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

Sibert BS, Patton JR - Nucleic Acids Res. (2011)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Microbiology and Immunology, University of South Carolina, School of Medicine, Columbia, SC 29208, USA.

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.

Show MeSH
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 tRNASer 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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3299991&req=5

gkr1017-F3: 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 tRNASer 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.

Mentions: These results suggest that the secondary structure of the tRNASer is very important for the modification at position 28, and a minimal substrate could be created by simply connecting the ASL and TΨC stem-loop as diagrammed (Figure 3A). The numbering on the diagram is consistent with full-length tRNASer to allow comparisons with the results reported in Figure 2 and Table 2. The level of Ψ formation at position 28 in this minimal substrate (wild-type ASL & TΨC mini-substrate) was ~0.60 mol Ψ/mol RNA after 2 h of incubation, a substantial level of modification (Table 3). A mini-substrate incorporating a 28C/42G double mutation was used in a separate experiment with little or no Ψ formation observed (Table 3), providing evidence that in this mini-substrate, the U at position 28 is the only one being converted to Ψ, as expected.Figure 3.


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

Sibert BS, Patton JR - Nucleic Acids Res. (2011)

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 tRNASer 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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299991&req=5

gkr1017-F3: 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 tRNASer 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.
Mentions: These results suggest that the secondary structure of the tRNASer is very important for the modification at position 28, and a minimal substrate could be created by simply connecting the ASL and TΨC stem-loop as diagrammed (Figure 3A). The numbering on the diagram is consistent with full-length tRNASer to allow comparisons with the results reported in Figure 2 and Table 2. The level of Ψ formation at position 28 in this minimal substrate (wild-type ASL & TΨC mini-substrate) was ~0.60 mol Ψ/mol RNA after 2 h of incubation, a substantial level of modification (Table 3). A mini-substrate incorporating a 28C/42G double mutation was used in a separate experiment with little or no Ψ formation observed (Table 3), providing evidence that in this mini-substrate, the U at position 28 is the only one being converted to Ψ, as expected.Figure 3.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Microbiology and Immunology, University of South Carolina, School of Medicine, Columbia, SC 29208, USA.

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.

Show MeSH