NEW YORK (GenomeWeb News) – In a paper appearing online today in Science, a team of New Jersey researchers provided evidence that abortive transcription initiation occurs in living bacterial cells.
As in previous in vitro experiments, the researchers found that RNA polymerase can generate and release numerous short nucleotides that are not extended into full-length transcripts. But this phenomenon was not limited to in vitro experiments. Escherichia coli cells also produced both full-length and abortive transcripts, suggesting the abortive transcription initiation might contribute to the process of transcription.
"The finding that abortive transcripts are generated in vivo and accumulate to detectable level in vivo raises the possibility that abortive transcripts may play functional roles," senior author Bryce Nickels, a geneticist at Rutgers University, and his team wrote.
Results from in vitro experiments suggest that the RNA polymerase-promoter initial transcribing complexes synthesize and release tiny RNA transcripts that are about two to 15 nucleotides long. Such abortive transcription cycles can occur dozens or hundreds of times in vitro, competing with productive initiation cycles that generate longer RNA transcripts.
Circumstantial evidence suggests abortive transcription also occurs in living cells. But direct evidence for such transcription has remained elusive. To explore the possibility that it occurs in bacterial cells, Nickels and his team transformed E. coli with a plasmid carrying a copy of N25anti — a promoter shown to produce a relatively high ratio of abortive to productive transcripts in vitro.
They then did parallel transcription experiments in vivo and in vitro, detecting in vivo transcripts by hybridization to locked nucleic acid probes and in vitro transcripts (generated using radioactive nucleotides) by autoradiography.
Using this approach, the team detected both full-length and abortive transcripts in both the in vitro and in vivo experiments. As in previous in vitro studies, they also showed that abortive transcription in living cells occurred more often when interactions between the RNA polymerase enzyme and promoter DNA or the transcription initiation factor sigma were stable or when the elongation factor GreA was missing.
Based on their results, the team concluded that abortive transcription occurs in living cells, likely influencing everything from promoter strength to RNA polymerase function. They also noted that such transcription probably occurs in other bacterial cells and in archea and eukaryotes.
In the future, they hope to tease apart abortive transcription's functional role in transcription, if any. "Key priorities will be to define the full set of abortive transcripts produced in vivo (the 'abortome') and to define functional roles of abortive transcripts in vivo," Nickels and his co-authors concluded.