Unexpected complexity in bacterial genomes

摘要 细菌基因组的教科书观点显示了一组离散的基因，单独转录或作为操纵子转录。 转录起始于这些基因/操纵子上游的启动子，主要产生蛋白质编码 mRNA 以及少量稳定的功能性 RNA（tRNA、rRNA、sRNA）。转录因子紧密结合 启动子并调节这些启动子的转录。转录终止于基因下游， 3’UTR。这一观点是数十年来基因表达和基因调控工作的基础， 我们对这些过程的理解取得了巨大进步。然而，我的团队和其他人的工作已经 表明细菌基因组要复杂得多。我们将利用我在遗传学、基因组学和 分子生物学，继续对与专业相关的四个重叠主题进行富有成效的研究 我小组的研究重点：细菌基因组的意外复杂性。我的实验室一直非常 自 2016 年以来，该主题已发表 19 篇论文，与本提案中描述的四个主题直接相关。 主题#1。普遍转录。我们和其他人已经证明，大多数细菌启动子并不存在于 基因间区域，基因上游。相反，它们位于基因内，有义或反义方向， 并参与“普遍转录”，即短的非编码 RNA 在被转录之前被转录 被 Rho 迅速终止并降解。大多数这些 RNA 被认为是无功能的，并且 维持细胞健康需要抑制普遍转录。 主题#2。非规范转录因子 (TF) 结合。我们绘制了直接和间接监管图 多种细菌物种中数百个转录因子的靶点。大多数 TF 结合位点位于 基因，而不是基因间区域。此外，大多数 TF 结合事件与可检测的调节无关 附近的基因。我们的数据还表明，体内结合谱通常不能用 DNA 序列很好地解释 基序，表明其他因素在确定 TF 结合的基因组位点中的作用。 主题#3。通过弱化进行广泛的基因调控。我们已经证明许多埃希氏菌的转录 大肠杆菌基因被保守的终止因子 Rho 过早终止，无论是在 5' UTR 或 ORF 中， 过程通常称为“衰减”。之前已经描述了衰减，但我们的数据表明 它发生的规模比以前想象的要大得多。我们对以下机制感兴趣 涉及 Rho 终止的衰减，特别关注发挥作用的上游 ORF (uORF) 的作用 作为顺式作用调节因子，因为我们已经在不同的细菌物种中鉴定出大量这些 ORF。 主题#4。进行性反终止。可以保护 RNA 聚合酶免受 Rho 的作用 称为“进行性反终止”的过程中的终止因素。我们将确定新的监管目标 已知的抗终止子蛋白，我们将确定抗终止子的机制，并且我们将发现新的 抗终止子蛋白。

SUMMARY 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 antiterminator proteins.