One third of the world’s population is affected by Tuberculosis (TB), a disease caused by infection with Mycobacterium tuberculosis (MtB). The emergence of multidrug-resistant MtB makes this disease a major public health concern. New agents are needed to treat TB infections in a manner that circumvents existing pathways of resistance. One strategy is to target the organism at the translational level by inhibiting vital modifications of RNA. One gene responsible for these modifications is the tRNA (guanosine-1)-methyltransferase, trmD, which has been shown to be essential in several bacteria. The eukaryotic and bacterial m1G methyltransferases are structurally dissimilar, making this enzyme an ideal target for selective anti-TB agents. One strategy for TrmD inhibitor design is to target the catalytic center of the enzyme. Existing inhibitors such as Sinefungin exhibit poor selectivity due to the substrate’s role, SAM, as a universal methyl donor in many biological processes. Structure/activity relationships for inhibitory compounds are sparse, impeding the design of novel antimicrobials. Crystallographic data would identify molecular features unique to TrmD, and allow design of agents complimentary to the TrmD active site with minimal differential toxicity. Presently, no crystal structure for Mycobacterium tuberculosis TrmD exists. As a first step in this direction, the MtB gene has been cloned and expressed by using a His-tagged T7 expression vector. The recombinant protein was characterized through kinetic and preliminary inhibitor assays. The native enzyme displays a mass of 50 kDa, proving this enzyme is a dimer of two identical subunits. This is similar to data found on other TrmD orthologs. Crystallization of MtB TrmD has been achieved and preliminary x-ray diffraction studies conducted.

Protein synthesis is a fundamental and ultimate process in living cells. Cells possess sophisticated machineries and continuously carry out complex processes. Monitoring protein synthesis in living cells not only inform us about the mechanism of translation but also deepen our insights about all aspects of life. Understanding the structure and mechanism of the ribosome and its associated factors helped us enlarge our knowledge on protein synthesis. Recently, with the dramatic advances of high-throughput sequencing and bioinformatics, a new technique called ribosome profiling emerged. By retrieving mRNA fragments protected by translating ribosomes, ribosome profiling reveals global ribosome occupancy along mRNAs in living cells, which can inform us with the identity and quantity of proteins being made. Easily adapted to other organisms, ribosome profiling technique is expanding its application in revealing various cellular activities as well as the knowledge on protein synthesis. Here, we report the mechanism of translating mRNA cleavage by endoribonuclease RelE in vivo. RelE is an endoribonuclease that is induced during nutrient deficiency stress and specifically cleaves translating mRNAs upon binding to the ribosomal A site. Overexpression of RelE in living cells causes growth arrest by inhibiting global translation. We monitored RelE activity in vivo upon overexpression using ribosome profiling. The data show that RelE actively cuts translating mRNAs whenever the ribosomal A site is accessible, resulting in truncated mRNAs. RelE causes the ribosome complexes to accumulate near the 5' end of genes as the process of ribosome rescue, translation, and cleavage by RelE repeats. RelE cleavage specific sub-codon level ribosome profiling data also represent reading frame in Escherichia coli and sequence specificity of RelE cleavage in vivo. We report another ribosome profiling study on a methyltransferase TrmD in E. coli. TrmD is known to methylate G37 (the residue at 3' side of anticodon) of some tRNAs and be responsible for codon-anticodon interaction. We constructed a TrmD depletion E. coli strain, whose deletion results in lethality of cells. Resulting depletion of m1G37 in the strain leads to growth arrest. Lack of m1G37 of some tRNAs whose codons start with C showed frequent frameshift when translating the gene message in vitro. By using ribosome profiling, we successfully observed significant difference on translation process when codons interact with anticodons of tRNAs lacking m1G37. The data reveal slow translation rate or pauses on the tRNAs when missing the appropriate methylation, which corresponds to the previous biochemical data in vitro.

Thermal stability of theG37 tRNA methyltransferase proteins from Thermotoga maritima and Aquifex aeolicus have been compared using Differential Scanning Calorimetry. It was shown that the Thermotoga protein is remarkably stable and is denatured at temperatures in excess of 100 degrees Centigrade. The Aquifex aeolicus protein was less stable, denaturing broadly at temperatures between 55oC and 100oC. In contrast, the mesophilic E. coli protein was completely denatured at 55oC. Enzymatic activity of the proteins was measured at various temperatures. Both the Thermotoga and Aquifex enzymes are active at ambient temperatures, and display a significant decrease in activity when the temperature is raised above 50oC. This may relate to subtle changes in protein structure causing an effect on the tRNA based assay. Both enzymes contain inter subunit disulfide bonds which might contribute to thermal stability. Assays of the enzymes in the presence of high concentrations of Dithiothreitol (DTT) did not significantly reduce activity at higher temperatures, but did stimulate activity at lower temperatures. Site directed mutagenesis of non -conserved protein sequences within Thermotoga maritima were initiated in order to determine what structures might confer heat stability on the protein. Alanine mutagenesis of lysine residues 103,104 led to reduced catalytic activity, but did increased activity at higher temperatures. Aspartate is the most common residue at the relative position 166 in the variable loop of most TrmD genes. It has been shown that in E. coli this is essential for catalytic activity and possibly the residue which carries out N1 deprotonation on residue G37 in tRNA. In Thermotoga glutamate is present at this position. Alanine mutagenesis of this residue did not eliminate activity suggesting another nearby residue may function in this capacity in the Thermotoga TrmD protein.

The bacterial enzyme t-RNA (m1G37) methyltransferase (TrmD) is an ideal anti-microbial drug target since it is found in all eubacteria, serves an essential role during protein synthesis, and shares very little sequence or structural homology with its eukaryotic counterpart, Trm5. TrmD, a homodimeric protein, methylates the G37 nucleotide of tRNA using S-adenosyl-L-methionine (SAM) as the methyl donor and thus enables proper codon-anticodon alignment during translation. The two deeply buried binding sites for SAM seen in X-ray crystallography suggest that significant conformational changes must occur for substrate binding and catalytic turnover. Results from molecular dynamics simulations implicate a flexible loop region and a halo-like loop which may be gating the entrance to the active site. Analysis of simulation trajectories indicates an alternating pattern of active site accessibility between the two SAM binding sites, suggesting a single site mechanism for enzyme activity. Isothermal titration calorimetry (ITC), demonstrates that binding of SAM to TrmD is an exothermic reaction resulting from sequential binding at two sites. A similar mode of binding at higher affinities was observed for the product, S-adenosyl-L-homocysteine (SAH) suggesting that product inhibition may be important in vivo. ITC reveals that tRNA binding is an endothermic reaction in which one tRNA molecule binds to one TrmD dimer. This further supports the hypothesis of a single site mechanism for enzyme function. However, mutational analysis using hybrid mutant proteins suggests that catalytic integrity must be maintained in both active sites for maximum enzymatic efficiency. Mutations impeding flexibility of the halo loop were particularly detrimental to enzyme activity. Noncompetitive inhibition of TrmD was observed in the presence of bis-ANS, an extrinsic fluorescent dye. In silico ligand docking of bis-ANS to TrmD suggests that dye interferes with mobility of the flexible linker above the active site. Because SAM is a ubiquitous cofactor in methyltransferase reactions, analogs of this ligand may not be suitable for drug development. It is therefore important to investigate allosteric modes of inhibition. These experiments have identified key, mobile structural elements in the TrmD enzyme important for activity, and provide a basis for further research in the development of allosteric inhibitors for this enzyme.