The textbook view of bacterial genomes shows a set of discrete genes, transcribed individually or as operons.
Transcription initiates at promoters upstream of these genes/operons, producing mostly protein-coding mRNAs
along with a smaller number of stable, functional RNAs (tRNAs, rRNAs, sRNAs). Transcription factors bind close
to promoters and regulate transcription from those promoters. Transcription terminates downstream of genes, in
3’ UTRs. This view has been the basis for decades of work on gene expression and gene regulation, with
enormous advances in our understanding of these processes. However, work from my group and others has
shown that bacterial genomes are far more complex. We will leverage my expertise in genetics, genomics, and
molecular biology, to continue productive lines of research on four overlapping topics that relate to the major
research focus of my group: the unexpected complexity of bacterial genomes. My lab has been very
productive on this topic, with 19 papers since 2016 directly relevant to the four themes described in this proposal.
Topic #1. Pervasive transcription. We and others have shown that most bacterial promoters are not in
intergenic regions, upstream of genes. Rather, they are located within genes, in sense or antisense orientations,
and are involved in “pervasive transcription”, whereby short, non-coding RNAs are transcribed before being
rapidly terminated by Rho and degraded. The majority of these RNAs are believed to be non-functional, and
suppression of pervasive transcription is required to maintain cell fitness.
Topic #2. Non-canonical transcription factor (TF) binding. We have mapped the direct and indirect regulatory
targets of hundreds of TFs across a wide range of bacterial species. Most TF binding sites are located within
genes, not intergenic regions. Moreover, most TF binding events are not associated with detectable regulation
of a nearby gene. Our data also show that in vivo binding profiles are often not well explained by a DNA sequence
motif, suggesting a role for other factors in determining the genomic sites of TF binding.
Topic #3. Widespread gene regulation by attenuation. We have shown that transcription of many Escherichia
coli genes is prematurely terminated by the conserved termination factor Rho, either in the 5’ UTR or ORF, a
process commonly referred to as “attenuation”. Attenuation has been described previously, but our data indicate
that it happens on a much larger scale than previously appreciated. We are interested in the mechanisms of
attenuation involving Rho termination, with a particular focus on the role of upstream ORFs (uORFs) that function
as cis-acting regulators, since we have identified large numbers of these ORFs in diverse bacterial species.
Topic #4. Processive antitermination. RNA polymerase can be protected from the action of the Rho
termination factor in a process known as “processive antitermination”. We will identify new regulatory targets of
known antiterminator proteins, we will determine the mechanisms of antitermination, and we will discover new