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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.

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Human tRNASer(UGA) sequence and secondary structure and ASL stem consensus sequence. (A) The sequence and proposed secondary structure of Human tRNASer(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.
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gkr1017-F1: Human tRNASer(UGA) sequence and secondary structure and ASL stem consensus sequence. (A) The sequence and proposed secondary structure of Human tRNASer(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.

Mentions: As an initial investigation into the substrate requirements for hPus1p, it was important to choose a known Pus1p substrate tRNA that has only one site modified by the enzyme (29,35), to simplify the analysis of the activity. A tRNA that is typical in structure and has a V stem-loop that was >6 nt, was preferred in order to be able to test the contribution of all structural components to substrate recognition by hPus1p. In addition, a tRNA with traditional Watson-Crick base-pairing in the ASL stem was desired. The tRNA sequence database, tRNAdb (56,57), was consulted to compile consensus sequences for the ASL stem where a Ψ or U is found at position 28 in sequenced tRNAs from the fungi and metazoan group in tRNAdb. At least 36 tRNA sequences were compiled to generate each consensus shown in Figure 1B. Several features are apparent in these two stems. First, with a Ψ at position 28, G30–C40 is the most conserved base pair (>90% conserved) but when a U is found at position 28, this sequence is not as well conserved. Therefore, one might expect that disruption of this base pair or replacement with the inverse (C30–G40) will affect modification. Second, for sequenced tRNAs that have a Ψ at 28, the preferred 27–43 bp is U27–A43, whereas with those tRNAs with a U at 28, the preferred base pair is C27–G43. Third, a A31–U39 base pair is more common in those ASL stems that have a Ψ at position 28 than those with a U at position 28. Finally, the base pairs 29–41 and 31–39, below the 28–42 bp, are Watson-Crick only but the rest of the base pairs in the stem can have G-U pairing in either consensus. In the selection of a tRNA, a good match to this consensus was preferred, and human tRNASer(UGA) meets most of the criteria. The only difference is that this tRNA has an A27–U43 base pair instead of a U–A base pair at that position (Figure 1A).Figure 1.


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

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

Human tRNASer(UGA) sequence and secondary structure and ASL stem consensus sequence. (A) The sequence and proposed secondary structure of Human tRNASer(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.
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Related In: Results  -  Collection

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

gkr1017-F1: Human tRNASer(UGA) sequence and secondary structure and ASL stem consensus sequence. (A) The sequence and proposed secondary structure of Human tRNASer(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.
Mentions: As an initial investigation into the substrate requirements for hPus1p, it was important to choose a known Pus1p substrate tRNA that has only one site modified by the enzyme (29,35), to simplify the analysis of the activity. A tRNA that is typical in structure and has a V stem-loop that was >6 nt, was preferred in order to be able to test the contribution of all structural components to substrate recognition by hPus1p. In addition, a tRNA with traditional Watson-Crick base-pairing in the ASL stem was desired. The tRNA sequence database, tRNAdb (56,57), was consulted to compile consensus sequences for the ASL stem where a Ψ or U is found at position 28 in sequenced tRNAs from the fungi and metazoan group in tRNAdb. At least 36 tRNA sequences were compiled to generate each consensus shown in Figure 1B. Several features are apparent in these two stems. First, with a Ψ at position 28, G30–C40 is the most conserved base pair (>90% conserved) but when a U is found at position 28, this sequence is not as well conserved. Therefore, one might expect that disruption of this base pair or replacement with the inverse (C30–G40) will affect modification. Second, for sequenced tRNAs that have a Ψ at 28, the preferred 27–43 bp is U27–A43, whereas with those tRNAs with a U at 28, the preferred base pair is C27–G43. Third, a A31–U39 base pair is more common in those ASL stems that have a Ψ at position 28 than those with a U at position 28. Finally, the base pairs 29–41 and 31–39, below the 28–42 bp, are Watson-Crick only but the rest of the base pairs in the stem can have G-U pairing in either consensus. In the selection of a tRNA, a good match to this consensus was preferred, and human tRNASer(UGA) meets most of the criteria. The only difference is that this tRNA has an A27–U43 base pair instead of a U–A base pair at that position (Figure 1A).Figure 1.

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.

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