The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.
The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.
Laboratory of Molecular Genetics, National Institutes of Child Health and Human Development, Bethesda, Maryland 20892, USA.
A
A
Atwood
J H
JH
Lin
H L
HL
Levin
D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015683:Gene Products, gag / Q000235:genetics* / Q000378:metabolism; D019426:Integrases; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D018626:Retroelements; D012190:Retroviridae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism / Q000821:virology*
D017931:DNA Primers; D004271:DNA, Fungal; D015683:Gene Products, gag; D018626:Retroelements; D004254:DNA Nucleotidyltransferases; D019426:Integrases