Research
My
laboratory has a general interest in the structure and function of
riboswitches. Specifically we are interested in how bacterial
riboswitch RNAs interact with a particular cellular metabolite in order
to modulate genetic control. The ultimate goal of my laboratory is to
understand riboswitch structure and function in order to design
non-natural ligands that target natural riboswitches and act as novel
antimicrobial agents against some of the hardest to treat human
pathogens.
INTRODUCTION TO RIBOSWITCHES
We are investigating the structure and function of naturally occurring riboswitches. Riboswitches are non-coding elements within messenger RNAs that directly bind to cellular metabolites and modulate gene expression. Approximately 20 metabolite-binding riboswitches have been characterized from bacteria, and many provide a mechanism of feedback regulation for gene products within the biosynthetic pathway of the cognate metabolite. In this manner, riboswitches afford an elegant mechanism of feedback regulation that allows bacterial cells to appropriately respond to metabolic supply and demand. All things considered, riboswitches represent pharmaceutical targets of considerable interest for the development of novel antibiotics.
To exert control over gene expression, riboswitches couple the task of metabolite recognition with that of modulating a requisite aspect of gene expression. Consequently, riboswitches typically reside in 5' untranslated regions of bacterial messenger RNAs, and are generally composed of two interdependent domains that include a natural ligand-binding or ‘aptamer’ domain and an ‘expression platform’ whose precise conformation impacts gene expression. Two common mechanisms of riboswitch function involve ligand-dependent conformational changes that modulate either terminator/anti-terminator stem formation thus impacting transcription, or Shine-Dalgarno sequence accessibility thus impacting translation.
INTRODUCTION TO RIBOSWITCHES
We are investigating the structure and function of naturally occurring riboswitches. Riboswitches are non-coding elements within messenger RNAs that directly bind to cellular metabolites and modulate gene expression. Approximately 20 metabolite-binding riboswitches have been characterized from bacteria, and many provide a mechanism of feedback regulation for gene products within the biosynthetic pathway of the cognate metabolite. In this manner, riboswitches afford an elegant mechanism of feedback regulation that allows bacterial cells to appropriately respond to metabolic supply and demand. All things considered, riboswitches represent pharmaceutical targets of considerable interest for the development of novel antibiotics.
To exert control over gene expression, riboswitches couple the task of metabolite recognition with that of modulating a requisite aspect of gene expression. Consequently, riboswitches typically reside in 5' untranslated regions of bacterial messenger RNAs, and are generally composed of two interdependent domains that include a natural ligand-binding or ‘aptamer’ domain and an ‘expression platform’ whose precise conformation impacts gene expression. Two common mechanisms of riboswitch function involve ligand-dependent conformational changes that modulate either terminator/anti-terminator stem formation thus impacting transcription, or Shine-Dalgarno sequence accessibility thus impacting translation.
A third mechanism of riboswitch function is uniquely represented by the catalytic glmS riboswitch. The glmS
riboswitch is a ribozyme that undergoes self-cleavage in response to
binding glucosamine-6-phosphate (GlcN6P), the metabolic product of the
GlmS enzyme required for cell wall biosynthesis. glmS
ribozyme self cleavage via internal RNA transesterification
substantially reduces messenger RNA translation by destabilizing the
transcript. Therefore, artificial agonists of glmS
ribozyme activity might in principal function as antibiotic agents for
a number of pathogens that harbor the riboswitch. This coupled with the
fact that the glmS ribozyme is a natural curiosity among catalytic RNAs has propelled intensive study of the RNA’s structure and function.
PAST GLMS RESEARCH PROJECTS
glmS Ribozyme Utilizes Metabolite as a Coenzyme
The most recent catalytic RNA (ribozyme) discovered is the bacterial glmS
riboswitch. While most riboswitch-metabolite interactions modulate
transcription termination or translation initiation of the associated
mRNA, the glmS riboswitch
uniquely utilizes its metabolite, glucosamine-6-phosphate (GlcN6P), as
a coenzyme to perform self-cleavage and promote mRNA degradation. Our
prior studies using GlcN6P and various analogs have shown that the
ligand amine is required for catalysis and that the apparent pKa for
the overall self-cleavage reaction largely reflects that of the ligand
amine. Additionally, the ligand phosphate affects the pKa of the amine
and is an important determinant for ligand recognition and affinity.
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A., Soukup, J.K. & Soukup, G.A. "Ligand requirements for glmS ribozyme self-cleavage." Chemistry & Biology 12: 1221-1226 (2005).
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A., Soukup, J.K. & Soukup, G.A. "Ligand requirements for glmS ribozyme self-cleavage." Chemistry & Biology 12: 1221-1226 (2005).
Backbone and Nucleobase Contacts to Glucosamine-6-phosphate (GlcN6P) in the glmS Ribozyme
We have utilized Nucleotide Analog Interference Mapping (NAIM) and Suppression (NAIS) to investigate backbone and nucleobase functional groups that are essential for ligand-dependent ribozyme function. In our studies we observed that the natural, full-length ribozyme is stabilized by peripherally folded domains within the RNA. Whereas a minimal ribozyme core has an increased need for metal ions as well as a differing group of essential nucleotide functional groups. NAIM experiments utilizing GlcN6P as ligand identified requisite structural features and potential sites of ligand and/or metal ion interaction, while NAIS experiments utilizing glucosamine (GlcN) as ligand analog revealed those sites that orchestrate recognition of the phosphate moiety of GlcN6P. These studies demonstrated that the ligand-binding site lies in close proximity to the cleavage site supporting a role for ligand within the catalytic core.
Subsequently X-ray crystallographic analyses have agreed with our biochemical analyses of the glmS ribozyme, where coenzyme binding does not alter the conformation of the ribozyme but positions the coenzyme amine adjacent to the scissile phosphodiester linkage in order to initiate self-cleavage by a presumed mechanism of acid-base catalysis.
Jansen J.A., McCarthy T.J., Soukup G.A., and Soukup J.K. “Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme.” Nature Structural & Molecular Biology 13: 517-523 (2006).
We have utilized Nucleotide Analog Interference Mapping (NAIM) and Suppression (NAIS) to investigate backbone and nucleobase functional groups that are essential for ligand-dependent ribozyme function. In our studies we observed that the natural, full-length ribozyme is stabilized by peripherally folded domains within the RNA. Whereas a minimal ribozyme core has an increased need for metal ions as well as a differing group of essential nucleotide functional groups. NAIM experiments utilizing GlcN6P as ligand identified requisite structural features and potential sites of ligand and/or metal ion interaction, while NAIS experiments utilizing glucosamine (GlcN) as ligand analog revealed those sites that orchestrate recognition of the phosphate moiety of GlcN6P. These studies demonstrated that the ligand-binding site lies in close proximity to the cleavage site supporting a role for ligand within the catalytic core.
Subsequently X-ray crystallographic analyses have agreed with our biochemical analyses of the glmS ribozyme, where coenzyme binding does not alter the conformation of the ribozyme but positions the coenzyme amine adjacent to the scissile phosphodiester linkage in order to initiate self-cleavage by a presumed mechanism of acid-base catalysis.
Jansen J.A., McCarthy T.J., Soukup G.A., and Soukup J.K. “Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme.” Nature Structural & Molecular Biology 13: 517-523 (2006).
CURRENT GLMS RESEARCH PROJECTS
Proton Hopping Mechanism of the glmS ribozyme
Building on the structural data we obtained from NAIM and NAIS studies as well as the crystal structures, my laboratory is investigating the mechanistic details of the glmS ribozyme self-cleavage reaction. A substantial body of biochemical and biophysical data relating the structure and function of the glmS ribozyme has been amassed, however, a precise and comprehensive mechanistic understanding of GlcN6P coenzyme function in glmS ribozyme self-cleavage has not been elaborated. We propose a comprehensive mechanistic model wherein the coenzyme, GlcN6P, functions both as the initial general base catalyst and consequent general acid catalyst within a proton-relay thus fulfilling the apparent biochemical requirements for activity. We have begun solvent isotope effect and proton inventory experiments to test our hypothesis. Further knowledge of the mechanism of acid-base catalysis will illustrate whether development of glmS ribozyme agonists as prospective antibiotic compounds needs to satisfy added chemical requirements for binding and activity.
Soukup, GA and Soukup, JK. Structure and mechanism of the glmS ribozyme. In: The New World of Non-Protein Coding RNAs. Batey, R., Walter, N., and S. Woodson (eds.), Springer, Berlin, Germany, 2009.
Metal Ion-Dependence in glmS Ribozyme
Our interest in glmS structure and function includes the role of metal ions in glmS ribozyme self-cleavage and coenzyme binding. Both X-ray crystallographic and biochemical analyses agree that metal ions do not directly interact with the scissile phosphodiester to promote catalysis. Nevertheless, metal ions are indeed required for the glmS ribozyme to perform coenzyme-dependent self-cleavage. Our Nucleotide Analog Interference Mapping (NAIM) and Suppression (NAIS) experiments have revealed important functional groups within the core ribozyme that appear to interact directly with metal ions. While such data support the location of metal ion binding sites in the glmS ribozyme core, it is difficult to distinguish whether metal ion binding serves only to coordinate the catalytically competent structural conformation or additionally promote catalytic mechanism. In regard to the latter, metal ions can play alternative roles in catalysis that include altering pKa values of RNA functional groups at a substantial distance. For example, direct coordination of a transition state metal ion to the N7 of Guanine can lower the N1 pKa by 2.3 pH units. We are interested in alternative means for metal ions to augment catalysis in the glmS ribozyme.
Our studies provide evidence for divalent metal ion preference in association with the ligand phosphate, but demonstrate that other metal ion binding sites can utilize monovalent or divalent metal ions that conform to appropriate physicochemical properties. While there is a strong correlation between ribozyme self-cleavage and the size and hardness of associated metal ions, there are nuances to metal ion coordination that are less intuitive. Nevertheless, our studies further demonstrate that metal ion identity can impact the overall apparent pKa of ribozyme self-cleavage, thus suggesting that metal ions can serve alternative roles supporting the mechanism of catalysis. We have previously proposed an acid-base mechanism of catalysis that invokes a proton relay within the catalytic core of the ribozyme. The identity of metal ions binding in the vicinity of such a proton relay could differently affect functional groups engaged in proton transfer and thus influence the efficiency of catalysis.
Our current work indicates that Mg2+ interaction with ligand phosphate in the context of the ribozyme core is essential to achieving rates of self-cleavage biologically relevant to regulation of glmS expression. In this regard, our work underscores the importance of preserving the phosphate group or another hard ligand for Mg2+ in coenzyme analogs designed to function as artificial agonist that elicit glmS ribozyme activity.
Klawuhn, K., Jansen, J.A., Souchek, J., Soukup, G.A. and Soukup, J.K. Analysis of Metal Ion-Dependence in glmS Ribozyme Self-Cleavage and Coenzyme Binding. (Submitted)
Our interest in glmS structure and function includes the role of metal ions in glmS ribozyme self-cleavage and coenzyme binding. Both X-ray crystallographic and biochemical analyses agree that metal ions do not directly interact with the scissile phosphodiester to promote catalysis. Nevertheless, metal ions are indeed required for the glmS ribozyme to perform coenzyme-dependent self-cleavage. Our Nucleotide Analog Interference Mapping (NAIM) and Suppression (NAIS) experiments have revealed important functional groups within the core ribozyme that appear to interact directly with metal ions. While such data support the location of metal ion binding sites in the glmS ribozyme core, it is difficult to distinguish whether metal ion binding serves only to coordinate the catalytically competent structural conformation or additionally promote catalytic mechanism. In regard to the latter, metal ions can play alternative roles in catalysis that include altering pKa values of RNA functional groups at a substantial distance. For example, direct coordination of a transition state metal ion to the N7 of Guanine can lower the N1 pKa by 2.3 pH units. We are interested in alternative means for metal ions to augment catalysis in the glmS ribozyme.
Our studies provide evidence for divalent metal ion preference in association with the ligand phosphate, but demonstrate that other metal ion binding sites can utilize monovalent or divalent metal ions that conform to appropriate physicochemical properties. While there is a strong correlation between ribozyme self-cleavage and the size and hardness of associated metal ions, there are nuances to metal ion coordination that are less intuitive. Nevertheless, our studies further demonstrate that metal ion identity can impact the overall apparent pKa of ribozyme self-cleavage, thus suggesting that metal ions can serve alternative roles supporting the mechanism of catalysis. We have previously proposed an acid-base mechanism of catalysis that invokes a proton relay within the catalytic core of the ribozyme. The identity of metal ions binding in the vicinity of such a proton relay could differently affect functional groups engaged in proton transfer and thus influence the efficiency of catalysis.
Our current work indicates that Mg2+ interaction with ligand phosphate in the context of the ribozyme core is essential to achieving rates of self-cleavage biologically relevant to regulation of glmS expression. In this regard, our work underscores the importance of preserving the phosphate group or another hard ligand for Mg2+ in coenzyme analogs designed to function as artificial agonist that elicit glmS ribozyme activity.
Klawuhn, K., Jansen, J.A., Souchek, J., Soukup, G.A. and Soukup, J.K. Analysis of Metal Ion-Dependence in glmS Ribozyme Self-Cleavage and Coenzyme Binding. (Submitted)
Glucosamine-6-phosphate ligand analogs act as artificial agonists through targeting of the bacterial glmS riboswitch
Building on the knowledge we have already gained in our studies of the ligand requirements of the glmS ribozyme, we have begun to examine the ability of non-natural ligands to stimulate glmS self-cleavage, affect gene expression and ultimately act as anti-microbial agents. We have formed a collaboration with Dr. David Berkowitz at University of Nebraska-Lincoln. He is an organic chemist with expertise in phosphonate and fluorinated phosphonate syntheses and an interest in modified sugar synthesis. He has begun to synthesize glucosamine-6-phosphate analogs that might act as novel antibiotics, targeting the glmS self-cleaving riboswitch. Promising results have already been obtained. The synthetic tools to make these studies possible have been under steady development in the Berkowitz laboratory for over a decade, with a particular emphasis on developing amino-sugar triflate displacement chemistry (glucosamine scaffold) with attention to appropriate N-protecting group.
We are currently preparing to test ligand analog prospects for their ability to affect bacterial growth by performing Kirby-Bauer antibiotic susceptibility assays. The antibiotic effects of the ligand on bacterial cell growth will be assessed first utilizing Bacillus subtilis or Bacillus cereus as model systems. The effectiveness of these ligand analogs as antibiotics is expected to translate to many of the other biomedically relevant bacterium that harbor the glmS ribozyme such as methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile, to name a few. Our collaborator Dr. Richard Goering (Creighton University) is an internationally-recognized leader in methicillin-resistant Staph. aureus research and we will have access to all the strains available in his laboratory.
Building on the knowledge we have already gained in our studies of the ligand requirements of the glmS ribozyme, we have begun to examine the ability of non-natural ligands to stimulate glmS self-cleavage, affect gene expression and ultimately act as anti-microbial agents. We have formed a collaboration with Dr. David Berkowitz at University of Nebraska-Lincoln. He is an organic chemist with expertise in phosphonate and fluorinated phosphonate syntheses and an interest in modified sugar synthesis. He has begun to synthesize glucosamine-6-phosphate analogs that might act as novel antibiotics, targeting the glmS self-cleaving riboswitch. Promising results have already been obtained. The synthetic tools to make these studies possible have been under steady development in the Berkowitz laboratory for over a decade, with a particular emphasis on developing amino-sugar triflate displacement chemistry (glucosamine scaffold) with attention to appropriate N-protecting group.
We are currently preparing to test ligand analog prospects for their ability to affect bacterial growth by performing Kirby-Bauer antibiotic susceptibility assays. The antibiotic effects of the ligand on bacterial cell growth will be assessed first utilizing Bacillus subtilis or Bacillus cereus as model systems. The effectiveness of these ligand analogs as antibiotics is expected to translate to many of the other biomedically relevant bacterium that harbor the glmS ribozyme such as methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile, to name a few. Our collaborator Dr. Richard Goering (Creighton University) is an internationally-recognized leader in methicillin-resistant Staph. aureus research and we will have access to all the strains available in his laboratory.
ANALYSIS OF OTHER RIBOSWITCHES
Structural Determination of Riboswitches using X-ray Crystallography
Structural studies of riboswitches are essential in order to gain detailed information about how the RNA interacts with its metabolite and to ultimately design non-natural metabolite analogs that can act as antibiotics. A number of riboswitch RNAs have already been crystallized. We are focused on crystallizing additional riboswitch class members as well as mutants of natural riboswitches in order to gain the information needed for rational design of potential anti-microbial agents.
X-ray crystallography is a technique that undergraduate students are rarely exposed to or involved with. It is exciting to be able to involve my students in this work and to be able to take some of my discoveries and new knowledge into the classroom where many students can learn about the power of this biophysical method. I spent my sabbatical year (2007-2008) learning about RNA crystallography from my colleague Dr. Robert Batey (Univ. Colorado-Boulder). Dr. Batey has remained a constant source of information to my research laboratory. We have access to an X-ray source in Omaha at University of Nebraska Medical Center in Dr. Gloria Borgstahl’s laboratory (less than 10 minutes from Creighton’s campus). Dr. Borgstahl is an expert protein crystallographer and she has given us open access to her equipment and personnel as needed.
Discovery of New Riboswitch Classes
My laboratory is also interested in discovering new classes of riboswitches. We are currently sequencing the genomes of some uncharacterized extremophiles isolated in Nebraska, and we will be searching for known and novel riboswitches. We will be aided by collaborations with the bioinformatics department at University of Nebraska – Omaha.
We are also interested in eukaryotic riboswitches, which have been discovered in plants, but not in animals. We are currently characterizing a putative mammalian riboswitch by in-line probing, equilibrium dialysis, and additional biochemical analyses.
Structural Determination of Riboswitches using X-ray Crystallography
Structural studies of riboswitches are essential in order to gain detailed information about how the RNA interacts with its metabolite and to ultimately design non-natural metabolite analogs that can act as antibiotics. A number of riboswitch RNAs have already been crystallized. We are focused on crystallizing additional riboswitch class members as well as mutants of natural riboswitches in order to gain the information needed for rational design of potential anti-microbial agents.
X-ray crystallography is a technique that undergraduate students are rarely exposed to or involved with. It is exciting to be able to involve my students in this work and to be able to take some of my discoveries and new knowledge into the classroom where many students can learn about the power of this biophysical method. I spent my sabbatical year (2007-2008) learning about RNA crystallography from my colleague Dr. Robert Batey (Univ. Colorado-Boulder). Dr. Batey has remained a constant source of information to my research laboratory. We have access to an X-ray source in Omaha at University of Nebraska Medical Center in Dr. Gloria Borgstahl’s laboratory (less than 10 minutes from Creighton’s campus). Dr. Borgstahl is an expert protein crystallographer and she has given us open access to her equipment and personnel as needed.
Discovery of New Riboswitch Classes
My laboratory is also interested in discovering new classes of riboswitches. We are currently sequencing the genomes of some uncharacterized extremophiles isolated in Nebraska, and we will be searching for known and novel riboswitches. We will be aided by collaborations with the bioinformatics department at University of Nebraska – Omaha.
We are also interested in eukaryotic riboswitches, which have been discovered in plants, but not in animals. We are currently characterizing a putative mammalian riboswitch by in-line probing, equilibrium dialysis, and additional biochemical analyses.
