightly packed
and less accessible for the transcriptional machinery
...
For example, if the DNA
sequence encoding lysines in the histone N-termini were mutated so that glycine
residues were substituted in their place, then repression of gene X would not

35

take place
...
This prevents binding by SIR3 and SIR4, preventing the
formation of heterochro-matin
...

16
...
Repressors contain two domains, one that binds DNA and a
second that represses transcription
...
The Sin3-interacting domainis likely to associate with a Sin3
containing histone deacetylase complex
...

17
...

18
...
These
constructs can be used to make transgenic organisms that can then be
analyzedfor GFP expression to identify the sequences capable of
activating reporter gene expression
...
The RNA-binding protein Tat binds to the RNA copy of a sequence
called TAR,which then binds cyclin T
...
In the
absence of Tat, the transcripts are terminated prematurely
...

20
...

21
...

bHLH proteins: contain an N-terminal a helix with basic residues that
interactwith DNA; bind to DNA as dimers
...

Zinc-finger proteins: 23- to 26-residue consensus sequence providing
proteinswith a region that folds around a central Zn2+ ion; found in

DNA and non- DNA–binding proteins

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