18 research outputs found
The Generation of Promoter-Mediated Transcriptional Noise in Bacteria
Noise in the expression of a gene produces fluctuations in the concentration
of the gene product. These fluctuations can interfere with optimal function or
can be exploited to generate beneficial diversity between cells; gene
expression noise is therefore expected to be subject to evolutionary pressure.
Shifts between modes of high and low rates of transcription initiation at a
promoter appear to contribute to this noise both in eukaryotes and prokaryotes.
However, models invoked for eukaryotic promoter noise such as stable activation
scaffolds or persistent nucleosome alterations seem unlikely to apply to
prokaryotic promoters. We consider the relative importance of the steps
required for transcription initiation. The 3-step transcription initiation
model of McClure is extended into a mathematical model that can be used to
predict consequences of additional promoter properties. We show in principle
that the transcriptional bursting observed at an E. coli promoter by Golding et
al. (2005) can be explained by stimulation of initiation by the negative
supercoiling behind a transcribing RNA polymerase (RNAP) or by the formation of
moribund or dead-end RNAP-promoter complexes. Both mechanisms are tunable by
the alteration of promoter kinetics and therefore allow the optimization of
promoter mediated noise.Comment: 4 figures, 1 table. Supplemental materials are also include
Negative Supercoiling Creates Single-Stranded Patches of DNA That Are Substrates for AID–Mediated Mutagenesis
Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID