M.Prasad Naidu discusses several types of polymerases including DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, RNA-dependent DNA polymerases, and RNA-dependent RNA polymerases. Reich et al. and Baltimore and Franklin demonstrated RNA-primed RNA synthesis using the enzyme RNA-dependent RNA polymerase, also called RNA replicase. Spiegelman et al. isolated RNA replicase from bacteriophage Qβ which requires an RNA template, magnesium ions, and ribonucleoside triphosphates to function.

1. M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

2.  DNA-dependent DNA polymerases
Eg. Prokaryotic and Eukaryotic DNA polymerases
 DNA-dependent RNA polymerases
Eg. RNA polymerases and DNA primase
 RNA-dependent DNA polymerases
Eg. Reverse transcriptase
 RNA-dependent RNA polymerases
Eg. RNA replicase

3. Reich et al. showed the RNA synthesis even in the
presence of actinomycin-D (that inhibits the DNA-
primed RNA synthesis)
Baltimore and Franklin showed the viral production
with specific cytoplasmic fractions
Thus, RNA-primed RNA synthesis was
demonstrated
This enzyme has been named RNA-dependent RNA
polymerase or RNA Replicase

4. Structure of Qβ replicase:
◦ Four subunits
 3 provided by E. coli host (Tu, Ts, and S1)
 1 in viral RNA genome (MW 65,000)
•Function:
– Synthesis in 5’ to 3’ direction
– Lacks proofreading function
– Requires RNA template
– Specific for RNA of the virus
All RNA viruses (except retroviruses) encode
a protein since the hosts do not possess it

5.  RNA Replicase was isolated by Spiegelman et al.
from Qβ bacteriophage
 This requires RNA template, Mg2+, rNTPs
 Qβ replicase is tetramer consisting of one Rep
molecule and three host proteins
 Replicase copies viral (+) strand to generate a (-)
strand

6.  During synthesis, (-) strand is in contact with
the (+) strand only at the polymerization site
 Initiation of several (-) strand occurs before the
first (-) is complete, and the replicative form is
branched
 The (-) strands are released and immediately
used by the replicase to form (+) strands only

9.  Temin and Baltimore (1970) independently
demonstrated the presence of RT in several
avian and mouse retroviruses
 RT is a RNA-dependent DNA polymerase
 It is used in rDNA technology to transcribe
mRNA into dsDNA

10.  RT has 3 enzymatic activities
It copies an RNA molecule to yield dsDNA-RNA,
using a primer and joining dNTPs in a 3’-5’
linkage
It copies a primed ssDNA to form dsDNA
It degrades RNA in a DNA-RNA hybrid; this is
called RNase H activity (3’ to 5’ exoribonuclease
activity)

13. (1) Viral genome
and reverse
transcriptase
enter cell.
Retrovirus - HIV

14. (1) Viral genome
and reverse
transcriptase
enter cell.
(2) DNA copy synthesized by
reverse transcriptase.
Site of action of AZT
and other reverse
Transcriptase
inhibitors
RNA
DNA

15. (1) Viral genome
and reverse
transcriptase
enter cell.
(2) DNA copy synthesized by
reverse transcriptase.
Site of action of AZT
and other reverse
Transcriptase
inhibitors
RNA
DNA
(3) RNA degraded;
second DNA strand
synthesized.
DNA
DNA

16. (1) Viral genome
and reverse
transcriptase
enter cell.
(2) DNA copy synthesized by
reverse transcriptase.
(3) RNA degraded;
second DNA strand
synthesized.
Host cell
nucleus
Host cell
genome
Site of action of AZT
and other reverse
Transcriptase
inhibitors
RNA
DNA
DNA
DNA
(4) DNA circularizes (unintegrated
provirus) or integrase functions
to incorporate DNA into host cell
genome (integrated provirus).
Site of action
of antiretroviral drugs
under development

17. (1) Viral genome
and reverse
transcriptase
enter cell.
(2) DNA copy synthesized by
reverse transcriptase.
(3) RNA degraded;
second DNA strand
synthesized.
(5) With host cell
activation, viral
DNA is transcribed,
yielding messenger RNAs
and viral genome RNA.
Host cell
nucleus
Host cell
genome
Site of action of AZT
and other reverse
Transcriptase
inhibitors
RNA
DNA
DNA
DNA
(4) DNA circularizes (unintegrated
provirus) or integrase functions
to incorporate DNA into host cell
genome (integrated provirus).
Site of action
of antiretroviral drugs
under development

18. (1) Viral genome
and reverse
transcriptase
enter cell.
(2) DNA copy synthesized by
reverse transcriptase.
(3) RNA degraded;
second DNA strand
synthesized.
(5) With host cell
activation, viral
DNA is transcribed,
yielding messenger RNAs
and viral genome RNA.
(6) Viral RNAs are
translated, yielding
viral enzymes
(including protease)
and structural
proteins.
Host cell
nucleus
Host cell
genome
Site of action of AZT
and other reverse
Transcriptase
inhibitors
Site of action
of protease
inhibitors
RNA
DNA
DNA
DNA
(4) DNA circularizes (unintegrated
provirus) or integrase functions
to incorporate DNA into host cell
genome (integrated provirus).
Site of action
of antiretroviral drugs
under development

19. (1) Viral genome
and reverse
transcriptase
enter cell.
(2) DNA copy synthesized by
reverse transcriptase.
(3) RNA degraded;
second DNA strand
synthesized.
(5) With host cell
activation, viral
DNA is transcribed,
yielding messenger RNAs
and viral genome RNA.
(6) Viral RNAs are
translated, yielding
viral enzymes
(including protease)
and structural
proteins.
Host cell
nucleus
Host cell
genome
Site of action of AZT
and other reverse
Transcriptase
inhibitors
Site of action
of protease
inhibitors
RNA
DNA
DNA
DNA
(7) Viral membrane proteins are
transported to host cell membrane.
(4) DNA circularizes (unintegrated
provirus) or integrase functions
to incorporate DNA into host cell
genome (integrated provirus).
Site of action
of antiretroviral drugs
under development

20. (1) Viral genome
and reverse
transcriptase
enter cell.
(2) DNA copy synthesized by
reverse transcriptase.
(3) RNA degraded;
second DNA strand
synthesized.
(8) Final viral assembly
and budding
take place.
(5) With host cell
activation, viral
DNA is transcribed,
yielding messenger RNAs
and viral genome RNA.
(6) Viral RNAs are
translated, yielding
viral enzymes
(including protease)
and structural
proteins.
Host cell
nucleus
Host cell
genome
Site of action of AZT
and other reverse
Transcriptase
inhibitors
Site of action
of protease
inhibitors
RNA
DNA
DNA
DNA
(7) Viral membrane proteins are
transported to host cell membrane.
(4) DNA circularizes (unintegrated
provirus) or integrase functions
to incorporate DNA into host cell
genome (integrated provirus).
Site of action
of antiretroviral drugs
under development