Enzymatic Formation of Modified Nucleosides in tRNA: Dependence on tRNA Architecture

The biosynthesis of 4-thiouridine (s 4 U) in Escherichia coli tRNA requires the action of both the thiamin pathway enzyme ThiI and the cysteine desulfurase IscS. IscS catalyzes sulfur transfer from L-cysteine to ThiI, which utilizes Mg-ATP to activate uridine 8 in tRNA and transfers sulfur to give s 4 U. In this work, we show through deletion analysis of unmodified E. coli tRNA Phe that the minimum substrate for s 4 U modification is a mini-helix comprising the stacked acceptor and T stems containing an internal bulged region. The size of the bulged loop must be at least 4 nucleotides and contain the target uridine as the first nucleotide. Replacement of the T loop sequence with a tetraloop in the deletion substrate increases activity and shows that the T⌿C primary sequence is not a recognition element. An unmodified tRNA Phe transcript in which the 3 -terminal ACCA sequence is removed to give a blunt terminus has <0.1% activity, although the addition of a single overhanging base essentially restores activity. In addition, reducing the distance of the 3 terminus relative to U8 by as little as 1 bp severely impairs activity. By dissecting a minimal RNA substrate in the T loop region, a two-piece system consisting of a substrate RNA and a "guide" RNA is efficiently modified. Our results indicate that outside of the modified U8, there is no primary sequence requirement for substrate recognition. However, the secondary and tertiary structure restrictions appear sufficient to explain why s 4 U modification is limited in the cell to tRNA.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

Lauhon

Erwin

Ton

2004

“…Suggestions have been put forward that, apart from their usual catalytic role, certain PUS enzymes (e.g. TruB) may also act as chaperones for RNA folding (11,13). Crystal structures have been determined for TruA (14), TruB (15,16), RsuA (17), and RluD (18,19) from E. coli and TruB from Thermatoga maritima (16).…”

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confidence: 99%

Crystal Structure of the Apo Forms of ψ 55 tRNA Pseudouridine Synthase from Mycobacterium tuberculosis

Chaudhuri

Chan

Perry

et al. 2004

The three-dimensional structure of the RNA-modifying enzyme, ⌿55 tRNA pseudouridine synthase from Mycobacterium tuberculosis, is reported. The 1.9-Å resolution crystal structure reveals the enzyme, free of substrate, in two distinct conformations. The structure depicts an interesting mode of protein flexibility involving a hinged bending in the central ␤-sheet of the catalytic module. Key parts of the active site cleft are also found to be disordered in the substrate-free form of the enzyme. The hinge bending appears to act as a clamp to position the substrate. Our structural data furthers the previously proposed mechanism of tRNA recognition. The present crystal structure emphasizes the significant role that protein dynamics must play in tRNA recognition, base flipping, and modification.

“…Finally, their origin in the different domains is associated with disparate tRNA recognition elements. In bacterial TGT, a UGU sequence at positions 33-35 of an anticodonlike stem-loop is the only essential structural element required for enzyme recognition (22,23), while the eukaryotic TGT appears to require higher order structure in the tRNA (24). The presence of archaeosine in a different region of the tRNA necessitates recognition elements that are unique to the archaeal enzyme.…”

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confidence: 99%

Hypermodification of tRNA in Thermophilic Archaea

Bai

Fox

Lacy

et al. 2000

tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified nucleosides. Two structural variants of the hypermodified nucleoside 7-deazaguanosine have been identified in tRNA: queuosine, which is found at the wobble position of the anticodon in bacterial and eukaryotic tRNA, and archaeosine, which is found at position 15 of the D-loop in archaeal tRNA. From homology searching of the Methanococcus jannaschii genome, a gene coding for an enzyme in the biosynthesis of archaeosine (tgt) was identified and cloned. The tgt gene was overexpressed in an Escherichia coli expression system, and the recombinant tRNA-guanine transglycosylase enzyme was purified and characterized. The enzyme catalyzes a transglycosylation reaction in which guanine is eliminated from position 15 of the tRNA and an archaeosine precursor (preQ 0 ) is inserted. The enzyme is able to utilize both guanine and the 7-deazaguanine base preQ 0 as substrates, but not other 7-deazaguanine bases, and is able to modify tRNA from all three phylogenetic domains. The enzyme shows optimal activity at high temperature and acidic pH, consistent with the optimal growth conditions of M. jannaschii. The nature of the temperature dependence is consistent with a requirement for some degree of tRNA tertiary structure in order for recognition by the enzyme to occur.