Pseudouridine synthase 1: a site-specific synthase without strict sequence recognition requirements

Abstract

Pseudouridine synthase 1 (Pus1p) is an unusual site-specific modification enzyme in that it can modify a number of positions in tRNAs and can recognize several other types of RNA. No consensus recognition sequence or structure has been identified for Pus1p. Human Pus1p was used to determine which structural or sequence elements of human tRNA(Ser) are necessary for pseudouridine (Ψ) formation at position 28 in the anticodon stem-loop (ASL). Some point mutations in the ASL stem of tRNA(Ser) had significant effects on the levels of modification and compensatory mutation, to reform the base pair, restored a wild-type level of Ψ formation. Deletion analysis showed that the tRNA(Ser) TΨC stem-loop was a determinant for modification in the ASL. A mini-substrate composed of the ASL and TΨC stem-loop exhibited significant Ψ formation at position 28 and a number of mutants were tested. Substantial base pairing in the ASL stem (3 out of 5 bp) is required, but the sequence of the TΨC loop is not required for modification. When all nucleotides in the ASL stem other than U28 were changed in a single mutant, but base pairing was retained, a near wild-type level of modification was observed.

Figure 1.

Human tRNA^Ser(UGA) sequence and secondary structure and ASL stem consensus sequence. (A) The sequence and proposed secondary structure of Human tRNA^Ser(UGA) (50). The major aspects of the secondary structure are labeled on the diagram and it is numbered without including most of the Variable stem-loop. The single site of Pus1p modification, position 28, is boxed. (B) Consensus sequences for sequenced tRNAs from the fungi and metazoa group in the tRNAdb (57) that have a Ψ or a U at position 28. The figure is presented in the output style from tRNAdb. A solid line between nucleotides indicates Watson-Crick pairs only and a dash line indicates G–U pairs (<50%) are also found at this position.

Figure 2.

Time course experiments with single and double mutants in the ASL of human tRNA^Ser. (A–G), ³H-labeled RNAs were incubated with hPus1p for the times indicated and the amount of Ψ formed assayed. Each panel has the same time course of wild-type (wt) tRNA^Ser values. Each mutant is plotted separately and standard deviation (SD) bars are displayed for all time points. (H) Diagram of ASL point mutations made in the context of full-length tRNA^Ser that are presented in panels A–G.

Figure 3.

ASL & TΨC stem-loop mini-substrate and mutations. (A) Predicted structure of the ASL & TΨC stem loop mini-substrate, retaining the numbering found on the full-length tRNA^Ser and indicating the position of the Ψ formed. The structure for the Y for Y R for R stem mini-substrate mutant and the C28 mutant are also shown. (B) Diagram of the possible base pairing in the ASL stem mutants in the mini-substrate listed in Table 3. The activities in percent of the level observed with wild-type ASL & TΨC stem-loop mini-substrate without the SD are listed above the diagrams. The mutations found in each stem are listed below the diagrams.