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Bacterial Wobble Modifications of NNA‐Decoding Trnas

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Bacterial Wobble Modifications of NNA‐Decoding Trnas Critical Review Bacterial Wobble Modi fications of NNA- Emil M. Nilsson Decoding tRNAs Rebecca W. Alexander * Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina Abstract Nucleotides of transfer RNAs (tRNAs) are highly modi fied, particularly (I34 or L34). The structural basis for many N34 modi fications in both at the anticodon. Bacterial tRNAs that read A-ending codons are espe- tRNA aminoacylation and ribosome decoding has been elucidated, and cially notable. The U34 nucleotide canonically present in these tRNAs is evolutionary conservation of modifying enzymes is also becoming modi fied by a wide range of complex chemical constituents. An addi- clearer. Here we present a brief review of the structure, function, and tional two A-ending codons are not read by U34-containing tRNAs but conservation of wobble modi fications in tRNAs that translate A-ending are accommodated by either inosine or lysidine at the wobble position codons. © 2019 IUBMB Life, 000(000):1 –9, 2019 tRNA MODIFICATIONS ribosome (3, 5), dictating aminoacyl-tRNA synthetase activity (6 –9), facilitating peptidyl-tRNA translocation (10), and fine-tuning Base pairing rules established in the context of the DNA double ribosomal kinetics (11, 12). P ost-transcriptional modi fications helix generally hold true in RNA, although the greater structural occur throughout tRNA; however, nucleotides in the anticodon and functional diversity of RNA is facilitated by a wide variety of loop are particularly highly decorated. Nucleotides N34, the wob- chemical modi fications. Transfer RNA (tRNA) serves a key func- ble position, and N37, just outside the anticodon, display a larger tion in genetic information transfer, accurately deciphering each diversity of modi fication chemistry than any other tRNA position trinucleotide codon of messenger RNA (mRNA) to produce the (1,4,13).Thisreviewwillconsidermodi fications at the N34 wob- encoded polypeptide product. tRNA is the most chemically modi- ble position in bacterial tRNAs decoding NNA codons. fied RNA species in any organism, with nucleotide modi fications impacting tRNA structure, dynamics, and function. To date over 100 unique RNA modi fications have been identi fied, more than NNA DECODING 90 of which are found in tRNA (1 –3). On average 17% of tRNA tRNAs translating NNA codons are typically encoded with a uri- nucleotides are post-transcriptionally modi fied, more than dine at position 34 (U34), as expected according to Watson-Crick 10 times the frequency observed in larger RNAs such as ribo- rules, and have the intrinsic potential to decode both NNA and somal RNA (rRNA), which is modi fied at 1 –2% (1, 4). Post- NNG codons by classical wobble pairing (14). Such expanded transcriptional modi fications have been shown to in fluence decoding can be advantageous for an organism, as a single tRNA translational accuracy by directly regulating base pairing on the can read both codons. In some cases, however, codon –anticodon wobble pairing could be detrimental to genetic code fidelity. In Abbreviations: AARS, aminoacyl-tRNA synthetase (individual AARS either situation organisms have evolved post-transcriptional enzymes are named using the 3-letter abbreviations for their cognate amino modi fication schemes to enforce the correct decoding function of acids); ASL, anticodon stem-loop; I, inosine; L, lysidine; N, any standard ribo- each tRNA (15, 16). The nature of these modi fications varies nucleotide (A, C, G, U); NMR, nuclear magnetic resonance © 2019 International Union of Biochemistry and Molecular Biology among tRNA isoacceptors and across the domains of life, but the Volume 000, Number 000, Pages 1 –9 high frequency of U34 modi fication is conserved. *Address correspondence to: Rebecca W. Alexander, Department of Chem- istry, Wake Forest University, Winston-Salem, NC 27109. E-mail: [email protected] ESCHERICHIA COLI U34 Received 3 June 2019; Accepted 21 June 2019 MODIFICATIONS DOI 10.1002/iub.2120 Published online 00 Month 2019 in Wiley Online Library There are 16 A-ending trinucleotides in the Universal Genetic (wileyonlinelibrary.com) Code, of which two are the ochre (UAA) and opal (UGA) stop IUBMB Life 1 IUBMB LIFE Gln highlighted in blue, Fig. 1) (21). tRNA UUG when fully modi fied is found with the 5-carboxymethylaminomethyl-2-thiouridine 5 2 Leu (cmnm sU) at position 34 while tRNA UAA presents a 5-carboxymethylaminomethyl-2´-O-methyluridine (cmnm 5Um) (reading codons highlighted in yellow, Fig. 1) (22, 23). Each cat- egory of modi fication will be addressed below in the context of bacterial translation, with respect to structure of the chemical moiety, the enzymatic pathway, how decoding is facilitated, and evolutionary conservation, if known. CMO 5U MODIFICATION All E. coli tRNAs with a cmo 5U modi fication also have a purine at nucleotide 35 and read codons from fourfold degenerate codon boxes (Fig. 1). The pathway for cmo 5U formation is incomplete, as the enzyme responsible fo r conversion of uridine to 5-hydroxyuridine (ho 5U) remains unknown (24). Installation of the The universal genetic code. Codons highlighted in FIG 1 carboxylmethyl group is achieved in E. coli by carboxy-S- orange are translated in bacteria by tRNAs containing adenosyl-L-methionine synth ase (CmoA), which generates an cmo 5U, those in green by tRNAs with mnm 5U, those 5 2 unusual Cx-SAM moeity, and tRNA U34 carboxymethyltransferase in blue by tRNAs with mnm sU, and those in yellow 5 by tRNAs with cmnm 5Um. Codons highlighted in (CmoB), which transfers Cx-SAM to ho U(Fig.2)(25 –27). The 5 gray are translated by tRNAs with L34 or I34. presence of the cmo U modi fication was originally proposed on theoretical grounds to expand the base pairing ability of the tRNA beyond Watson-Crick and wobble pairing so as to include the codons. Escherichia coli encodes 29 U34-containing tRNAs to ability to form the cmo 5U:U base pair (18). Subsequent in vivo translate 12 sense codons (highlighted in Fig. 1) (17), leaving work using knockouts of individual tRNA or modifying enzyme two A-ending sense codons to be decoded through adaptation of genes demonstrated that some but not all cmo 5U34-containing other tRNAs. All U34-containing E. coli tRNAs are post- tRNAs are able to ef ficiently decode even NNC codons (16, 27). transcriptionally modi fied at the wobble position with a total For example, Salmonella enterica decodes its four CCN proline of five unique species (15). The most frequent modi fication is codons with three tRNAs, but all four codons can be read with the 5 uridine 5-oxyacetic acid (cmo U), which is observed in single tRNA Pro cmo 5UUG species to maintain robust cell growth in Leu Val Ser Thr Ala tRNA UAG , tRNA UAC , tRNA UGA , tRNA UGU , tRNA UGC , the absence of G34- and C34-containing isoacceptors (27). Simi- Pro 5 Val Ala 5 and tRNA UGG (16, 18). Codons read by cmo U-containing larly, E. coli tRNA and tRNA species with cmo U34 can par- tRNAs are highlighted in orange on the Universal Genetic Code tially rescue growth phenotypes arising from knockdown of the Arg Gly table (Fig. 1). Next, tRNA UCG and tRNA UCC both contain a other cellular valine and alan ine tRNAs (16). However, the 5-methylaminomethyluridine (mnm 5U) to translate codons CGA expanded decoding ef ficiency is not used equally, even within a Glu and GGA (highlighted in green in Fig. 1) (19, 20). tRNA UUC given species. A simila r attempt to decode all S. enterica ACN Lys 5 Thr 5 and tRNA UUU also have mnm U34 but are further differenti- threonine codons with a single tRNA cmo UGU was not suc- ated through the addition of a 2-thio group, forming cessful (16). Depletion of the S. enterica cmoB gene results in 5-methylaminomethyl-2-thiouridine (mnm 5s2U) (reading codons tRNAs devoid of cmo 5U; inef ficient decoding of G-ending proline, Biosynthesis of uridine 5-oxyacetic acid (cmo 5U). R represents the 5 0-phosphoribosyl group of the tRNA backbone. The enzyme FIG 2 responsible for hydroxylation remains unknown. Carboxy-S-adenosyl-Lmethionine (Cx-SAM) is synthesized by carboxy-S- adenosyl-L-methionine synthase (CmoA) and transferred to ho 5U by tRNA U34 carboxymethyltransferase (CmoB). 2 BACTERIAL WOBBLE MODIFICATIONS OF NNA-DECODING TRNAS valine, and alanine codons demonstrated that cmo 5Uisimportant even for the predicted “normal ”wobble pairing to occur (16). The structural basis for enhanced decoding by cmo 5U incorporation was observed in crystal structures of the Thermus thermophilus 30S subunit in complex with GUN-containing mRNA oligonucleotides and a cmo 5U tRNA Val anticodon stem- loop (ASL Val ) (28). Surprisingly, both the cmo 5U:A and cmo 5U: G-containing complexes exhibit Watson-Crick geometry; this indicates that the modi fied uridine adopts the enol tautomer in the cmo 5U:G pair. The cmo 5U:U and cmo 5U:C pairs exhibit a single hydrogen bond in each case (Fig. 3). The nonwobble geometry enables stacking of cmo 5U with ASL Val A35 (position 35 is always a purine for cmo 5U-containing tRNAs) (28). While earlier NMR studies on nucleotide monophosphates suggested the ribose of cmo 5U would adopt a C2 ’-endo conformation (18), all four crystal structures exhibit a C3 0-endo conformation for Val the modi fied uridine (28). A parallel solution study of ASL UAC in the absence and presence of cmo 5U revealed that the modi fi- cation serves to preorder the anticodon loop for codon bind- ing (29). XM 5U MODIFICATIONS This class of modi fications in bacteria include mnm 5U, cmnm 5Um, and the thiolated derivatives mnm 5s2U and cmnm 5s2U, which are generated by a network of modifying enzymes in multiple steps (Fig. 4) (30 –33). These modi fications are parallel to the mcm 5U and mcm 5s2U nucleotides found in eukaryotic tRNAs (2, 32).
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