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.

项目摘要/摘要这项工作的长期目标是了解细菌的病毒和热冲击反应生存环境变化。细菌通过调节基因表达的变化迅速反应主动且复杂的转录组。细菌基因通常受温度引起的变化调节 RNA结构，称为“ RNA温度计”（RNAT）。 RNAT通常通过形成结构来发挥作用该测序者是Shine-Dalgarno（SD）序列，从而防止了核糖体结合。发现和历史上，RNAT的验证是通过计算方法和体外研究进行的。差距知识存在于理解体内rnat折叠的知识，以及这些结构控制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局基因组在体内结构探测最近在该实验室中开发的称为结构seq2。该技术将用于探测枯草芽孢杆菌的转录组，一种模型革兰氏阳性细菌物种。开创性的工作导致对第一个RNAT的描述，使用体内基因组全基因组技术拟议的工作将识别RNAT 通过转录组，不仅是近SD序列。使用结构seq2以下目标将完成：（1）探测低和高的枯草芽孢杆菌转录组的结构景观发现RNA温度计的温度。已知RNA结构会因响应而改变温度。该现象将在枯草芽孢杆菌转录组中进行探讨。由于枯草芽孢杆菌是土壤细菌，高温和低温将用于复制自然生长的极端环境。测试将从23°C和44°C的低温和高温开始，以鉴定RNA结构在这些不同的条件下变化。这项工作由初步数据支持。（2）对潜水员进行分类细菌RNA温度计在一系列温度下全基因组。没有RNA温度计从枯草芽孢杆菌中进行了实验表征，尽管在整个基因组中都预测了许多。经过将在一系列的生长温度下进行结构seq2，发现具有不同的RNAT 熔化温度，狭窄和宽温度范围的功能，并具有瞬时或等级回复。 AIM 1的结果将用作可能包含RNAT的地区的指南。（3）表征B。枯草型功能。响应温度变化的RNA结构将被测试为使用BGAB记者测定法。感兴趣的区域将被克隆为BGAB翻译融合到基因表达的报告和转录融合将用于体外转录衰减分析。这项工作将根据温度中点和灵敏度进行首次对RNAT的分类，并将揭示新的调节策略将在其他细菌物种（包括人类病原体）中鉴定出来。