Unexpected complexity in bacterial genomes

细菌基因组的意外复杂性

• 批准号: 10559673
• 负责人: Joseph Thomas Wade
• 金额: $ 42.72万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10559673
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Bacterial Genome; Binding; Binding Sites; Cells; Code; Complex; DNA Sequence; DNA-Directed RNA Polymerase; Data; Escherichia coli; Event; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genomics; Individual; Intercistronic Region; Maps; Messenger RNA; Molecular Biology; Open Reading Frames; Operon; Paper; Process; Productivity; Proteins; RNA; Regulation; Research; Rho Factor; Ribosomal RNA; Role; Site; Textbooks; Transcription Initiation; Transfer RNA; Untranslated RNA; Work; antitermination; attenuation; fitness; in vivo; interest; premature; promoter; rho; termination factor; transcription factor; transcription termination

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.

总结 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，产生主要是蛋白质编码的mRNA 沿着少量稳定的功能性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。这一观点是几十年来基因表达和基因调控研究的基础， 我们对这些过程的理解有了巨大的进步。然而，我的团队和其他人的工作 细菌基因组要复杂得多。我们将利用我在遗传学，基因组学， 分子生物学，继续生产线的研究四个重叠的主题，涉及的主要 我的团队的研究重点是：细菌基因组出乎意料的复杂性。我的实验室 自2016年以来，有19篇论文与本提案中描述的四个主题直接相关。 主题#1。普遍的转录。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，基因的上游。相反，它们以有义或反义方向位于基因内， 并参与“普遍转录”，即短的非编码RNA在被转录之前被转录。 迅速被Rho终止并降解。这些RNA中的大多数被认为是非功能性的， 需要抑制普遍的转录以维持细胞适应性。 主题#2非典型转录因子（TF）结合。我们已经绘制了直接和间接的监管 在广泛的细菌物种中的数百个TF的靶标。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，在体内结合概况往往不能很好地解释了DNA序列 基序，表明其他因素在确定TF结合的基因组位点的作用。 主题#3通过衰减进行广泛的基因调节。我们已经证明，许多大肠杆菌的转录 大肠杆菌基因被保守的终止因子Rho提前终止，无论是在5' UTR还是ORF中， 这一过程通常被称为“衰减”。前面已经描述了衰减，但我们的数据表明， 它发生的规模比以前想象的要大得多。我们感兴趣的是 涉及Rho终止的衰减，特别关注上游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了大量的这些ORF。 主题#4进行性抗终止。RNA聚合酶可以被保护免受Rho的作用， 终止因子的过程称为“进行性抗终止”。我们将确定新的监管目标， 已知的抗终止蛋白，我们将确定抗终止的机制，我们将发现新的 抗终止子蛋白。