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Chap. 7 Problem 1
In glucose media without lactose, the
lac repressor is bound to the lac
operator, and the CAP protein is not
bound to its control site near the
promoter due to low cAMP level. As a
result, transcription of the lac operon
is shut off. In lactose media lacking
glucose, the operon is turned on and
transcription occurs at the highest
rate. cAMP is synthesized in the
absence of glucose, and the CAP-cAMP
complex binds to its control element
stimulating transcription initiation by
RNA polymerase. Due to binding of
lactose to the lac repressor, the
complex leaves the operator and
transcription no longer is blocked by
the repressor (Fig. 7.3).
Chap. 7 Problem 3
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Part 1: Classes of RNA transcribed by RNA Pols I, II, and III, that are
important to know, are marked with asterisks (Table 7.2).
Part 2: RNA polymerase II is very sensitive to inhibition by the Amanita
phalloides poison called -amanitin. The activity of RNA Pol II, but not Pols
I & III is inhibited at a 1 g/ml concentration. Therefore, one can
determine if a particular gene is transcribed by RNA Pol II by determining
if 1 g/ml -amanitin inhibits transcription of the gene.
Chap. 7 Problem 5
TATA boxes, CpG islands, and initiators all serve as promoters, which set the
transcription start site for genes. The TATA box is found in genes that are
strongly expressed, and therefore was the first promoter element to be
identified by in vitro transcription assays. In addition, the transcription start
site occurs at a fixed location downstream of a TATA box (Fig. 7.14).
Chap. 7 Problem 6
A commonly used method to detect promoter-proximal control elements is linkerscanning mutagenesis (Fig. 7.21). In this technique, a segment of random DNA is
substituted for DNA sequences across the control region. Reporter gene assays
are used to determine if the substitutions block transcription, indicating a control
region is present.
Chap. 7 Problem 7
Promoter-proximal elements typically are functional only when close to the
promoter, whereas distal enhancers often can function at variable distances
from the promoter (Fig. 7.22). Distal enhancers sometimes can be moved to
the other side of the gene and still regulate transcription. Both types of
sequences are bound by transcription activators that help RNA Pol II load
onto promoters.
Chap. 7 Problem 8
In DNase I footprinting, DNA labeled on one strand
is incubated with a transcription factor (TF), and the
complex is treated with a small amount of DNase I,
which cleaves DNA where it is not masked by the TF
(Fig. 7.23a). A control DNA sample lacking the TF is
treated under parallel conditions. The banding
patterns from the two samples are compared by gel
electrophoresis to locate the "footprint" region where
the TF has shielded the DNA from cleavage.
In gel-shift assays (electrophoretic mobility
shift assays) (Fig. 7.24), a labeled DNA
fragment (200-300 bp) containing the binding
site is incubated with the TF and then is run on
a polyacrylamide or agarose gel. A sample of the
DNA lacking the protein is run in parallel. Bound
DNA fragments run more slowly and are shifted
to a higher position on the gel.
Chap. 7 Problem 9
Transcription factors have a modular structure consisting of at least two
domains. Transcriptional activators contain a DNA binding domain and an
activation domain. Transcriptional repressors contain a DNA binding domain and
a repression domain. Some TFs also contain a ligand binding domain that
regulates activity. Domains typically are joined together in a single polypeptide
by flexible linker sequences that serve as hinges and allow conformational
changes needed for activation/repression. Some examples of transcriptional
activators are shown in Fig. 7.27.
Chap. 7 Problem 12
The sequential assembly of Pol II
transcription pre-initiation complex is
shown in Fig. 7.17. TBP (TATA boxbinding protein) binds first and
determines where transcription will
initiate. TFIIH is the last factor to
bind. TFIIH has a helicase activity
that is important in melting DNA and
generating an open complex wherein
the template strand is located within
the active site of the polymerase.
TFIIH also phosphorylates the CTD
of Pol II making the enzyme highly
processive.
Chap. 7 Problem 16
UASs are comparable to promoter-proximal elements and enhancers
in higher eukaryotes (Fig. 7.22).
Chap. 7 Problem 22
The four main classes of DNA-binding proteins we have discussed
are the 1) helix-loop-helix proteins (Fig. 7.28), 2) basic zipper
(bZIP) proteins (Fig. 7.29c), 3) basic helix-loop-helix (bHLH)
proteins (Fig. 7.29d), and zinc-finger proteins (Fig. 7.29a & b). A
detailed description of their structural features is presented in
the text and lecture slides. Zinc finger TFs are the most common
type of DNA binding protein encoded by the human genome.