SII-facilitated transcript cleavage in RNA polymerase II complexes stalled early after initiation occurs in primarily dinucleotide increments.
RNA polymerase II ternary complex cleaves its nascent transcript in a 3'-->5' direction in the presence of elongation factor SII (Izban, M. G., and Luse, D. S. (1992) Genes & Dev. 6, 1342-1356; Reines, D. (1992) J. Biol. Chem. 267, 3795-3800). We have characterized the cleavage products generated during the truncation process with a variety of stalled RNA polymerase II ternary complexes containing uniformly labeled transcripts. These complexes, which remain elongation competent, had stopped transcription because one nucleoside triphosphate was missing from the reaction mixture. Using a novel assay system, we demonstrate that cleavage occurs in predominantly dinucleotide increments, liberating 5'-phosphodinucleotides (pNpNs). In one instance with a particular C20 complex, the first cleavage event was equally partitioned between either a di-or trinucleotide increment with all subsequent truncations occurring by the preferred dinucleotide step. Our data indicate that both the kinetics and the exact increment of SII-facilitated transcript cleavage are influenced by transcript sequence.
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
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Izban
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Luse
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013950:Thymus Gland / Q000378:metabolism; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D035165:Transcription Factors, General; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
D015226:Dinucleoside Phosphates; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014157:Transcription Factors; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D012319:RNA Polymerase II