The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription

Ning Ke, Xiang Gao, Jill B. Keeney, Jef D. Boeke, Daniel F. Voytas

Research output: Contribution to journalArticle

Abstract

Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated.

Original languageEnglish (US)
Pages (from-to)929-938
Number of pages10
JournalRNA
Volume5
Issue number7
DOIs
StatePublished - 1999

Fingerprint

RNA, Transfer, Met
Anticodon
Retroelements
Methionine
Reverse Transcription
Yeasts
Binding Sites
Transfer RNA
Base Pairing
Messenger RNA
Mutation
Schizosaccharomyces
Retroviridae
Arabidopsis
RNA
DNA

Keywords

  • cDNA synthesis
  • Copia
  • Priming
  • Retrotransposition
  • Retrovirus

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

Cite this

The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription. / Ke, Ning; Gao, Xiang; Keeney, Jill B.; Boeke, Jef D.; Voytas, Daniel F.

In: RNA, Vol. 5, No. 7, 1999, p. 929-938.

Research output: Contribution to journalArticle

Ke, Ning ; Gao, Xiang ; Keeney, Jill B. ; Boeke, Jef D. ; Voytas, Daniel F. / The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription. In: RNA. 1999 ; Vol. 5, No. 7. pp. 929-938.
@article{82fd18ab79a3467088050eae8e5537bf,
title = "The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription",
abstract = "Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated.",
keywords = "cDNA synthesis, Copia, Priming, Retrotransposition, Retrovirus",
author = "Ning Ke and Xiang Gao and Keeney, {Jill B.} and Boeke, {Jef D.} and Voytas, {Daniel F.}",
year = "1999",
doi = "10.1017/S1355838299990015",
language = "English (US)",
volume = "5",
pages = "929--938",
journal = "RNA",
issn = "1355-8382",
publisher = "Cold Spring Harbor Laboratory Press",
number = "7",

}

TY - JOUR

T1 - The yeast retrotransposon Ty5 uses the anticodon stem-loop of the initiator methionine tRNA as a primer for reverse transcription

AU - Ke, Ning

AU - Gao, Xiang

AU - Keeney, Jill B.

AU - Boeke, Jef D.

AU - Voytas, Daniel F.

PY - 1999

Y1 - 1999

N2 - Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated.

AB - Retrotransposons and retroviruses replicate by reverse transcription of an mRNA intermediate. Most retroelements initiate reverse transcription from a host-encoded tRNA primer. DNA synthesis typically extends from the 3'-OH of the acceptor stem, which is complementary to sequences on the retroelement mRNA (the primer binding site, PBS). However, for some retrotransposons, including the yeast Ty5 elements, sequences in the anticodon stem-loop of the initiator methionine tRNA (IMT) are complementary to the PBS. We took advantage of the genetic tractability of the yeast system to investigate the mechanism of Ty5 priming. We found that transposition frequencies decreased at least 800-fold for mutations in the Ty5 PBS that disrupt complementarity with the IMT. Similarly, transposition was reduced at least 200-fold for IMT mutations in the anticodon stem-loop. Base pairing between the Ty5 PBS and IMT is essential for transposition, as compensatory changes that restored base pairing between the two mutant RNAs restored transposition significantly. An analysis of 12 imt mutants with base changes outside of the region of complementarity failed to identify other tRNA residues important for transposition. In addition, assays carried out with heterologous IMTs from Schizosaccharomyces pombe and Arabidopsis thaliana indicated that residues outside of the anticodon stem-loop have at most a fivefold effect on transposition. Our genetic system should make it possible to further define the components required for priming and to understand the mechanism by which Ty5's novel primer is generated.

KW - cDNA synthesis

KW - Copia

KW - Priming

KW - Retrotransposition

KW - Retrovirus

UR - http://www.scopus.com/inward/record.url?scp=0033031015&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033031015&partnerID=8YFLogxK

U2 - 10.1017/S1355838299990015

DO - 10.1017/S1355838299990015

M3 - Article

VL - 5

SP - 929

EP - 938

JO - RNA

JF - RNA

SN - 1355-8382

IS - 7

ER -