Transcript How does Pol II

Hannah Baer
Hawley Lab
DNA
Pol II
RNA
ribosome
protein
How does Pol II “know” when to
terminate transcription?
Termination is linked to 3’ end
processing.
3’ end processing
Pol II
5’
5’
AAAAAAA 3’
Anti-terminator vs. torpedo models
Pol II
Anti-terminator: recognition of the poly(A) sequence
causes a conformational change
Torpedo: transcript cleavage allows degradation to occur
How is termination linked to
polyadenylation?
Termination-defective mutants
What is the nature of the defects?
What interactions are there that could have
been disrupted by the mutations?
The Yeast Two-Hybrid System
GAL4
activating
domain
?
?
?
Rpb2
GAL4
DNA-binding
domain
Reporter gene
Experiments
• Screen 1 million transformants from
each of three libraries
• Test for expression of all three
reporter genes: ADE2, HIS3, and lacZ
• Test any “positive” preys against four
mutant baits
Assays for reporter gene expression
left = HIS3; right = lacZ
Results
• Fourteen colonies expressed all three reporters, but
only four were real proteins
– RSC2, CIN5, YRF1, and YLR035C-A
• None of the preys interacted with any of the mutant
baits
• Two TFIIF prey constructs failed to interact with the
mutant or wild-type baits
Summary
• Three million transformants screened; three
interactions found
• When these were tested against the mutant
baits, results were inconclusive

Verify that the fusion proteins are being
expressed

Screen more from the three libraries

Characterize the remaining 36 mutants
Acknowledgments
•
•
•
•
Diane Hawley
Susan Strachan
Brittney Lilly
SPUR