Incoming Graduate Student 2013Paul 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

Transcription reads throughTrp Genes are Expressed

Transcription terminatesTrp genes are NOT expressed

1988

Limiting Tryptophan Excess Tryptophan

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Intrinsic terminator

TRAP

A. Antson et al. Nature 401; 235-242 1999

Crystal structure of TRAP in complex with RNA

Transcription Attenuation Control of the B. subtilis Tryptophan Operon

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Antiterminator

Terminator

Isn’t an 11 subunit protein and an RNA binding site with 11 repeatsA 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

RNAPRNAP

TRAP

mRNA

TRAP plays an additional Role in Attenuation besides altering RNATRAP plays an additional Role in Attenuation besides altering RNA

TRAP binding induces TRAP binding induces forward translocation forward translocation of RNAPof RNAP

Kristine Potter Nucleic Acids Res. 2011

How does TRAP cause RNA Polymerase to Terminate Transcription?

By Protein-Protein interactionsBy Protein-Protein interactions

A.A. non-specific “car crash”non-specific “car crash”

B. Specific protein-protein interactionsB. Specific protein-protein interactions Involving specific amino acidsInvolving specific amino acids on each proteinon 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

Natalie Merlino

Suggests Glu60 interacts with RNAP

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?

Future Projects