Paul Gollnick

Molecular Genetics: RNA-Protein Interactions and Prokaryotic Gene Expression

Professor

PhD 1986 Iowa State University
Postdoctoral work 1986 Stanford University
(American Cancer Society and American Heart Association Fellow)
Assistant Professor 1990 SUNY/Buffalo
PEW Scholar (93-98)
Associate Professor 1996 University at Buffalo
Professor 2002 University at Buffalo


Address Information

Paul Gollnick
Department of Biological Sciences
609 Hochstetter Hall
State University of New York at Buffalo
Buffalo, NY 14260

(716) 645-4972

To send e-mail: gollnick@buffalo.edu


RESEARCH SUMMARY

 

RNA-protein interactions are crucially important in many biological processes. My research involves investigating RNA-protein interactions involved in regulating gene expression. The model system we are studying is the tryptophan biosynthetic (trp) operon from Bacillus subtilis and several other related Bacilli. A novel form of attenuation controls transcription this operon (Figure 1). Two alternative RNA secondary structures regulate transcription termination in an untranslated leader region upstream of the structural genes. A trans-acting regulatory protein called TRAP (trp RNA-binding Attenuation Protein) controls which RNA structure forms. In the presence of tryptophan, TRAP is activated to bind to a specific target in the leader RNA. This binding results in formation of the transcription terminator, thereby halting expression of the operon. In the absence of tryptophan, TRAP does not bind RNA and the alternative antiterminator structure forms allowing expression of the operon. We have shown that TRAP protein has a unique structure consisting of 11 identical 75 amino acid subunits arranged in a ring (Figure 2). This is the first example of an 11 subunit protein, and one of few RNA-binding proteins whose three dimensional structure is known. TRAP is activated by binding 11 tryptophan molecules in clefts between each subunit. The TRAP binding site in the trp leader consists of 11 repeated GAG and UAG trinucleotide repeats. The discoveries that TRAP contains 11 subunits and that its RNA target contains 11 triplet repeats suggested a model for RNA binding to TRAP with each subunit of the protein interacts with on repeat in the RNA (Fig. 1). We used alanine scanning mutagenesis to identify three amino acid residues in each TRAP subunit (Lys37, Lys56 and Arg58) that are directly involved in RNA binding. The location of these residues on the structure of TRAP suggested that RNA binds by wrapping around the protein ring.

We have recently collaborated with Dr. Alfred Antson at the University of York to solve the crystal structure of B. stearothermophilus TRAP complexed with a 53 nucleotide RNA containing 11 GAG repeats, (GAGAU)10GAG to 1.9 resolution (Figure 3). The RNA binds to TRAP by wrapping around the outside edge of the protein ring with the bases on the inside interacting with the protein and the sugar-phosphate backbone on the outside. As predicted, Lys37, Lys56 and Arg58 form Hydrogen-bonds with the RNA. In addition Glu36 and Asp39 also Hydrogen bond with the RNA and Phe32 stacks on the 3rd G of each repeat. We have used nucleoside analogs to probe the TRAP/RNA interaction and to investigate the contributions of each interaction predicted in the crystal structure to the stability of the complex.

We are also studying the mechanism by which tryptophan activates TRAP to bind RNA by NMR in a collaboration with Dr. Peter Flynn using the 750 MHz NMR instrument here at UB. Future studies will involve characterizing the effect of TRAP binding on trp leader RNA structure and further investigating the mechanism by which TRAP regulates gene expression in vivo as well as investigating the possibility that other organisms use this type of gene regulation.


PUBLICATIONS

 

  • Kleckner, I.R., GOLLNICK, P. and Foster, M. (2011) Mechanisms of Allosteric Gene Regulation by NMR Quantification of Microsecond–Millisecond Protein Dynamics. J. Mol. Biol. (In Press)
  • Tate, J. and GOLLNICK, P. (2011) The Role of Forward Translocation in NPH I-mediated Transcription Termination of Vaccinia Early Genes. J. Biol. Chem. 286, 44764–44775.
  • Chen, C.-S., Smits, C., Dodson, G. G., Shevtsov, M. B., Merlino, N., GOLLNICK, P. Antson, A. A. (2011) How to Change the Oligomeric State of a Circular Protein Assembly: Switch from 11-Subunit to 12-Subunit TRAP Suggests a General Mechanism PLOS One 6(10): e25296 doi:10.1371/journal.pone.0025296
  • Potter, K.D., Merlino, N.M. Jacobs, T. and GOLLNICK, P. (2011) TRAP Binding to the Bacillus subtilis trp Leader Region RNA Causes Efficient Transcription Termination at a Weak Intrinsic Terminator. Nucl. Acids.Res. 39, 2092-2012.
  • Sachleben JR, McElroy CA, GOLLNICK P, Foster MP. (2010) Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti-trp RNA-binding attenuation protein. Proc. Natl. Acad. Sci. 107, 15385-15390.
  • Shevtsov, M.B., Chen, Y. Isupov, M.N. Leech, A., GOLLNICK, P. and Antson, A.A. (2010) B.licheniformis Anti- TRAP can assemble into two types of dodecameric particles with the same symmetry but inverted orientation of trimers. J. Structural Biol. 170 127-133.
  • Fairman, M.E., Wang, W., Maroney, P.A., GOLLNICK, P., Luhrmann, R., Nilsen, T.W., and Jankowsky, E. (2004)
    Active Remodeling of Ribonucleoprotein Complexes by a DexH/D ATPase Independent of Duplex Unwinding
    Science 304, 730-734
  • Snyder, D., Lary, J., Chen, Y., GOLLNICK, P., and Cole, J.L. (2004)
    Interaction of the Trp RNA-binding Attenuation Protein
    J. Mol. Biol. 338, 669-682.
  • Hopcroft, N.H., Manfredo, A., Wendt, A.L., Brzozowski, A.M., GOLLNICK, P., and Antson, A.A. (2004)
    The Interaction of RNA with TRAP: Role of Triplet Repeats and Spacer Nucleotides
    J. Mol. Biol. 338, 43-53.
  • Li, P.T., and GOLLNICK, P. (2004)
    Characterization of a trp RNA-binding Attenuation Protein (TRAP) Mutant with Tryptophan Independent RNA Binding Activity
    J. Mol. Biol. 335, 707-722.
  • Szigeti, R., Milescu, M. and GOLLNICK, P. (2004)
    Regulation of the Tryptophan Biosynthetic Genes in Bacillus halodurans: Common Elements, Different Strategies as Compared to Bacillus subtillus.
    J. Bacteriology 186, 818-828.
  • Li, P. and GOLLNICK, P. (2002)
    Using Hetero-11-mers Composed of Wild-type and Mutant Subunits to Study Tryptophan Binding to TRAP and its Role in Activating RNA Binding
    J. Biol. Chem. 277, 35567-35573.
  • McElroy, C., Wendt, A., Manfredo, A., GOLLNICK, P. and Foster, M. (2002)
    TROSY-NMR of the 91 kDa TRAP Protein Reveals Allosteric Control of Gene Regulatory Protein by Ligand-Altered Flexibility
    J. Mol. Biol. 323, 463-473.
  • Li, P., Scott, D. and GOLLNICK P. (2002)
    Creating hetero-11-mers Composed of Wild-type and Mutant Subunits to study RNA binding to TRAP
    J. Biol. Chem. 277, 11838-11844.
  • Valbuzzi, A., GOLLNICK, P. Babitzke, P. and Yanofsky, C. (2002)
    The anti-trp RNA-binding protein (anti-TRAP), AT, recognizes the tryptophan-activated RNA binding domain of the TRAP regulatory protein J.
    J. Biol. Chem. 277, 10608-10613.
  • Elliott, M., Gottlieb, P. A. and GOLLNICK, P. (2001)
    The mechanism of RNA binding to TRAP: Initiation and Cooperative Interactions
    RNA 7, 85-93.
  • Antson, A.A., Chen,X.-P., Dodson, E. J., Dodson, G. G. and GOLLNICK, P. (1999)
    Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA
    Nature 401, 235-242
  • Elliott, M.B., Gottilieb, P.A. and GOLLNICK, P. (1999)
    Probing the TRAP-RNA interaction with nucleoside analogs.
    RNA 5, 1277-1289.
  • Xirasagar, S, Elliott, M.B., Bartolini, W., GOLLNICK, P. and Gottlieb, P. (1998)
    RNA Structure Inhibits the TRAP (trp RNA-binding attenuation protein)-RNA interaction.
    J. Biol. Chem 273, 27146-27153.
  • Yang, M., Chen, X.-P., Millitello, R., Hoffman, R., Fernandez, B., Bauman, C., and Gollnick, P. (1997)
    Alanine-scanning mutagenesis of Bacillus subtilis trp RNA-binding attenuation protein (TRAP) reveals residues involved in tryptophan binding and RNA binding.
    J. Mol. Biol. 270: 696-710
  • Bauman, C., Otridge, J., and Golnick, P.(1996)
    Kinetic and thermodynamic analysis ofthe interaction between TRAP (trp RNA-binding Attentuation Protein) and trp leader RNA from Bacillus subtilis.
    J. Biol. Chem. 271:12269-12274
  • Antson, A.A., Otridge, J.B., Brzozowski, A.M., Dodson, E.J., Dodson, G.G., Wilson, K.S., Smith, T.M., Yang, M., Kurecki, T. and GOLLNICK, P. (1995)
    The three dimensional structure of trp RNA-binding attenuation protein.
    Nature 374, 693-700.
  • Gollnick, P. (1994)
    Regulation of the Bacillus subtilis trp operon by an RNA binding protein
    Molecular Microbiology 11:991-997
  • J. Otridge and P. Gollnick (1993)
    MtrB from Bacillus subtilis binds specifically to trp leader RNA in a tryptophan-dependent manner
    Proc. Natl. Acad. Sci. USA. 90:128-132

  • Figure 1. Model of transcription attenuation of the B. subtilis trp operon. Large boxed letters designate the complementary strands of the terminator and antiterminator RNA structures. Small rectangles represent the GAG and UAG repeats involved in TRAP binding; these triplet repeats are also outlined in the sequence of the antiterminator structure. Numbers indicate the residue positions relative to the start of transcription. Nucleotides 108-111 overlap between the antiterminator and terminator structures and are shown as outlined letters. The TRAP protein is represented as an 11 segmented ring and the bound RNA is shown forming a matching circle upon binding to TRAP with each triplet repeat interacting with one subunit.


    Figure 2. The crystal structure of TRAP depicted by computer graphics using the program MOLSCRIPT. Eleven identical subunits form a ring containing 11 seven-stranded beta-sheets containing 4 strands from one subunit and 3 strands from the adjacent subunit. Each subunit is shown as a different color.The eleven bound tryptophan molecules are shown as van der Waals spheres.



    Figure 3. Ribbon diagram of Bacillus stearothermophilus TRAP 11mer bound toa 53 base RNA containing 11 GAG repeats separated by AU spacers, (GAGAU)10-GAG. Each TRAP subunit is a different color and the RNA is shown as ball-and-stick models. The 11 bound L-tryptophan molecules are shown in van der Waals spheres. This structure reveals how the TRAP protein functions in regulating both transcription and translation of the trp genes by arresting a segment of the RNA and preventing it from participating in RNA stem-loop structures or from interacting with ribsomes. This figure was featured on the cover of Nature volume 401 issue 6750 September 16 1999


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