M.

Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

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

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

Structure of Q 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
RNA Replicase was isolated by Spiegelman et al.
from Q bacteriophage

This requires RNA template, Mg
2+
, rNTPs

Q replicase is tetramer consisting of one Rep
molecule and three host proteins

Replicase copies viral (+) strand to generate a (-)
strand
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

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