Dihydrouridine , tRNA structure

Figure 4. Ribbon diagram of the tRNA structure, here the yeast tRNA (29), on the right and its decomposition into two domains made of co-axially stacked helices on the left. Notice how the 3 -end of the anticodon helix (AC) runs over the deep groove of the dihydrouridine helix (D). Note the rotation angle between the AC and D helices is large, while the one between the acceptor helix (AA) and the thymine (T) helix is usual for RNA helices.

The base sequence and the tertiary structure of the yeast tRNA specific for phenylalanine (tRNA " ) is typical of all tRNAs. The molecule (see also p.86) contains a high proportion of unusual and modified components (shaded in dark green in Fig. 1). These include pseudouridine (T), dihydrouridine (D), thymidine (T), which otherwise only occurs in DNA, and many methylated nucleotides such as 7-methylguanidine (m G) and—in the anticodon—2 -0-methylguanidine (m G). Numerous base pairs, sometimes deviating from the usual pattern, stabilize the molecule s conformation (2). 

The results of these efforts show that no method of tRNA recognition is universal.2443 In some cases, e.g., for methionine- or valine-specific tRNAs, the synthetase does not aminoacylate a modified tRNA if the anticodon structure is incorrect. Although the anticodon is 7.5 ran away from the CCA end of the tRNA, the synthetases are large enzymes. Many of them are able to accommodate this large distance between a recognition site and the active site (Fig. 29-9A). For some other tRNAs the anticodon is not involved in recognition 245 For yeast tRNAphe residues in the stem of the dihydrouridine loop and at the upper end of the amino acid acceptor stem seem to be critical.241... 

Figure 5 Structural features of the tRNA from E. coli. Elements involved in recognition by seryl-tRNA synthetase are shaded, antideterminants against recognition by EF-Tu are hatched. The modified bases are D dihydrouridine F pseudouridine i A isopentenyl-adenosine T ribothymidine. Tertiary interactions involving base pairing are indicated by lines, those with intercalations by arrows...

Figure 29.3. Alanine-tRNA Sequence. The base sequence of yeast alanyl-tRNA and the deduced cloverleaf secondary structure are shown. Modified nucleosides are abbreviated as follows methylinosine (ml), dihydrouridine (UH2),...

Figure 3-12. The cloverleaf structure of tRNA. Bases that commonly occur in a particular position are indicated by letters. Base-pairing in stem regions is indicated by lines between strands, xy = pseudouridine T = ribothymi-dine D = dihydrouridine.

Transfer RNA (tRNA) molecules transport amino acids to ribosomes for assembly into proteins. Comprising about 15% of cellular RNA the average length of a tRNA molecule is 75 nucleotides. Because each tRNA molecule becomes bound to a specific amino acid, cells possess at least one type of tRNA for each of the 20 amino acids commonly found in protein. The three-dimensional structure of tRNA molecules, which resembles a warped cloverleaf (Figure 17.22), results primarily from extensive intrachain base pairing. tRNA molecules contain a variety of modified bases. Examples include pseudouridine, 4-thiouridine, 1-methylguanosine, and dihydrouridine ... 

Little mechanistic work has been published on these enzymes to date. The four DUS enzymes in S. cerevisiae each show distinct site specificity on tRNA, with each enzyme reducing either one or two specific uridines in tRNA. An in vivo DUS-complementation assay has been developed to screen for residues important in catalysis in which an E. coli strain that has all the DUS genes knocked out is complemented with a plasmid-borne DUS and the dihydrouridine content of the tRNA is then determined. This approach was used to identity a cysteine residue that is essential for DUS activity. Mapping this cysteine onto the crystal structure of a DUS from Thermatoga maritimc shows that it is positioned in the active site to act as a catalytic acid. Presumably, the mechanism of uridine reduction involves hydride transfer from N5 of the flavin to C6 of uridine and protonation of C5 of uridine by an active site acid. The actual substrate of DUSs has not been identified. The enzymes reduce in transcribed tRNAs several orders of magnitude slower than naturally modified tRNA. ° The critical modification for DUS activity has not yet been identified. 

FIGURE 25.15 (a) Structure of a tRNA isolated from yeast that has the speoifio function of transferring alanine residues. Transfer RNAs often oontain unusual nuoleosides. PSU = pseudouridine, RT = ribothymidine, Ml = 1 -methylinosine, I = inosine, DMG = A/ -methylguanosine, DHU = 4,5-dihydrouridine, 1 MG = 1 -methylguanosine. (b) The X-ray crystal structure of a phenylalanine-... 

The 2-D structure of tRNA, which is generally described by a cloverleaf model, consists of five elements a CCA acceptor stem, a D (dihydrouridine)-loop, an anticodon loop, a variable loop, and a T (ribothymidine)-loop. Tertiary interactions among the secondary structural motifs contribute to the folding that gives rise to the typical L-shaped molecule (Fig. 1.20). X-ray crystallographic structures... 

---