Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

Nucleic Acids Research
Sumit HandaPartho Ghosh

Abstract

Diversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelity in vitro for the prototypical Bordetella bacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules, as observed in vivo. We identified RNA-cDNA molecules to be branched and most plausibly linked through 2'-5' phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream of TR, which specifically bound Avd and...Continue Reading

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Citations

May 3, 2019·Nucleic Acids Research·Fatemeh Sharifi, Yuzhen Ye
Oct 11, 2019·Nucleic Acids Research·Anna J SimonIlya J Finkelstein
Dec 29, 2020·Nucleic Acids Research·Sumit HandaPartho Ghosh
May 14, 2021·FEMS Microbiology Reviews·Alejandro González-DelgadoNicolás Toro
May 26, 2021·Nature Communications·Simon RouxEmiley A Eloe-Fadrosh

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Methods Mentioned

BETA
electrophoresis
gel filtration
in vitro runoff
PCR

Software Mentioned

ImageQuant TL
align
bowtie2
Python
samtools
cutadapt
bRT
Avd

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