RESUMEN
Group II introns are usually removed from precursor RNAs as lariats comprised of a circular component and a short 3' tail. We find that group II introns can also be excised as complete circles. Circle formation requires release of the 3' exon of a splicing substrate, apparently by a trans splicing mechanism. After 3' exon release, the terminal uridine of the intron attacks the 5' splice site, releasing the 5' exon and joining the first and last intron residues by a 2'-5' phosphodiester bond. RNA isolated from yeast mitochondria also contains circles, indicating that at least one group II intron (aI2) forms circles in vivo. Furthermore, analysis of RNA and DNA from certain mutant yeast strains shows that circular DNA introns exist and are produced by reverse transcription of RNA, rather than by ectopic homing.
Asunto(s)
Intrones/genética , Precursores del ARN/metabolismo , Empalme del ARN , ADN Ligasas/metabolismo , ADN Mitocondrial/química , Células HeLa , Humanos , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , ARN/química , Precursores del ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Levaduras/genéticaRESUMEN
The spectrum of tantalum emitted by a subpicosecond laser-created plasma, was recorded in the regions of the 3d-5f, 3d-4f, and 3d-4p transitions. The main difference with a nanosecond laser-created plasma spectrum is a broad understructure appearing under the 3d-5f transitions. An interpretation of this feature as a density effect is proposed. The supertransition array model is used for interpreting the spectrum, assuming local thermodynamic equilibrium (LTE) at some effective temperature. An interpretation of the 3d-4f spectrum using the more detailed unresolved transition array formalism, which does not assume LTE, is also proposed. Fitted contributions of the different ionic species differ slightly from the LTE-predicted values.
RESUMEN
Group II introns self-splice via a two-step mechanism: cleavage at the 5' splice site followed by exon ligation at the 3' splice site. The second step has been difficult to study in vitro because it is generally faster than the first. Herein we describe development and partial kinetic characterization of a novel assay for studying the second step in isolation. In this system, a truncated linear intron (nucleotides 1-881) mediates exon ligation between two oligonucleotide substrates: a 19 nt 5' exon and a 3' substrate consisting of the last 6 nucleotides of the intron plus a 6 nucleotide 3' exon. We found that neither the exact structure of domain 6 nor the identity of nucleotides flanking the 3' splice site is critical for accurate 3' splice site choice by the ai5gamma group II intron. The multiple turnover k(cat) (0.14 min(-)(1)) is slower than the single turnover k(obs) (0.6-0.7 min(-)(1)), consistent with rate-limiting product release under steady-state conditions. Decreased single turnover rates at lower pHs were more consistent with loss of catalytic activity than with rate-limiting chemistry. Binding of the 3' substrate (K(m) = 2.6 microM) could be improved by changing a long-range A:U base pair involving the last intronic nucleotide (the gamma-gamma' interaction) to G:C (K(m(3)(')(substrate)) = 1 microM).