Binding synergy as an essential step for tRNA editing and modification enzyme codependence in Trypanosoma brucei

• Published in: RNA (Cambridge) Vol. 24; no. 1; pp. 56 - 66
• Main Authors: McKenney, Katherine M, Rubio, Mary Anne T, Alfonzo, Juan D
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01.01.2018
• Subjects: Active sites; Adenosine; Adenosine deaminase; Deamination; Enzymes; Gene expression; Kinetics; Methyltransferase; Post-transcription; Protein Binding; Protein biosynthesis; Protozoan Proteins - chemistry; Protozoan Proteins - physiology; RNA Editing; RNA, Protozoan - chemistry; RNA, Transfer - chemistry; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - physiology; Substrate specificity; Thermodynamics; tRNA; tRNA Ala; Trypanosoma brucei; Trypanosoma brucei brucei - enzymology; T. brucei; RNA editing; tRNA; modification
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.062893.117

Transfer RNAs acquire a variety of naturally occurring chemical modifications during their maturation; these fine-tune their structure and decoding properties in a manner critical for protein synthesis. We recently reported that in the eukaryotic parasite, , a methylation and deamination event are unexpectedly interconnected, whereby the tRNA adenosine deaminase (TbADAT2/3) and the 3-methylcytosine methyltransferase (TbTrm140) strictly rely on each other for activity, leading to formation of m C and m U at position 32 in several tRNAs. Still however, it is not clear why these two enzymes, which work independently in other systems, are strictly codependent in Here, we show that these enzymes exhibit binding synergism, or a mutual increase in binding affinity, that is more than the sum of the parts, when added together in a reaction. Although these enzymes interact directly with each other, tRNA binding assays using enzyme variants mutated in critical binding and catalytic sites indicate that the observed binding synergy stems from contributions from tRNA-binding domains distal to their active sites. These results provide a rationale for the known interactions of these proteins, while also speaking to the modulation of substrate specificity between seemingly unrelated enzymes. This information should be of value in furthering our understanding of how tRNA modification enzymes act together to regulate gene expression at the post-transcriptional level and provide a basis for the interdependence of such activities.