How are tRNAs synthesized and matured? How are they selectively charged with amino acids? How do they enter the ribosome, and how does their dynamics affect the decoding specificity? These questions have direct impact on human health and disease.
We aim to investigate these questions at the fundamental level: What does it take to achieve the specificity of tRNA at each step of decoding? How does each step affect the biology of a cell? How does the dysfunction of a specific step lead to a disease state? To address this fundamental question, we focus on the following features of tRNA and enzymes:
- tRNA methyl transferases TrmD and Trm5, which synthesize the essential m1G37 methylation in bacteria and in eukaryotes and archaea, respectively, for accuracy of reading frame maintenance
- Aminoacyl-tRNA synthetases, which charge an amino acid onto the cognate tRNAs to synthesize the charged aminoacyl-tRNAs
- CCA-Adding enzyme, which adds the CCA sequence to the tRNA 3' end to complete the 3’ end maturation
- The ribosome, which uses the charged aminoacyl-tRNAs as the substrates for protein synthesis.
Our major projects are described below and each answers important questions:
How does tRNA methylation regulate gene expression?
Methylation of tRNA is the most common form of post-transcriptional modification. With the addition of just one methyl group to a nucleobase or a backbone group, tRNA can gain structural stability in a particular region or decoding specificity upon pairing with an mRNA codon. However, how each methylation confers a unique strength to tRNA is poorly understood.
Recent advance in tRNA biology has opened a new era, in which the primary challenge is no longer to simply know what methylation is expressed and where, but rather to determine how each methylation impacts translation of codons and expression of genomes. We have focused on the m1G37-tRNA methylation, which is necessary to maintain protein synthesis reading frame. Loss of m1G37-tRNA leads to accumulation of +1 frameshift errors, resulting in pre-mature termination of translation and cell death. We are interested in the biology of this methylation and how it regulates gene expression.
Methylation is the major form of tRNA modifications
How is a tRNA specifically methylated?
The active site of TrmD, the bacteria-specific m1G37-tRNA methyl transferase, uses a knotted protein fold to catalyze methyl transfer. Christian et al., NSMB (2016). The specificity of anticodon-codon base pairing cannot be achieved simply by 3 base pairs. Methylation and modifications of bases adjacent to the anticodon-codon region are necessary.
The m1G37-tRNA methylation is essential for bacterial cell growth. The TrmD enzyme that synthesizes the methylation is unrelated to its human counterpart Trm5, suggesting that TrmD is an attractive target for high throughput screening of novel antibiotics. Other methylation and modifications are essential to protect against oxidative damage on tRNA.
How is a tRNA specifically charged with an amino acid?
While all tRNA molecules are similar in both the secondary and tertiary structure, how is each specifically charged with an amino acid?
How does impaired tRNA charging and aminoacylation cause human disease? Human Charcot-Marie-Tooth disease is an example where mutations in charging enzymes cause peripheral neuropathy. Other examples involving mutations in tRNA charging enzymes include epileptic encephalopathy. What is the molecular basis that links impaired tRNA charging to these disease phenotypes?
How is a tRNA matured at the 3’ end?
While the CCA sequence at the 3’ end is essential for tRNA biology, this sequence is typically not encoded in the genes and must be added post-transcriptionally after the tRNA transcripts are made. The enzyme that catalyzes the 3’ end CCA synthesis does not use a nucleic acid template. How does it achieve the specificity?
The human CCA enzyme is a model of de novo synthesis enzyme that is linked to cardiovascular disorder associated with early infant death and cardiomyopathy. Additional mutations found in this enzyme are linked to immunodeficiencies and periodic fever syndrome. What is the molecular basis that links mutations in this enzyme to the diseases?
Frameshift on the ribosome
How does a tRNA move through the ribosome one triplet codon at a time?
The tRNA movement must be fast, yet it must retain the reading frame, in order to synthesize correct proteins to support cell growth. How does a tRNA interact with the ribosome to make sure that it does not slip during the process of translation?
Frameshift of tRNA on the ribosome can occur by a programmed or non-programmed mechanism, both of which are relevant to human health. The programmed frameshifts expand the capacity of the genome, while the non-programmed shifts can create premature termination of protein synthesis, leading to cell death and a disease state. tRNA modifications are essential to control the level of frameshifts.
We are using kinetic tools to address these questions. These are unique tools that give us special insights into the problems.