Incoming Graduate Student 2013
Paul Gollnick Hoch. 613
• Two major research areas in my lab
• Bacterial gene regulation (transcription)
– Tryptophan genes in Bacilli (Gram positive
bacteria)
– TRAP (RNA binding gene regulatory protein)
– Anti-TRAP (regulator of TRAP function)
– Natalie Merlino and Shraddha Sharma (Both
graduated! in 2013!)
• Eukaryotic viral gene regulation
– vaccinia virus transcription
– Ryan Hindman and Rachel Boldt
Transcription termination in bacteria occurs by two mechanisms
1) intrinsic termination: Involves folding of the nascent RNA transcript
into a G + C-rich helix followed by a U-rich track.
2) Rho-dependent termination: Involves a protein factor called Rho
Rho
Transcription Attenuation Control of the B. subtilis Tryptophan Operon
10
Limiting Tryptophan
9
Excess Tryptophan
11
91
8
7
TRAP
116
6
36
1
2
3
4
5
109
130
109
136
140
Intrinsic terminator
Transcription reads through
Trp Genes are Expressed
Transcription terminates
Trp genes are NOT expressed
1988
Crystal structure of TRAP in complex with RNA
A. Antson et al. Nature 401; 235-242 1999
Transcription Attenuation Control of the B. subtilis Tryptophan Operon
Antiterminator
10
9
11
Terminator
91
8
7
116
6
36
1
2
3
4
5
109
130
109
136
140
Isn’t an 11 subunit protein and an RNA binding site with 11 repeats
A bit complicated just to influence the base-paring of 3 nucleotides?
Maybe there’s more to this story?
Does TRAP have an additional role in promoting termination?
Used several
strategies to prevent
the antiterminator but
didn’t see termination
Kristine Potter Nucleic Acids Res. 2011
Then we found that we can Disrupt or Delete the Terminator
Structure and still get TRAP-mediated Termination in
The trp leader region in vivo
Natalie Merlino
TRAP plays an additional Role in Attenuation besides altering RNA
TRAP binding induces forward translocation of RNAP
TRAP
mRNA
RNAP
Kristine Potter Nucleic Acids Res. 2011
How does TRAP cause RNA Polymerase to Terminate Transcription?
By Protein-Protein interactions
A. non-specific “car crash”
B. Specific protein-protein interactions
Involving specific amino acids
on each protein
Natalie Merlino
Recent Evidence Suggests that TRAP makes Specific
Interactions with RNAP occur to induce Transcription Termination.
Mutant TRAP with Glu60 changed to Lys Binds
RNA and Tryptophan like WT but fails to induce Termination
Suggests Glu60 interacts with RNAP
Natalie Merlino
Future Projects
How Does TRAP interact with RNA Polymerase?
Is this interaction species specific?
Can TRAP only work with B. subtilis RNAP or
will it work with E. coli RNAP?
Which subunit(s) and what Amino Acids residues are involved
On RNA Polymerase?
How does this interaction induce Transcription Termination?
Is this a fundamentally new Termination mechanism?