GENE EXPRESSION

GSND 5113Q

Previous Exam 2 used in Spring 2018

Emanuel Goldman, PhD

New Jersey Medical School
Rutgers Biomedical Health Sciences (RBHS)
Rutgers, The State University of New Jersey
Department of Microbiology, Biochemistry & Molecular Genetics
International Center for Public Health (ICPH)
225 Warren Street, room E450T
Newark, NJ 07103 USA
Telephone: 973-972-4367
Fax: 973-972-3644 or -8982
Email: [email protected]
1. Briefly explain your answers to the following questions. (18 points total)
A mutation (Mutation A) in the lac operon of E. coli leads to an inability to ferment lactose, and
the expression of the operon is always off. Introduction into the mutant of an F' factor containing
the wild type lac operon does NOT restore the ability to ferment lactose, i.e., there is no
expression of the operon from the plasmid either.
a. What is the probable nature of Mutation A? Is the mutation cis- or trans-acting?

b. A second site (in another gene) mutation (Mutation B) in the original mutant strain causes a
change of phenotype to constitutive expression of the operon. What kind of second site mutation
could account for this?

c. If Mutation B were genetically isolated in a fresh strain (i.e., it is the only mutation in this
strain), what lac phenotype would you expect? Is the mutation cis- or trans-acting?

d. Another mutation is found (Mutation C) that leads to constitutive expression of the lac operon.
To your surprise, Mutation C genetically maps within the first structural gene of the operon, the z
gene coding for beta-galactosidase. Nevertheless, the beta-galactosidase synthesized in this
mutant is full length and exhibits normal activity. Suggest an explanation for the constitutive
phenotype of Mutation C. (Hint: recall the regulatory features of other catabolic operons such as
gal and ara.)
2. (16 points)
Predict the phenotype for expression of the E. coli trp operon enzymes for the following mutants:
a) G to A mutation corresponding to position 56 of the trp operon leader RNA.
b) G nucleotide insertion between nucleotides 63 and 64 of the trp operon leader RNA.
Explain your predictions taking into account the alternate stem-loop structures of the
leader RNA.
Assume the strain is trpR- (i.e., no repressor). A diagram of the trp operon leader
sequence and alternate stem-loop structures is shown on the accompanying sheet.
Remember that the bulk of a ribosome covers about 15 nucleotides on either side of the triplet
that the ribosome is sitting on.

think about this but changing the

answer a little

https://www.chegg.com/homewo
rk-help/questions-and-
answers/2-16-points-predict-
phenotype-expression-e-coli-trp-
operon-enzymes-following-
mutants-g-mut-q43774865
 trp operon leader RNA 1

70 100

90

80

110
60

80

120 120

90 130 Trp limited

130
No translation
60 70 120 130 Excess Trp
30 40 50 100 110 140 1 2
80 90 100 110 140
30 40 50 60 70
3. (16 points)
If Egr-1 is a transcription factor that regulates expression of the galK gene in eukaryotic cells, then it
would be expected that the galK gene would possess upstream promoter regions to which Egr-1 would
bind. Analysis of the published sequence of the galK gene promoter region from liver cells revealed
that it does indeed contain such regions, along with other potential transcription factor binding sites.
To investigate the ability of Egr-1 binding to the galK promoter to stimulate transcription of the galK
gene, fragments of different lengths of the promoter region were cloned upstream of the firefly
luciferase reporter gene into a plasmid vector (pGL3-b). Expression of this reporter gene results in a
fluorescent signal that can be measured. The resulting plasmids were then introduced (by transfection)
into Hep3B cells, which express Egr-1. Cell extracts were later assayed for lucerifase activity. The
fragments used and the results are shown in the following Figure. (Identity of the binding sites for each
transcription factor in each plasmid follows the same pattern as indicated on the top line.)

Top panel: Schematic diagrams showing the galK promoter fragments that were cloned upstream of
the luciferase reporter and introduced into the Hep3B cells. Regions of the promoter that may bind the
other transcription factors, AP-4, Nkx-2.5, and TATA are also shown. Each box represents a binding
site for a factor. pGK(-) indicates promoter luciferase constructs of the lengths indicated.
Bottom panel: Relative levels of luciferase activity in Hep3B cells containing the different fragment
lengths or the pGL3-b control plasmid alone.
(questions follow on the next page)
3. (continued)
(a) How many regions that bind Egr-1 are present in each of the different length galK promoter
fragments?
pGK(-425):
pGK(-272):
pGK(-155):
pGK(-83):

(b) Describe the QUANTITATIVE differences in relative luciferase activity in cells transfected with
promoter fragments of different lengths compared with cells transfected with control plasmid (pGL3-b).

(c) Considering the data from the (-155) and (-83) fragments only, in comparison to the control, do the
data support a role for Egr-1 as stimulating transcription of the galK gene? Explain your answer.

(d) What might the data from the cells receiving the (-272) and (-155) fragments suggest about the
possible nature of the transcriptional control of Nkx-2.5 on galK transcription?
4. (16 points) Select the letter from the key which BEST fits each statement below, regarding
eukaryotic post-transcriptional and translational control. (Any one answer may be used more than
once and some may not be used at all; but only one letter is the BEST answer to each question).

A. Diphtheria toxin
B. mRNA degradation
C. translation initiation
D. iron homeostasis
E. Ricin
F. signal peptide
G. splicosome
H. RNA editing
I. RNA polymerase II carboxy-terminal domain
J. Poly(A) binding protein

___ Nonsense-mediated decay

___ Responsible for circularizing mRNA

___ Binding of polyadenylation factors for synthesis of poly(A) tails

___ Signal recognition particle (SRP)

___ snRNP

___ Internal Ribosome entry site (IRES)

___ Ferritin

___ Ribosome scanning to first AUG

___ RNAi

___ Transferrin receptor

___ Secretion through membranes

___ Intron

___ Phosphorylated eIF2

Binding of capping factors for addition of 5' cap to mRNA

___ CAP-binding protein (eIF4E)

___ Exon skipping

5. (16 points)
The alpha operon of E. coli can be diagramed as follows, with the relative amounts of each
protein product produced in the cell indicated:
P S13 S11 S4 alpha L17
S))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))
1 1 1 0.2 1
(alpha is an RNA polymerase subunit; S13, S11, and S4 are 30S ribosomal subunit proteins; and
L17 is a 50S ribosomal subunit protein.)
The coding sequences of each of the genes (i.e., beginning with the ATG start codon) were
subcloned individually onto plasmids, under lac repressor transcriptional control with the beta-
galactosidase translation start signals, and the following measurements were obtained in cells
following induction with IPTG: (Note: expression from the plasmid copy of each cloned gene is
100-fold greater than expression of the chromosomal copy of the gene.)

cells containing
plasmids Relative amount of proteins synthesized following IPTG induction
encoding: S13 S11 S4 alpha L17
none (control) 1 1 1 0.2 1
S13 100 1 1 0.2 1
S11 1 100 1 0.2 1
S4 0.01 0.01 100 0.2 0.01
alpha 1 1 1 100 1
L17 1 1 1 0.2 100

a. Which protein is the regulator for this operon? Why?

b. Can you determine if control of operon expression is at the level of transcription or

translation? Why?

c. Would any of these plasmid-containing cells not be viable in the presence of IPTG? Why?

d. Suggest a mechanism of control (consistent with the data in the table) which could account for
the 5-fold lower levels of alpha protein (compared to the ribosomal proteins) normally found in
this operon. Explain.
6. (18 points)
The sequence of the slippery site for the HIV translational -1 frameshift from the gag reading
frame to the gag-pol fusion reading frame is given below, with spaces inserted between the
codons in the gag reading frame, the amino acids encoded noted above the codons, and the
nucleotide residues numbered (arbitrarily) below the sequence:

Phe Leu Gly Lys

U UUU UUA GGG AAG
1 5 9

It is known that the ribosome changes its reading frame to the left (-1) by one nucleotide, within
the run of the 6 U residues, to enter the gag-pol reading frame. (Note that this requires an
unorthodox wobble pairing in the P-site of the new frame, after the UUA codon has been
decoded as leucine, in which the U in the anticodon wobble position of tRNALeu pairs opposite U
in the wobble position of the Phe codon.)

Predict the effect of the following mutations on the translational frameshift. (Phe codons are
UUU and UUC.) Explain your reasoning.

a) U to G mutation at nucleotide 1

b) U to C mutation at nucleotide 4

c) U to A mutation at nucleotide 6

d) A to U mutation at nucleotide 7

e) A to C mutation at nucleotide 7

f) G to C mutation at nucleotide 9