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RNA thermometers and their role in regulating Bacillus subtilis gene expression

RNA温度计及其在调节枯草芽孢杆菌基因表达中的作用

基本信息

批准号：
10311971
负责人：
elizabeth jolley
金额：
$ 5.28万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-01 至 2021-09-30
项目状态：
已结题

项目摘要

Project Summary/Abstract The long-term goal of this work is to understand the virulence and heat shock responses by which bacteria survive environmental changes. Bacteria respond rapidly to changes by regulating gene expression through an active and complex transcriptome. Bacterial genes are often regulated by temperature-induced changes in RNA structure, which are termed `RNA thermometers' (RNATs). RNATs often function by forming a structure that sequesters the Shine-Dalgarno (SD) sequence, thereby preventing ribosome binding. Discovery and verification of RNATs has historically been conducted by computational methods and in vitro studies. A gap in knowledge exists for understanding RNAT folding in vivo, and the dynamic ability of these structures to control gene expression. To fill this gap, global genome-wide in vivo structure probing will be applied using a method recently developed in this laboratory called Structure-seq2. This technique will be used to probe the transcriptome of Bacillus subtilis, a model Gram-positive bacterial species. While pioneering work led to the description of the first RNATs, using in vivo genome-wide techniques the proposed work will identify RNATs throughout the transcriptome, not just those near SD sequences. Using Structure-seq2 the following Aims will be accomlished: (1) Probe the structural landscape of the B. subtilis transcriptome at low and high temperatures to discover RNA thermometers. RNA structure is known to change in response to temperature. This phenomenon will be explored in the B. subtilis transcriptome. As B. subtilis is a soil bacterium, high and low temperatures will be used to replicate the extremes of natural growth in the environment. Testing will begin at low and high temperatures of 23°C and 44°C to identify RNA structures that change under these different conditions. This work is supported by preliminary data. (2) Classify diverse bacterial RNA thermometers genome-wide at a series of temperatures. No RNA thermometers have been experimentally characterized from B. subtilis, although many are predicted throughout the genome. By conducting Structure-seq2 at a series of growth temperatures, RNATs will be discovered that have different melting temperatures, function at narrow and wide temperature ranges, and have an instantaneous or gradual response. Results from Aim 1 will be used as a guide for regions likely to contain RNATs. (3) Characterize B. subtilis RNAT function. RNA structures that change in response to changing temperature will be tested as thermometers using bgaB reporter assays. Regions of interest will be cloned as bgaB translational fusions to report on gene expression, and transcriptional fusions will be used for in vitro transcription attenuation assays. This work will result in the first classification of RNATs by temperature midpoints and sensitivity, and will reveal novel regulatory strategies that will be identified in other bacterial species, including human pathogens.

项目总结/摘要这项工作的长期目标是了解细菌的毒力和热休克反应，在环境变化中生存。细菌通过调节基因表达对变化迅速作出反应，活跃而复杂的转录组。细菌基因通常受温度诱导的基因表达变化的调节。 RNA结构，被称为“RNA温度计”（RNAT）。RNAT通常通过形成一种结构来发挥作用其隔离Shine-Dalgarno（SD）序列，从而防止核糖体结合。发现和 RNAT的验证历来是通过计算方法和体外研究进行的。中的间隙存在理解体内RNAT折叠的知识，以及这些结构的动态能力，基因表达。为了填补这一空白，将使用一种方法应用全球全基因组体内结构探测，最近在这个实验室里开发了一种叫做Structure-seq 2的技术。这项技术将用于探测枯草芽孢杆菌的转录组，一种革兰氏阳性细菌的模型。虽然开创性的工作导致了第一个RNAT的描述，使用体内全基因组技术，拟议的工作将确定RNAT 在整个转录组中，不仅仅是那些靠近SD序列的。使用Structure-seq 2，以下目标将（1）探讨B的结构景观。枯草杆菌转录组在低和高发现RNA温度计。已知RNA结构会响应于温度这一现象将在B中探讨。枯草杆菌转录组作为B。subtilis是一种土壤细菌，高温和低温将被用来复制极端的自然增长，环境测试将在23°C和44°C的低温和高温下开始，以鉴定在这些不同的条件下变化。这项工作得到了初步数据的支持。(2)分类多样细菌RNA温度计在一系列温度下全基因组。没有RNA温度计从B实验表征。枯草芽孢杆菌，虽然许多是预测整个基因组。通过在一系列生长温度下进行Structure-seq 2，将发现RNAT具有不同的熔融温度，在窄和宽的温度范围内起作用，并且具有瞬时或逐渐的反应目标1的结果将被用作可能包含区域核试验的区域的指南。(3)描述B。 subtilis RNAT功能。响应于温度变化而变化的RNA结构将被测试为使用bgaB报告基因测定的温度计。将感兴趣的区域克隆为bgaB翻译融合物，关于基因表达的报告，并且转录融合体将用于体外转录衰减测定。这项工作将导致RNAT的第一次分类的温度中点和灵敏度，并将揭示新的调控策略，将在其他细菌物种，包括人类病原体中确定。