Correlation Between Termination Phenotype and Elongation Speed in the Rpb2 Subunit of RNA

Polymerase II

Christian Henry BurnsDr. Diane Hawley Research Group

Summer Program for Undergraduate ResearchAugust 17th 2012

The Role of RNA Polymerase II in the Central Dogma

Transcription

Translation

DNA

mRNA

Protein

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

AT

CG

TA

(1) Initiation (2) Elongation

AU

C

(3) Termination

STOP

Transcription Activity of RNAP II

AT

CG

TA

(1) Initiation (2) Elongation

A

UC

(4) Termination

CUT STOP

(3) mRNA Cleavage

mRNA

Determination of Termination Phenotype

RPB2

5-FOASD - LEUSD –TRP -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 CUT

5’ Intron 3’ Intron

LacZ ORF

Cutting at the Poly-A site results in no lacZ protein produced and

thus a “white” termination phenotype

Splicing of the intron results in translatable lacZ RNA and thus a “blue” termination

phenotype

X-GAL Filter Lift Termination Phenotype AssayLeu+ and Trp+ colonies grown for several days on S-gal medium. S-gal is the same as and selective SD medium

but with galactose in place of glucose as the carbon source, the galactose is an inducer for the lacZ promoter.

Colonies transferred from S-gal medium to a filter circle to be exposed to X-GAL chemical solution where “blue” or “white”

phenotype of the polymerase will develop. Wild-type colonies will show a faint green coloring

The Big Question

Is there a defined correlation between the elongation speed of a polymerase and its termination

phenotype?

The T35 Mutant Investigation

S45L

K537R

Active Site

The T35 mutant is a double mutant polymerase that has a fast

transcriptional speed as well as a “blue” termination phenotype

T35 Mutant Investigation

What do each of the mutations in the T35 RNA Polymerase II mutant bring to the overall termination phenotype of the polymerase?

K537R MutantS45L Mutant

Two other sets of mutations bring to light the potential input of each T35

mutation to the overall phenotype of the polymerase.

Another mutation at the same residue, K537E, caused a white

phenotype. This mutant is a slow elongator. Mutations at the same

residue in other polymerases cause changes to the polymerase elongation speed and have termination phenotypes.

Mutations at the adjacent residues, Q46R and Q47R, both cause a blue phenotype. The

RNAP II isolated from Q46R was not detectably fast in an initial assay.

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

Dr. Diane Hawley Rita Aulie

Opher KornfeldAmber Bonds Haobo WangCharles Kubicek

Thank you to the NIH, SPUR, Peter O’Day, Adam Unger, and my fellow R25 and REU

colleagues