tRNA in codon usage

PROJECT SUMMARY: Codon usage is an important regulator of gene expression of each genome. In the degeneracy of the genetic code, proteins can be coded in multiple ways using different sets of synonymous codons, which are not translated equally. Each codon makes distinct demands on the supply of the tRNAs with the matching anticodons. The quality of a codon-anticodon pairing is determined not only by the availability of the tRNA, but also by the epigenetic modifications to the tRNA, to the mRNA codon, and to the rRNA that reads the pairing interaction. Importantly, epigenetic modifications to tRNA, mRNA, and rRNA, the three major types of RNAs, have emerged as the frontier for understanding expression of the genetic code. Each modification in an RNA is catalyzed by a dedicated enzymatic pathway post-transcriptionally without altering the genome. While all modifications must converge at the ribosome during protein synthesis, this concept is under-appreciated and rarely tested. In the past 5 years, we have developed tools and reagents to test this concept. In the next 5 years, we will focus on three areas, using the experimentally trackable E. coli as a model, where post-transcriptional modifications in all three major types of RNAs have been identified. Notably, while modifications to tRNA and rRNA are well known in E. coli, modifications to mRNA were just discovered, representing an unexplored frontier that will impact on our understanding of bacterial survival and resistance to antibiotics. In the first area, we will extend our study of the N1-methylation of guanosine at position 37 (m1G37) on the 3'-side of the tRNA anticodon. While m1G37 is conserved in evolution and is essential for life, we have successfully isolated E. coli mutants that suppress the loss of viability upon deletion of m1G37. Mechanistic studies show that these suppressors now require a minor isoacceptor tRNA for survival. We will determine whether this is a general principle of bacterial survival upon loss of m1G37 by testing two pathogenic bacterial species of importance (Vibrio cholerae and Acinetobacter baumannii). We will also define the genetic mechanism of survival of one of these suppressors. In the second area, we will study the adenosine-to-inosine (A-to-I) editing, which occurs both at the wobble position of bacterial Arg(ACG) tRNA and at Y29 (tyrosine at position 29) of hokB-mRNA, which codes for a self-killing toxin that guides bacteria to persistence in nutrient starvation. We will determine how the A-to-I editing in Arg(ACG) changes the decoding specificity on the ribosome, how the editing in hokB-mRNA is regulated, and whether E. coli virulent strains exhibit a different editing profile than a non-virulent strain. In the third area, we will study the di-methylation marks m62A1518- m62A1519 in the 16S rRNA of the E. coli ribosome as a checkpoint for quality control of protein synthesis. We will address how loss of the checkpoint confers resistance to the antibiotic kasugamycin (KSG) and how the checkpoint interacts with m1G37-tRNA and with the inosine-modified Arg(ACG). We aim to obtain an integrated view of how post-transcriptional modifications in these RNAs confer the quality of life at the limit of cell survival.