The global regulatory architecture of transcription during the Caulobacter cell cycle.

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5' RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle.

Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation.

During cell division, multiple processes are highly coordinated to faithfully generate genetically equivalent daughter cells. In bacteria, the mechanisms that underlie the coordination of chromosome replication and segregation are poorly understood. Here, we report that the conserved replication initiator, DnaA, can mediate chromosome segregation independent of replication initiation. It does so by binding directly to the parS centromere region of the chromosome, and mutations that alter this interaction result in cells that display aberrant centromere translocation and cell division. We propose that DnaA serves to coordinate bacterial DNA replication with the onset of chromosome segregation.

The essential genome of a bacterium.

Caulobacter crescentus is a model organism for the integrated circuitry that runs a bacterial cell cycle. Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of a bacterial cell. Using hyper-saturated transposon mutagenesis coupled with high-throughput sequencing, we determined the essential Caulobacter genome at 8 bp resolution, including 1012 essential genome features: 480 ORFs, 402 regulatory sequences and 130 non-coding elements, including 90 intergenic segments of unknown function. The essential transcriptional circuitry for growth on rich media includes 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor. We identified all essential promoter elements for the cell cycle-regulated genes. The essential elements are preferentially positioned near the origin and terminus of the chromosome. The high-resolution strategy used here is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species.

Comparative TFIIS-mediated transcript cleavage by mammalian RNA polymerase II arrested at a lesion in different transcription systems.

Upon prolonged arrest at a cyclobutane pyrimidine dimer (CPD), RNAPII can reverse-translocate, misaligning the 3'-end of the RNA from its active site. Transcription factor SII (TFIIS) is required for cleavage of the disengaged 3'-end and restoration of its correct positioning. We have previously shown in vitro that when RNAPII is arrested at a CPD, TFIIS-induced cleavage results in shortened transcripts. Here, we hypothesized that the pattern of transcript cleavage does not depend solely upon TFIIS itself, but also on some other general transcription factors (GTFs) and/or their effects on RNAPII. To test this hypothesis we compared three in vitro transcription systems which differ with respect to the mode of initiation and the requirement for GTFs. The first consisted of RNAPII and GTFs from rat liver, and required a eukaryotic promoter for initiation. The other two supported transcription in the absence of any GTFs or promoter sequences. In each case, a CPD on the transcribed strand was a complete block for RNAPII translocation. However, the effect of TFIIS on transcript cleavage varied. In the promoter-initiated system, distinct transcripts up to about 20 nucleotides shorter than the uncleaved original one were produced. In the other two systems, the transcripts were degraded nearly completely. Introduction of GTFs partially interfered with cleavage, but failed to reproduce the pattern of transcript lengths observed with the promoter-initiated system. Our results suggest that the extent of TFIIS-mediated transcript cleavage is a well-orchestrated process, depending upon other factors (or their effects on RNAPII), in addition to TFIIS itself.

Transcription arrest at a lesion in the transcribed DNA strand in vitro is not affected by a nearby lesion in the opposite strand.

Cis-syn cyclobutane pyrimidine dimers (CPDs) are the most frequently formed lesions in UV-irradiated DNA. CPDs are repaired by the nucleotide excision repair pathway. Additionally, they are subject to transcription-coupled DNA repair. In the general model for transcription-coupled DNA repair, an RNA polymerase arrested at a lesion on the transcribed DNA strand facilitates repair by recruiting the repair machinery to the site of the lesion. Consistent with this model, transcription experiments in vitro have shown that CPDs in the transcribed DNA strand interfere with the translocation of prokaryotic and eukaryotic RNA polymerases. Here, we study the behavior of RNA polymerase when transcribing a template that contains two closely spaced lesions, one on each DNA strand. Similar DNA templates containing no CPD, or a single CPD on either the transcribed or the nontranscribed strand were used as controls. Using an in vitro transcription system with purified T7 RNA polymerase (T7 RNAP) or rat liver RNAP II, we characterized transcript length and efficiency of transcription in vitro. We also tested the sensitivity of the arrested RNAP II-DNA-RNA ternary complex, at a CPD in the transcribed strand, to transcription factor TFIIS. The presence of a nearby CPD in the nontranscribed strand did not affect the behavior of either RNA polymerase nor did it affect the reverse translocation ability of the RNAP II-arrested complex. Our results additionally indicate that the sequence context of a CPD affects the efficiency of T7 RNAP arrest more significantly than that of RNAP II.