Transcription elongation in the human c-myc gene is governed by overall transcription initiation levels in Xenopus oocytes.

Abstract

Both transcription initiation and transcription elongation contribute to the regulation of steady-state c-myc RNA levels. We have used the Xenopus oocyte transcription assay to study premature transcription termination which occurs in the first exon and intron of the human c-myc gene. Previous studies showed that after injection into Xenopus oocytes transcription from the c-myc PI promoter resulted in read-through transcripts whereas transcription from the stronger P2 promoter resulted in a combination of prematurely terminated and read-through transcripts. We now demonstrate that this promoter-specific processivity results from the overall amount of RNA polymerase TI transcription occurring from either promoter. Parameters that reduce the amount of transcription from P1 or P2, such as decreased concentration of template injected or decreased incubation time, result in a reduction in the ratio of terminated to read-through c-myc transcripts. Conversely, when transcription levels are increased by higher concentrations of injected template, increased incubation time, or coinjection with competing template, the ratio of terminated to read-through transcripts increases. We hypothesize that an RNA polymerase IT processivity function is depleted above a threshold level of transcription initiation, resulting in high levels of premature transcription termination. These findings account for the promoter-specific effects on transcription elongation previously seen in this assay system and suggest a mechanism whereby limiting transcription elongation factors may contribute to transcription regulation in other eukaryotic cells.

Distinct properties of c-myc transcriptional elongation are revealed in Xenopus oocytes and mammalian cells and by template titration, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), and promoter mutagenesis.

“…At 1 ng of injected template, the low signal obtained with the E intron probe most likely reflects premature termination of transcription at the Ti and T2 sites near the end of the first exon. This suggests that at low injection levels, transcriptional arrest within the first c-myc exon is less efficient, as described recently (52). To investigate this possibility further, we determined the efficiency of 3' end formation at various template injection concentrations.…”

The Poly(A) Signal, without the Assistance of Any Downstream Element, Directs RNA Polymerase II to Pause in Vivo and Then to Release Stochastically from the Template

“…However, our plasmids carry the SV40 origin of replication (8), which is active in COS cells (52). Perhaps the high proportion of abortives reflects a template copy number effect as previously reported for Xenopus oocyte injection experiments (13,14).…”

“…Relief of pausing and the establishment of processivity is brought about by the action of P-TEFb (positive transcription elongation factor b), which phosphorylates both DSIF and the C-terminal domain (11,12). However, the efficiency with which processivity is established varies with the promoter and the physiological context (3,13,14). The promoterproximal termination of those polymerases on which high processivity has not been conferred has been called abortive elongation (15).…”

Mitotic Repression of RNA Polymerase II Transcription Is Accompanied by Release of Transcription Elongation Complexes

“…The GAPDH (glyceraldehyde-3-phosphate dehydrogenase gene) probe was a 979-bp fragment from ϩ44 to ϩ1023 of a human GAPDH cDNA (2). The histone H2b probe was a 302-bp BstEII fragment from ϩ110 to ϩ412 of the chicken histone H2b gene (64). The Pol 5Ј probe was a 471-bp BsaHI-SphI fragment from Ϫ26 to ϩ445 of the mouse RNA polymerase II large subunit gene, encompassing most of exon 1 (1).…”