Download Homework #10: Transcription and Post

Homework #10: Transcription and Post-transcriptional Regulation (MMG409)
1. Molecular Mechanisms of Eukaryotic Transcriptional Control
You have constructed a set of plasmids containing a series of nucleotide insertions spaced along
the length of the glucocorticoid-receptor gene. Each insertion encodes three or four amino
acids. The map positions of the various insertions in the coding sequence of the receptor gene
are as follows:
0 Glucocorticoid-receptor coding sequence 783
Insertion:
A
B
C
D
E G I
F H
K M O Q
S
J L
N P R
The plasmids containing the receptor gene can be functionally expressed in CV-1 and COS cells,
which contain a steroid-responsive gene. Using these cells, you determine the effect of each of
these insertions in the receptor on the induction of the steroid-responsive gene and on binding of
the synthetic steroid dexamethasone. The results of these analyses are summarized in Table 1.
a. From this analysis, how many different functional domains does the glucocorticoid
receptor have? Indicate the position of these domains on the insertion map.
b. Which domain is the steroid-binding domain?
c. Does this experiment give you information about: 1) the DNA binding domain? 2) the
transcription activation domain?
Insertion
Induction
Dexamethasone binding
A
++++
++++
B
++++
++++
C
++++
++++
D
0
++++
E
0
++++
F
0
++++
G
++++
++++
H
++++
++++
I
+
++++
J
++++
++++
K
0
++++
L
0
++++
M
0
++++
N
+
++++
O
++++
++++
P
++++
++++
Q
0
0
R
0
0
S
0
0
2. The segmentation of eukaryotic genes into exons and introns presents an opportunity for the
production of multiple gene products from a single gene by alternative RNA processing.
Developmental programs often use differential splicing or differential polyadenylation to
produce tissue-specific variants from one transcription unit.
The gene encoding the small peptide hormone calcitonin is one such differentially utilized
gene. The calcitonin gene contains six exons. In thyroid cells an mRNA that encodes calcitonin
is produced; it contains exons 1, 2, 3, and 4 and uses a polyadenylation site at the end of exon 4.
In neuronal cells no calcitonin is produced from this gene. In neuronal cells calcitonin generelated peptide (CGRP) is produced from this gene; its mRNA consists of exons 1,2,3,5, and 6.
The gene and its tissue-specific pattern of processing are diagrammed in Figure 1. In both cell
types transcription begins in the same place and extends beyond exon 6.
The mechanism of differential processing of the calcitonin/CGRP transcript is not
understood. Because different poly-A sites and different splice sites are utilized in the two
processing pathways, the tissue-specific factors that regulate calcitonin and CGRP expression
could be involved either in poly-adenylation or in splicing. There are two popular models. One
is that thyroid cells produce calcitonin because they contain a specific factor that recognized the
poly-A site in exon 4 with high efficiency and causes cleavage of the precursor RNA before
splicing of exon 3 to exon 5 can occur. Neuronal cells lack this factor with the result that
splicing of exon 3 to exon 5 predominates, leading to CGRP mRNA production. A second
model is that splice-site selection determines which RNA is produced. Thyroid cells produce
calcitonin because they splice exon 3 to exon 4; neuronal cells produce CGRP because they
splice exon 3 to exon 5. Presumably, one or both cell types produce a factor that favors one
splice over the other.
To test these hypotheses, the splicing and polyadenylation signal at the ends of exon 4 were
altered by mutation (see Fig. 1). The altered genes were transfected into a lymphocyte cell line,
which produces only calcitonin from the wild-type gene. The mutant lacking the exon-4
polyadenylation site produced no mRNA at all; the mutant lacking the exon-4 splice site
produced only CGRP mRNA.
A. Does the lymphocyte cell line contain the splicing and polyadenylation factors necessary to
produce both calcitonin and CGRP mRNA? Why?
B. If differential processing results from polyadenylation-site selection, which mutant would
you expect to produce CGRP mRNA when transfected into the lymphocyte cell line?
Why?
C. If differential processing results from splice-site selection, which mutant would you expect
to produce CGRP mRNA when transfected into the lymphocyte cell line? Why?
D. Which model for differential processing best explains the ability of the lymphocyte cell line
to produce calcitonin mRNA but not CGRP mRNA. Why?
Figure 1: Structure and tissue-specific splicing of the gene encoding calcitonin and CGRP.
Black boxes indicate exons. The splicing/polyadenylation choices used to produce calcitonin are
diagrammed above the line; those used to produce CGRP, below the line. Arrows mark the
positions of the splice-site and polyadenylation mutations.