Elucidation of Nucleotide Addition Cycle of SARS-CoV-2 Polymerase

1. The Nucleotide Addition Cycle of the Sars - CoV 2 Polymerase
(Authors: Subhas Chandra Mona.. Pauline van,
(Authors: Dr. Subhas Chandra Bera, Dr. Mona Seifert, Dr. Robert N. Kirchdoerfer, Dr. Pauline van Nies, Dr. Yibulayin
Wubulikasimu, Dr. Salina Quack, Dr. Flavia S. Papini, Dr. Jamie J. Arnold, Dr. Bruno Canard, Dr. Craig E. Cameron, Dr.
Martin Depken, and Dr. David Dulin)
Presenter: Sandipan Saha
Prospective PhD Student
Masters in Chemistry, IIT Madras
Presentation to:
Dr. David Dulin
Assistant Professor
Vrije Universiteit Amsterdam
Date: 5th August, 2022

2. Contents
• Introduction: SARS-CoV-2 and its Salient Features
• Investigation of SARS-CoV-2 RNA Polymerase Kinetics by Magnetic Tweezers
- The Kinetics & Dynamics of Nucleotide Addition in SARS-CoV-2
- Dependence of Polymerase Activity on Cofactors & Salts
- Dependence of Polymerase Activity on NTP Concentration
- Effect of Force on Polymerase Activity
- Force Dependence in dsRNA elongation
• Key Outcomes & Conclusion
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3. Introduction: SARS-CoV-2 and its Salient Features
• Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is
responsible for the ongoing COVID-19 pandemic. It has infected 575 million
people globally with more than 6 million deaths to date.
• It has a 30 kb long single stranded RNA (ssRNA) which codes for many
structural and non-structural proteins (nsp). The nsp comprise the RNA-
dependent RNA polymerases (RdRp) essential for genome replication and
transcription.
• The RdRp consists of a nsp12 core, which gets associated with accessory
factors nsp7 and nsp8 to generate the active polymerase unit.
• The rates of replication and transcription of its viral genome is one of the
fastest (170 nt/s at 37ᵒC).
• Remdesivir being a nucleotide analogue forms one of the most important
drugs for the treatment of COVID-19 till date.
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4. Investigation of SARS-CoV-2 RNA
Polymerase Kinetics by Magnetic Tweezers
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5. The Kinetics and Dynamics of Nucleotide Addition in SARS-CoV-2
• A suitable RNA hairpin biotinylated to a magnetic
bead and fused to glass by digoxigenin was
constructed.
• Following elongation, the polymerase detaches itself
from the dsRNA and resets conformation before
adding to another ssRNA.
• The different steps involved in the pathway consist
of:
A Nucleotide Addition Burst (NAB), <0.5 sec
Pauses representing slow addition of nucleotides,
wherein
- Pause 1: Slow Nucleotide Addition (SNA), <1 sec
- Pause 2: Very Slow Nucleotide Addition (VSNA),
1-5 sec
A long lived pause, characterized by a t-3/2 power law.
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6. Dependence of Polymerase Activity on Cofactors and Salts
• The SARS CoV-2 RNA Polymerase is a Processive RNA Polymerase.
The rate of polymerase activity is independent of the pre-assembly of the polymerase cofactors, as both the pre and non pre-assembled
polymerases individually exhibit comparable activity.
Adjusting cofactor concentrations produce little change in the rate of polymerase activity.
The length of the product is also invariant in the presence of salts like KGlu and NaCl.
Hence, the variations in the rate are not due to any possible viral protein disassembly or exchange with the polymerase unit; it is the
exclusive characteristic of the polymerase itself!
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7. Dependence of Polymerase Activity on NTP concentration
• A decrease in the NTP concentration leaves the product length unchanged, but increases the pause probability. The rate of nucleotide
addition is hence decreased.
• The pause exit rates are dependent on the NTP concentration according to
𝑘𝑎
𝑁𝑇𝑃 =
𝑘𝑚𝑎𝑥
𝑎
[𝑁𝑇𝑃]
𝐾𝑀
𝑎
+ [𝑁𝑇𝑃]
, 𝑎 = 1,2
𝐾𝑀
𝑎
is the Michaelis Menten constant for the ath pause step.
• Pause 1 and 2 predominate at lower NTP concentrations. Pause 1 probability increases almost 6 fold, while pause 2 increases 2.3 fold
upon reduction in [NTP] from 500μM to 20μM. The long lived pause also follows a similar pattern.
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8. Effect of Force on Polymerase Activity
• The nucleotide addition rate to ssRNA is affected by the force, which obeys the following Arrhenius equation:
𝑘𝑠𝑠 𝐹 = 𝑘𝑠𝑠
0
𝑋 exp(−
𝐹𝛿𝑥
𝑘𝐵𝑇
) + 𝐴
The rate of addition decreases exponentially from 20pN to 30pN, following which it becomes stable.
• The pause exit rates are dependent on the force according to
𝑘𝑎 𝐹 =
1
1
𝑘𝑎( 𝑁𝑇𝑃 ∗)
+
1
𝑘𝑠𝑠,𝑎(𝐹)
, 𝑎 = 1,2
where 𝑘𝑠𝑠,𝑎(𝐹) is the Arrhenius relation followed for the ath pause step.
• With an increase in the force, the probability of the pauses increase as implied by the nucleotide addition rate. Further, the experimental observations conclude
that the pause exit step is fast and irreversible, and controlled by two sub-steps, one being NTP dependent and the other being a faster force-dependent step.
• The long lived pause probability is only increased in the larger force regime (>45 pN).
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9. Force dependence in dsRNA elongation
• If the applied force < hairpin opening force, polymerase elongation is found to occur with hairpin opening.
• Decrease in slope of the position-time trace implies polymerase backtracking, predominantly at high forces.
• The pause 1 and pause 2 probabilities dominate at lower forces with exit rates peaking at higher forces, contrary to the ssRNA
elongation. This justifies that the polymerase activity alone is inefficient, and assisted by cofactors like nsp13 helicase.
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10. Key Outcomes & Conclusion
• The polymerase can adopt either the NAB, SNA (Pause 1) or VSNA (Pause 2) at a particular instant, whose transition occurs prior to
nucleotide translocation and NTP binding.
• Following the NTP binding, two irreversible rate limiting steps exist:
- the first one is the addition of the nucleotide to the existing chain by a phosphodiester bond formation (kcat).
- the second step is the faster, force dependent step involving a polymerase conformational reset (kss).
A backtracking of the polymerase also contributes to a decrease in the nucleotide addition, leading to a long pause owing to polymerase
inactivity.
• The replication and transcription processes form the core of the coronavirus life-cycle, and hence serves as an important target for the
characterization of drugs.
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