REGULATION OF A 'MUTATION FREQUENCY SWITCH' IN E. COLI

该实验室的新结果表明， ms 216 A-37 tRNA修饰和突变在E.杆菌 这些发现 已经导致这样的假设，即ms 2 i6 A-37到i6 A-37的修饰不足 或A-37作为“生理开关”增加自发突变 频率. 这种修饰不足发生在miaA+的铁限制期间 细菌或在ms 2 i6 A-37缺陷的miaA（以前的trpX）突变体中 阵 因此，这种特定的tRNA修饰不足使细胞能够 增加它们的突变频率以应对某些环境 不暴露于诱变剂的应力。 长期目标是 一项为期五年的建议是批判性地评估后一种假设， 描述这种tRNA的遗传成分、靶点和生理学特征- 链接开关 为实现这一目标，将实现以下六个具体目标： 完成 (I)转座子插入突变将在基因中分离 参与ms 216 A-37生物合成，而不是miaA。 根据 上述假设，这些新的突变体应该知道增加自发性 突变频率 (II)DNA修复的步骤或 将识别直接或间接受到以下因素阻碍的复制 ms 216 A-37 tRNA修饰不足。 各种生理和遗传 将进行表征以满足这一目标。 (III)分析将 继续miaA的结构和调控，这可能会形成一个 复合操纵子与mutL。 重点将放在有前途的遗传 分离调节突变体的方法以及确定为什么2- 氨基嘌呤诱导染色体miaA转录。 (IV)突变 频率将在细菌中测量， ms 216 A-37 tRNA修饰不足，而不是铁限制。 根据 上述假设，生理应激条件，如半胱氨酸 剥夺，应该会增加自发突变的频率。 （五） 生化和遗传分析将进行的机制， 导致ms 216 A-37 tRNA修饰不足，以确定是否有活性 发生ms 216 A-37修饰的酶促去除。 (VI)的 目标I中鉴定的新基因的结构和调控将 确定，因为这些基因很可能是复杂的 操纵子，并且它们与复杂的mutL-miaA共享调节模式。 操纵子 总之，这项建议的结果将提供重要的， 关于适应、突变、 频率和进化，细胞代谢的遗传整合， 复杂操纵子的组成和控制，以及生物合成和 tRNA分子中修饰碱基的功能。

New results from this laboratory indicate four novel relationships between the ms2i6A-37 tRNA modification and mutagenesis in E. coli. These findings have lead to the hypothesis that undermodification of ms2i6A-37 to i6A-37 or A-37 acts as a "physiological switch" to increase spontaneous mutation frequency. Such undermodification occurs during iron limitation of miaA+ bacteria or in miaA (formerly trpX) mutants defective in ms2i6A-37 formation. Thus, this specific tRNA undermodification allows cells to increase their mutation frequencies in response to certain environmental stresses without exposure to mutagens. The long-term objective of this five-year proposal is to evaluate critically the latter hypothesis and to characterize the genetic components, targets, and physiology of this tRNA- linked switch. To meet this goal, the following six Specific aims will be completed. (I) Transposon insertion mutations will be isolated in genes involved in ms2i6A-37 biosynthesis, other than miaA. According to the above hypothesis, these new mutants should know increased spontaneous mutation frequencies. (II) The steps in DNA repair or the fidelity of replication will be identified that are directly or indirectly hindered by ms2i6A-37 tRNA undermodification. A variety of physiological and genetic characterizations will be performed to meet this aim. (III) Analysis will be continued of the structure and regulation of miaA, which likely forms a complex operon with mutL. Emphasis will be placed on promising genetic approaches to isolate regulatory mutants and on determining why 2- aminopurine induces chromosomal miaA transcription. (IV) Mutation frequencies will be measured in bacteria stressed by conditions that cause ms2i6A-37 tRNA undermodification, other than iron limitation. According to the above hypothesis, physiological stress conditions, such as cysteine deprivation, should increase spontaneous mutation frequency. (V) Biochemical and genetic analyses will be performed on the mechanism that leads to ms2i6A-37 tRNA undermodification to determine whether active enzymatic removal of the ms2i6A-37 modification takes place. (VI) The structure and regulation of new genes identified in Aim I will be determined, since it is likely that these genes are members of complex operons and that they share modes of regulation with the complex mutL-miaA operon. Taken together, results from this proposal will provide important, basic information about the relationship between adaptation, mutation frequency and evolution, the genetic integration of cellular metabolism, the composition and control of complex operons, and the biosynthesis and functions of modified bases in tRNA molecules.