Authors:

Naomi Nunez

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About Paper:

Transposon associated protein B (TnpB) is a transposon-associated, RNA guided endonuclease regarded as an evolutionary ancestor of the type V CRISPR-Cas family of effectors. These enzymes, like Cas12s, are part of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) systems, a natural adaptive immune system in bacteria. Cas12 proteins use a guide RNA to recognize a matching DNA sequence next to a Protospacer Adjacent Motif (PAM) and cleave it causing a double-stranded DNA break. Similarly, TnpB proteins recognize Transposon Adjacent Motif (TAM) sequence via their REC lobe and cleave DNA using their NUC lobe. Recent studies have identified a class of protein that evolved from TnpB, known as TnpB-like nuclease-dead repressors (TldRs). Together with their associated ?RNA, these proteins form an ancient RNA-guided system that regulates gene expression, not destroy DNA. The guide sequence of ?RNA pairs with the promoter regions probably inhibits transcription. We've obtained a cryo-EM structure of TldR-?RNA-target DNA ternary complex. With this structure, we hypothesized that by binding to the promoter region, TldR could block the binding of RNA polymerase competitively, interrupting transcription. This project aims to understand the mechanism of TldR-mediated inhibition of transcription using cryo-EM and T7 transcription assay. Additionally, we identified the interaction between TAM sequence and TldR-?RNA complex besed on the structure. Therefore, site-directed mutagenesis will be employed to understand the binding mechanism of TldRs-?RNA and target DNA. Keywords: Structural Biology; TldR; Protein; Transcription Inhibition

Source:

Purdue University / 2025

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Co-authors:

Naomi Nunez