# Untangling the mechanisms of initiation and discontinuous RNA synthesis by COVID-19 RdRp

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2023 · $227,841

## Abstract

SUMMARY/ ABSTRACT
 The broad goal of this collaborative project is to understand the molecular mechanisms of initiation and
discontinuous RNA synthesis by the coronavirus SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and
establish determinants that regulate these processes. To this end, the laboratories of Dr. Sergei Borukhov at
Rowan University SOM and of Dr. Shimon Weiss at UCLA will: (i) reconstitute an in vitro minimal SARS-CoV2
transcription/replication system capable of primer-independent (de novo) and protein-primed initiation of (-) and
(+) RNA strand synthesis using viral RdRp, 5’-UTR and 3’-UTR RNA elements, and nucleotidylated (uridylated
and guanylated) viral non-structural proteins nsp8 and nsp9; (ii) characterize the process of viral
transcription/replication initiation biochemically and determine the contribution of viral protein factors (nsp9,
nsp10, nsp13, nsp14, and N protein), using gel-based ensemble assays as well as recently developed single-
molecule RdRp activity assays; (iii) reconstitute SARS-CoV-2 discontinuous transcription system in which
RdRp pauses during RNA synthesis at transcription-regulating sequences (TRSs) and switches RNA
templates to produce nested sets of sub-genomic mRNAs; (iv) develop both single-molecule and ensemble
level assays such as single-molecule FRET based template switching assay, RNA-protein cross-linking,
exonuclease-footprinting and localized Fe2+ -induced hydroxyl-radical mapping to characterize the
discontinuous transcription intermediates (e.g., paused, paused-backtracked, and template-switched RdRp
complexes); (v) identify viral and human host cell protein factors that are required for template-switching.

## Key facts

- **NIH application ID:** 10629420
- **Project number:** 5R21AI171702-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** SERGEI BORUKHOV
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $227,841
- **Award type:** 5
- **Project period:** 2022-06-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10629420

## Citation

> US National Institutes of Health, RePORTER application 10629420, Untangling the mechanisms of initiation and discontinuous RNA synthesis by COVID-19 RdRp (5R21AI171702-02). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10629420. Licensed CC0.

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