Correlation Between Termination
Phenotype and Elongation Speed in the
Rpb2 Subunit of RNA Polymerase II
Christian Henry Burns
Dr. Diane Hawley Research Group
Summer Program for Undergraduate Research
August 17th 2012
The Role of RNA Polymerase II in the Central Dogma
Translation
Protein
DNA
mRNA
Transcription
RNA Polymerase II
The Structure of RNA Polymerase II
Rpb2
Rpb1
Rpb1 and Rpb2 are not only the
largest subunits, but they
contain the active site of the
enzyme where RNA
polymerizing activity occurs
This second
largest subunit is
the primary focus
of research
Transcription Activity of RNA Polymerase
(1) Initiation
(2) Elongation
(3) Termination
A
T
C
G
STOP
T
A
A
C
U
Transcription Activity of RNAP II
(1) Initiation
(2) Elongation
(3) mRNA
Cleavage
(4) Termination
CUT
A
T
C
G
T
A
U
A
mRNA
C
STOP
Determination of Termination Phenotype
SD –TRP
5-FOA
SD - LEU
-LEU
We have now selected for yeast cells with both plasmids we
need to determine the termination phenotype of the polymerase
The LacZ Termination System
CUT
5’ Intron
LacZ ORF
3’ Intron
Cutting
SplicingatofthethePoly-A
intron
site
results
in no lacZ
results
in translatable
protein
produced
anda
lacZ RNA
and thus
thus atermination
“white”
“blue”
termination
phenotype
phenotype
CUT
X-GAL Filter Lift Termination Phenotype Assay
Colonies
transferred
from grown
S-gal medium
to adays
filteroncircle
Leu+ and
Trp+ colonies
for several
S-galto be
exposed
to X-GAL
where “blue”
or “white”
medium.
S-gal is chemical
the same solution
as and selective
SD medium
phenotype
of the
polymerase
will of
develop.
colonies will
but with
galactose
in place
glucoseWild-type
as the carbon
show is
a faint
green for
coloring
source, the galactose
an inducer
the lacZ promoter.
The Big Question
Is there a defined correlation
between the elongation speed of a
polymerase and its termination
phenotype?
The T35 Mutant Investigation
S45L
The T35 mutant is a double mutant
polymerase that has a fast
transcriptional speed as well as a
“blue” termination phenotype
K537R
Active Site
T35 Mutant Investigation
Two
other
bringin
tothe
light
What
dosets
eachofofmutations
the mutations
the
potential
input of each
T35 mutation
T35
RNA Polymerase
II mutant
bring
to the
overall
phenotypephenotype
of the
to the
overall
termination
polymerase.
of the
polymerase?
S45L Mutant
K537R
Another mutation at the same
residue, K537E, caused a white
Mutations
at the adjacent
residues,
phenotype.
This mutant
is a Q46R
slow and
Q47R,
both cause
a blue phenotype.
elongator.
Mutations
at the sameThe
RNAP
II isolated
Q46R was
not
residue
in otherfrom
polymerases
cause
detectably
fast
initial assay.
changes
to in
theanpolymerase
elongation speed and have
termination phenotypes.
Creation of a Mutant Polymerase
via Site-Directed Mutagenesis
T35 Objectives
The primary objective of the T35 study is to separate the two single
point mutations of the T35 mutant polymerase and determine if the
isolated rpb2 mutations confer any elongation or termination
phenotypes.
Thus far we have been able to confirm
the two single point mutations in the
smaller plasmid vectors and they have
been recombined with the larger sum
of the rpb2 subunit plasmid.
Sequencing and termination phenotype
determination is underway.
T35 Mutant Analysis
If it is found that the K537R
or S45L mutation confers a
phenotype, the course of
action would be as follows:
Combine this mutation with alternative mutations, for
example mutations with opposing phenotypes to that of the
T35 mutation, and determine the effect on the phenotype of
the T35 based mutation.
Determine if there is an “enhancing” effect on
elongation or termination phenotypes of the T35
based mutations from combination with
alternative single point mutations.
Future Direction of T35 Study
• Confirm the presence of K537R mutation and test to
determine if it has a termination/ elongational speed
phenotype.
• Confirm the presence of S45L mutation and test to see
if the mutation has a termination/ elongational speed
phenotype.
• Determine if the termination/ speed phenotype of the
T35 polymerase mutant is associated with one of the
singular mutations or if there is a combinatory effect
from the presence of both mutations, which will help
determine the relationship between speed and
termination phenotype in the polymerase.
Special Thank You
Thank you to the NIH, SPUR, Peter O’Day,
Adam Unger, and my fellow R25 and REU
colleagues
Dr. Diane Hawley
Charles Kubicek
Rita Aulie
Amber Bonds
Haobo Wang
Opher Kornfeld