Ty1 reverse transcriptase does not read through the proposed 2',5'-branched retrotransposition intermediate in vitro.
2',5'-branched RNA was recently proposed as a key Ty1 retrotransposition intermediate, for which cleavage by lariat debranching enzyme (Dbr1p) enables reverse transcription to continue synthesizing the complete Ty1 cDNA. Because dbr1 cells can produce substantial Ty1 cDNA despite lacking Dbr1p, the obligatory intermediacy of branched RNA would require that Ty1 reverse transcriptase (RT) can read through the proposed branch site with considerable efficiency. Here we have used deoxyribozyme-synthesized 2',5'-branched RNA corresponding exactly to the proposed Ty1 branch site for a direct test of this read-through ability. Using an in vitro assay that incorporates all components known to be required for Ty1 cDNA synthesis (including the TyA chaperone protein), Ty1 RT can elongate up to the branch site. Strand transfer from the 2'-arm to the 3'-arm of the branch is observed when the Ty1 RT is RNase H+ (i.e., wild-type) but not when the Ty1 RT is RNase H-. When elongating from either the 2'-arm or the 3'-arm, Ty1 RT reads through the branch site with <or=0.3% efficiency. This is at least 60-fold lower than would be necessary to explain in vivo Ty1 cDNA synthesis in dbr1 cells, because others have reported 18% cDNA synthesis relative to wild-type cells. Our finding that Ty1 RT cannot efficiently read through the proposed Ty1 branch site is inconsistent with the hypothesis that branched RNA is an obligatory Ty1 retrotransposition intermediate. This suggests that Dbr1p acts as other than a 2',5'-phosphodiesterase during Ty1 retrotransposition.
Pratico
Elizabeth D
ED
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Silverman
Scott K
SK
D019426:Integrases / Q000737:chemistry / Q000235:genetics; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics*; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D018626:Retroelements / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics
D012331:RNA, Fungal; D018626:Retroelements; D019426:Integrases; D012316:RNA Nucleotidyltransferases; C505387:RNA debranching enzyme Dbr1, S cerevisiae; D012194:RNA-Directed DNA Polymerase