Molecular Aspects and Applications of RNA Function

The focus of my research group is to study the structural, functional, and evolutionary capabilities of RNA and use this as a foundation to develop new biotechnological reagents. In recent years, the importance of RNA has reemerged in the post-genomic era, when it was discovered that mammalian genomes display an order of magnitude more transcripts than genes. Much of this ‘non-coding’ RNA was subsequently shown to adopt complex, globular conformations with the ability to display catalytic activities, some of which are responsible for essential cellular processes. My group uses the prokaryotic 70S ribosome from E. coli as a model system for studying the macromolecular functions of RNA/protein complexes. Current projects include: (1) performing solution-based structure probing of ribosomal complexes at different steps in translation, (2) using site-directed mutagenesis to construct in vivo-assembled ribosomes to test our structure-based hypotheses, and (3) developing in vitro evolution protocols to construct novel RNA molecules that inhibit the function of therapeutic targets.

Ribosome Structure and Function

The ribosome is a universally conserved macromolecular machine that performs protein synthesis and is essential to all forms of life. During protein synthesis, or ‘translation’, messenger RNA (mRNA) and transfer RNA (tRNA) move in a coordinated manner through the ribosome, translating genetic information into a series of peptidyl transferase reactions that are responsible for polypeptide synthesis. Various x-ray crystal structures of the 30S, 50S and 70S ribosome complexes have provided snapshots of the interactions between tRNA, mRNA, and the ribosome (Figure 1). The ribosome binds to tRNA at three different sites: the A- (aminoacyl), P- (peptidyl), and E- (exit) sites. Each binding site can be split into two interactions between the tRNA and each subunit of the ribosome. The 50S subunit is primarily responsible for the peptidyl transferase reaction, which is where the acceptor ends of each tRNA bind. The 30S subunit binds to the mRNA and the anticodon ends of each tRNA. Each phase of translation (initiation, elongation, termination, and recycling) involves various protein factors that bind to the ribosomal subunit interface, and it appears that different conformations of tRNA and the ribosome are responsible for the specificity of each translation factor, although the nature of these changes is not well understood.

Figure 1: The 70S ribosome crystal structure from T. thermophilus

Ribosome Structure Probing: Chemical Footprinting

The chemical footprinting methodology enables a unique opportunity to measure tRNA and factor binding sites, conformational changes in ribosomal RNA (rRNA), and changes in equilibrium of different conformations and/or binding states in solution. In contrast, other methods used to study ribosome structure are limited to static visualization of stable conformations that do not provide information about equilibrium and/or nucleotide identity. Chemical footprinting involves the use of base-specific reagents to covalently modify rRNA in positions that are solvent-accessible. Once the ribosome is modified and RNA is extracted, the naked rRNA can be used as a template for primer extension analysis (a technique in which a primer is annealed to the rRNA and a reverse transcriptase (RT) synthesizes a complementary DNA strand). When the RT encounters a base modification, the reaction stops and can be visualized on a denaturing polyacrylamide gel. Previous chemical footprinting results have assigned specific rRNA bases that interact with tRNA at each position in the 70S ribosome by examining differences in chemical protection for various ribosome·tRNA complexes. Combining chemical footprinting data with the atomic resolution x-ray crystal structure of the 70S ribosome provides an experimental system to: (1) unequivocally determine the binding sites of previously uncharacterized translation factors, and (2) study the movement of ribosome-bound tRNA during various steps of translation.

Figure 2: Chemical Footprinting Protocol. (A) ribosome functional complex; (B) chemical modification; (C) RNA extraction; (D) primer annealing; (E) primer extension.

Figure 2: Chemical Footprinting Protocol. (A) ribosome functional complex; (B) chemical modification; (C) RNA extraction; (D) primer annealing; (E) primer extension.

Identification of Compounds that Inhibit Ribosome Function

Appearance and spread of antibiotic resistance remains a growing problem and is the subject of active research in many laboratories. In some cases, resistance is attained by mutations in the rRNA. Additionally, many pathogens have inherent resistance to established antibiotic compounds, also possibly due to sequence differences at the rRNA level. Recent mutational data have mapped regions of the ribosome that are essential, potentially elucidating regions to target for antibiotic development. We propose to use a specialized translation system to construct mutant ribosomes conferring antibiotic resistance and use a cell-based assay to screen for compounds that specifically inhibit these mutants.

The specialized ribosome system is constructed by making mutations in a 16S rRNA plasmid containing a modified anti-Shine-Dalgarno sequence (5’-GGGGU-3’). The 16S rRNA plasmid is then transformed into a cell line that carries a chromosomally encoded lacZ reporter gene with a complementary modified Shine-Dalgarno sequence (5’AUCCC-3’). When cells carrying the mutant plasmid are subjected to a compound library, inhibitors will be distinguished by a decrease in b-galactosidase production. Compounds that target the expression of b-galactosidase will be eliminated by determining if they cause inhibition in cells transformed with a wild-type lacZ reporter gene. Confirmation of ribosome inhibition will be tested with in vitro translation and ribosome function assays.

Evolving RNA Aptamers: Developing Inhibitors of Human Factor VIII

Factor VIII is an essential plasma glycoprotein that functions as a cofactor in the blood coagulation cascade. The C-terminal, or ‘C2’ domain of factor VIII is primarily responsible for the ability of factor VIII to bind activated platelet surfaces that contain phosphatidylserine (PS) as well as its circulatory partner, von Willebrand Factor (vWF). Elevated levels of factor VIII are often associated with venous thrombosis and many efforts have been made to disrupt its cofactor function for therapeutic purposes. Recently, inhibitory antibodies and small molecules have been shown to directly interact with the factor VIII C2 domain and effectively block its coagulant function. We propose to use SELEX (Systematic Evolution of Ligands by EXponential enrichment) to construct RNA aptamers that specifically bind to the C2 domain of factor VIII with the hypothesis that blocking the C2 domain will inhibit the coagulant activity of factor VIII.

The SELEX method is a well-established protocol, where 10¹⁴ – 10¹⁶ synthetic, random-sequence nucleic acid molecules are generated for in vitro selection. The segment of random RNA sequences is flanked by primer-binding sequences to facilitate amplification. The SELEX selection cycle is performed by iteratively partitioning RNAs that display the desired binding properties with subsequent amplification. This enriches the nucleic acid population for sequences that display the desired biochemical property. Once the RNA pools have been sufficiently enriched, the binding RNAs are sequenced to identify classes of RNA molecules that perform the desired biochemical function. Subsequent biochemical and biophysical characterization of each class of RNA molecules will elucidate the nature of the RNA aptamer/factor VIII C2 domain interaction. Furthermore, classes of RNAs that bind with high affinity to the C2 domain will be used for cocrystallization experiments. We have already developed a method for high-level expression and purification of an affinity-tagged factor VIII C2 domain. Additionally, we have established protocols for examining the structure and cofactor function of the factor VIII C2 domain.

In my research group, students will gain experience in the fields of molecular biology, protein and RNA biochemistry, ribosome biochemistry, and structural biology. More specifically, we use techniques such as PCR, site-directed mutagenesis, FPLC/HPLC, SDS-PAGE, chemical footprinting, filter-binding, UV/Vis and CD spectroscopies, ITC, and x-ray crystallography. All student researchers will also be expected to communicate their research findings by written reports and oral presentation.