1. 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

2. 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

3. Transcription Attenuation Control of the B. subtilis Tryptophan Operon Transcription reads through Trp Genes are Expressed Transcription terminates Trp genes are NOT expressed 1988 Limiting Tryptophan Excess Tryptophan 36 91 109 116 130 136 140 12345 6 7 8 9 10 11 Intrinsic terminator TRAP

4. A. Antson et al. Nature 401; 235-242 1999 Crystal structure of TRAP in complex with RNA

5. Transcription Attenuation Control of the B. subtilis Tryptophan Operon 36 91 109 116 130 136 140 12345 6 7 8 9 10 11 Antiterminator Terminator 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?

6. 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

7. 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

8. RNAP TRAP mRNA TRAP plays an additional Role in Attenuation besides altering RNA TRAP binding induces forward translocation of RNAP Kristine Potter Nucleic Acids Res. 2011

9. 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 Involving specific amino acids on each protein on each protein Natalie Merlino

10. 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

11. 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