Structural Requirements for Reverse Transcription by a Diversity-generating Retroelement.

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• Diversity-generating retroelements (DGRs) are mobile genetic elements found in microbes.
• DGRs generate protein sequence diversity through a unique reverse transcription process.
• The role of flanking RNA sequences in DGR function was previously unknown.

Purpose of the Study:

• To elucidate the function of upstream and downstream RNA segments in the Bordetella bacteriophage DGR.
• To determine the structural basis for RNA-mediated control of reverse transcription in DGRs.

Main Methods:

• Cryo-electron microscopy (Cryo-EM) to determine the structure of the DGR ribonucleoprotein (RNP) complex.
• Biochemical assays to analyze the roles of RNA elements in reverse transcription.

Main Results:

• Cryo-EM revealed an intricate ribonucleoprotein (RNP) complex formed by bRT, Avd, and the surrounding RNA.
• Specific RNA structural elements interact with bRT and Avd, positioning an RNA homoduplex for cis-priming.
• The surrounding RNA is crucial for initiating, promoting processivity, and terminating reverse transcription, while limiting mutagenesis.

Conclusions:

• The surrounding RNA acts as a scaffold and guide for bRT and Avd, controlling key steps in DGR-mediated cDNA synthesis.
• These findings explain the mechanism of cis-priming and mutagenesis control in this DGR.
• The identified mechanisms are likely conserved across diverse DGRs in various taxa.