The 2-thiouridine (s2U) at the wobble position of certain bacterial and eukaryotic tRNAs enhances aminoacylation kinetics, assists proper codon-anticodon base pairing at the ribosome A-site, and prevents frameshifting during translation. By mass spectrometry of affinity-purified native E. coli tRNA1GlnUUG, we show that the complete modification at the wobble position 34 is 5-carboxyaminomethyl-2-thiouridine (cmnm5s2U). The crystal structure of E. coli GlnRS bound to native tRNA1Gln and ATP demonstrates that cmnm5s2U34 improves the order of a previously unobserved 11 amino acid surface loop in the distal β-barrel domain of the enzyme, and imparts other local rearrangements of nearby amino acids that create a binding pocket for the 2-thio moiety. Together with previously solved structures, these observations explain the degenerate recognition of C34 and modified U34 by GlnRS. Comparative pre-steady state aminoacylation kinetics of native tRNA1Gln, synthetic tRNA1Gln containing s2U34 as sole modification, and unmodified wild-type and mutant tRNA1Gln and tRNA2Gln transcripts demonstrates that the exocyclic sulfur moiety improves tRNA binding affinity to GlnRS 10-fold compared with the unmodified transcript, and that an additional four-fold improvement arises from the presence of the cmnm5 moiety. Measurements of Gln-tRNAGln interactions at the ribosome A-site show that the s2U modification enhances binding affinity to the glutamine codons CAA and CAG, and increases the rate of GTP hydrolysis by E. coli EF-Tu by five-fold.

Adenosine to inosine editing at the wobble position allows decoding of multiple codons by a single tRNA. This reaction is catalyzed by adenosine deaminases acting on tRNA (ADATs) and is essential for viability. In bacteria, the anticodon-specific enzyme is a homodimer that recognizes a single tRNA substrate (tRNA Arg ACG ) and can efficiently deaminate short anticodon stem-loop mimics of this tRNA in vitro. The eukaryal enzyme is composed of two nonidentical subunits, ADAT2 and ADAT3, which upon heterodimerization, recognize seven to eight different tRNAs as substrates, depending on the organism, and require a full-length tRNA for activity. Although crystallographic data have provided clues to why the bacterial deaminase can utilize short substrates, residues that provide substrate binding and recognition with the eukaryotic enzymes are not currently known. In the present study, we have used a combination of mutagenesis, binding studies, and kinetic analysis to explore the contribution of individual residues in Trypanosoma brucei ADAT2 (TbADAT2) to tRNA recognition. We show that deletion of the last 10 amino acids at the C terminus of TbADAT2 abolishes tRNA binding. In addition, single alanine replacements of a string of positively charged amino acids (KRKRK) lead to binding defects that correlate with losses in enzyme activity. This region, which we have termed the KR-domain, provides a first glance at key residues involved in tRNA binding by eukaryotic tRNA editing deaminases.

Human tRNALys3UUU is the primer for HIV replication. The HIV-1 nucleocapsid protein, NCp7, facilitates htRNALys3UUU recruitment from the host cell by binding to and remodeling the tRNA structure. Human tRNALys3UUU is post-transcriptionally modified, but until recently, the importance of those modifications in tRNA recognition by NCp7 was unknown. Modifications such as the 5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34 and 2-methylthio-N6-threonylcarbamoyladenosine, adjacent to the anticodon at position-37, are important to the recognition of htRNALys3UUU by NCp7. Several short peptides selected from phage display libraries were found to also preferentially recognize these modifications. Evolutionary algorithms (Monte Carlo and self-consistent mean field) and assisted model building with energy refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify the in silico results and elucidate a 15-amino acid signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic) that recognized the tRNA’s fully modified anticodon stem and loop domain, hASLLys3UUU. Peptides of this sequence specifically recognized and bound modified htRNALys3UUU with an affinity 10-fold higher than that of the starting sequence. Thus, this approach provides an effective means of predicting sequences of RNA binding peptides that have better binding properties. Such peptides can be used in cell and molecular biology as well as biochemistry to explore RNA binding proteins and to inhibit those protein functions.

Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by adenosine deaminases acting on tRNA (ADATs). This essential reaction in bacteria and eukarya permits a single tRNA to decode multiple codons. Bacterial ADATa is a homodimer with two bound essential Zn 2؉ . The ADATa crystal structure revealed residues important for substrate binding and catalysis; however, such high resolution structural information is not available for eukaryotic tRNA deaminases. Despite significant sequence similarity among deaminases, we continue to uncover unexpected functional differences between Trypanosoma brucei ADAT2/3 (TbADAT2/3) and its bacterial counterpart. Previously, we demonstrated that TbADAT2/3 is unique in catalyzing two different deamination reactions. Here we show by kinetic analyses and inductively coupled plasma emission spectrometry that wild type TbADAT2/3 coordinates two Zn 2؉ per heterodimer, but unlike any other tRNA deaminase, mutation of one of the key Zn 2؉ -coordinating cysteines in TbADAT2 yields a functional enzyme with a singlebound zinc. These data suggest that, at least, TbADAT3 may play a role in catalysis via direct coordination of the catalytic Zn 2؉ . These observations raise the possibility of an unusual Zn 2؉ coordination interface with important implications for the function and evolution of editing deaminases.

In all organisms, precursor tRNAs are processed into mature functional units by post-transcriptional changes. These involve 5′ and 3′ end trimming as well as the addition of a significant number of chemical modifications, including RNA editing. The only known example of non-organellar C to U editing of tRNAs occurs in trypanosomatids. In this system, editing at position 32 of the anticodon loop of tRNAThr(AGU) stimulates, but is not required for, the subsequent formation of inosine at position 34. In the present work, we expand the number of C to U edited tRNAs to include all the threonyl tRNA isoacceptors. Notably, the absence of a naturally encoded adenosine, at position 34, in two of these isoacceptors demonstrates that A to I is not required for C to U editing. We also show that C to U editing is a nuclear event while A to I is cytoplasmic, where C to U editing at position 32 occurs in the precursor tRNA prior to 5′ leader removal. Our data supports the view that C to U editing is more widespread than previously thought and is part of a stepwise process in the maturation of tRNAs in these organisms.

The primary sequence of all nucleic acids in a cell contain 4 canonical nucleotides (G, A, T, and C for DNA and G, A, U, and C for RNA). However, post-transcriptionally, nucleic acids can undergo a number of chemical modifications, which may change their structure and function. tRNAs contain the most diverse array of post-transcriptionally added chemical groups that involve both editing and modification. Because editing and modification events can serve vital roles in cell function, it is important to develop techniques that allow for fast and accurate analysis of these events. This chapter describes the methods used to purify tRNAs from total native RNA pools and for subsequent analysis of their edited and modified states using reverse transcriptase-based approaches. These techniques, in combination with 2D-TLC, allow for the routine analysis and quantitation of edited and modified nucleotides in a fast, cost effective manner and without the need for special equipment such as HPLC or a mass spectrometer. Admittedly, the techniques described here are only applicable to a subset of post-transcriptional changes occurring in a tRNA such as C to U and A to I editing as well as modifications that prevent reverse transcriptase elongation; these have been highlighted throughout the chapter.

With the growing popularity of probiotics in dietary supplements, foods, and beverages, it is important to substantiate not only the health benefits and efficacy of unique strains but also safety. In the interest of consumer safety and product transparency, strain identification should include whole-genome sequencing and safety assessment should include genotypic and phenotypic studies. Bacillus subtilis MB40, a unique strain marketed for use in dietary supplements, and food and beverage, was assessed for safety and tolerability across in silico, in vitro, and in vivo studies. MB40 was assessed for the absence of undesirable genetic elements encoding toxins and mobile antibiotic resistance. Tolerability was assessed in both rats and healthy human volunteers. In silico and in vitro testing confirmed the absence of enterotoxin and mobile antibiotic resistance genes of safety concern to humans. In rats, the no-observed-adverse-effect level (NOAEL) for MB40 after repeated oral administration for 14 days was determined to be 2000 mg/kg bw/day (equivalent to 3.7 × 1011 CFU/kg bw/day). In a 28 day human tolerability trial, 10 × 109 CFU/day of MB40 was well tolerated. Based on genome sequencing, strain characterization, screening for undesirable attributes and evidence of safety by appropriately designed safety evaluation studies in rats and humans, Bacillus subtilis MB40 does not pose any human health concerns under the conditions tested.

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