Fidelity of Transcription

转录保真度

• 批准号: 7965911
• 负责人: Jeffrey Strathern
• 金额: $ 65.33万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965911
• 关键词: Affect; Back; Bacteria; Biological; Biological Assay; Catalytic Domain; Cell Aging; Cells; DNA; DNA-Directed RNA Polymerase; Defect; Frequencies; Genes; Genetic Transcription; HIV; HIV-1; HIV-1 Reverse Transcriptase; Human; Hybrids; Lead; Malignant Neoplasms; Methods; Molecular and Cellular Biology; Monitor; Mutation; Organism; Polymerase; Process; Proteins; Retroelements; Retrotransposition; Retrotransposon; Retroviridae; Reverse Transcription; Role; System; Translations; Variant; Yeasts; inhibitor/antagonist; mutant

While the central role of transcription in cell and molecular biology has been recognized for over 50 years, there has been no successful approach to the study of what controls the fidelity of the process or the consequences of infidelity in transcription. We developed methods to monitor the fidelity of transcription and the functions that contribute to the accuracy of that process. One such method involves monitoring the fidelity of retrotransposition. We isolated mutations in a subunit of RNA polymerase that reduce the fidelity of retrotransposition and demonstrated that they directly affect the accuracy of transcription. These are the first eukaryotic mutations known to reduce the fidelity of transcription. We also developed a screen for RNA polymerase mutants that increase the frequency of slippage during transcription. This class of transcription error has been shown to occur in bacteria and humans, but the features that avoid such errors have not been determined. With that screen we isolated the first mutations that increase errors of this type. We are investigating the biological consequences of increased transcription error rates. We have demonstrated that the combination of an error prone RNA polymerase with a defect in editing transcription errors is a lethal combination. We used a related assay that monitors the accuracy of retrotransposition to identify mutations that alter the fidelity of the HIV-1 reverse transcriptase. Our system uses a hybrid retrotransposon that uses the HIV-1 RT to propagate a yeast retroelement. We developed an assay that detects errors made during retrotransposition and applied it as a screen of variants of the HIV-1 RT gene. This approach identified a new class of mutations that reduce the accuracy of HIV-1 RT. These mutations, when put back into the HIV virus reduce its ability to replicate. They may define a new target for retroviral inhibitors.

虽然转录在细胞和分子生物学中的核心作用已被认可了50多年，但没有成功地研究控制过程的忠诚度或转录中不忠的后果的方法。我们开发了监测转录保真度以及有助于该过程准确性的功能的方法。一种这样的方法涉及监测逆转录置位的保真度。我们在RNA聚合酶的亚基中分离了突变，该突变降低了转载置的忠诚度，并证明它们直接影响转录的准确性。这些是已知降低转录保真度的第一个真核突变。我们还开发了一个RNA聚合酶突变体的屏幕，该筛选增加了转录过程中滑动的频率。这类转录误差已显示出在细菌和人类中发生，但是避免这种误差的特征尚未确定。在该屏幕上，我们隔离了增加此类误差的第一个突变。我们正在研究提高转录错误率的生物学后果。 我们已经证明，在编辑转录误差中，容易发生RNA聚合酶与缺陷的组合是致命的组合。我们使用了一种相关的测定，该测定法可以监视反转词的准确性，以识别改变HIV-1逆转录酶的保真度的突变。我们的系统使用使用HIV-1 RT来传播酵母菌元素的混合逆转录座子。我们开发了一种检测逆转录置次置换误差的测定，并将其应用于HIV-1 RT基因的变体屏幕。这种方法确定了一种新的突变，可降低HIV-1 RT的准确性。这些突变恢复到HIV病毒时会降低其复制能力。它们可能定义逆转录病毒抑制剂的新靶标。