Reverse

Transcription
Giriel Rose F. Voluntad
I-AI BS BIOLOGY
 REVERSE TRANSCRIPTION
¤ It is the process of making a double
stranded DNA molecule from a single
stranded RNA template.
¤ It is called reverse transcription as it
acts in the opposite or reverse direction to
transcription.
¤ It is a trancription of a single stranded
RNA to complementary DNA (cDNA) with
the help of the enzyme called Reverse
Transcriptase.
¤ It also involves the presence of a reverse
transcriptase enzyme, a primer, and a RNase
inhibitor (RI).
¤ RNase is an enzyme that degrades RNA molecules
into smaller products. (i.e.- oligoribonucleotide and
ribonucleotide).
¤ The same sequence of reactions is widely used in
the laboratory to convert RNA to DNA for use in
molecular cloning, RNA sequencing, polymerase
chain reaction (PCR), or genome analysis.
 REVERSE TRANSCRIPTASE
T h e N e w
C o r also
¤ A reverse transcriptase, o n a v i
known as RNA dependent
r u s
DNA polymerase, is a DNA polymerase
enzyme that transcribes single-stranded RNA into
double-stranded DNA.
¤ Normal transcription involves the synthesis of RNA
from DNA; hence, reverse transcription is the reverse of this.
¤ Reverse Transcriptase was discovered by Howard
Temin and Baltimore in 1970 independently.
¤ Shared Nobel Prize in Physiology or Medicine in 1975
for their discovery.
¤ The other names of Reverse Transcriptase is
RNA directed DNA Polymerase, DNA Nucleotidyl
transferase (RNA directed), or Revertase.
¤ Reverse transcriptase common in Retrovirus.
¤ Reverse Transcriptase have fallowing activity:
1- RNA dependent DNA polymerase.
2- RNase H.
3- DNA dependent DNA polymerase.
 Mechanism
of reverse
transcription
T h e N e w
C o r o n a v i
r u s
T h e N e w
C o r o n a v i
r u s
 Mechanism of reverse
1.A retrovirus-specific
T h e N ecellular
transcription. w tRNA
C o r o n a v i
hybridizes with a
r u s
complementary region
called the primer-binding site (PBS).
2.A DNA segment is extended from tRNA
based on the sequence of the retroviral
genomic RNA.
3.The viral R and U5 sequences are
removed by RNase H.
4.First jump: DNA hybridizes with the
remaining R sequence at the 3' end.
6. Most viral RNA is removed
T h e N e w
by RNase
H. C o r o n a v i
r u s
7. A second DNA strand is extended
from the viral RNA.
8. Both tRNA and the remaining viral
RNA are removed by RNase H.
9. Second jump: The PBS region of the
second strand hybridizes with the PBS
region of the first strand.
10. Extension on both DNA strands.
 RETROVIRUSES
Is a type of RNA virus that inserts a copy
of its genome into the DNA of a host cell
that it invades, thus changing the genome
of that cell.
Typical retrovirus has three or four genes.
Gag-pol- env
Retrovirus are called + strand because
viral RNA itself code for protein products.
R : Redundant or repeating seq. – 10-80
U5: 80 – 100
U3: 170-1350
R E P L I C AT I O
N FIDELITY
 There are three different replication
systems during T h etheN e wlife cycle of a
C o r o n a v i
retrovirus. r u s
1. RT synthesizes viral DNA from viral
RNA, forming cDNA
2. Second replication process occurs
when host cellular DNA polymerase
replicates the integrated viral DNA.
3. RNA polymerase II transcribes the
proviral DNA into RNA, which will be
packed into virions.
 RT has a high error rate when
T h e N e w
transcribing CRNA into
o r o n a v i
DNA since,
unlike most other
r u s DNA polymerases,
it has no proofreading ability.
 Allowing mutations to accumulate at
an accelerated rate relative to
proofread forms of replication.
 The commercially available RT
produced by Promega are quoted by
their manuals as having error rates
Other than creating single-nucleotide
T h e N e w
polymorphisms, RT have also
C o r o n a v i been shown to be
involved in processes
r u s such as transcript fusions,

exon shuffling and creating artificial antisense

transcripts.
 It has been speculated that this template
switching activity of RT may have been one of
the causes for finding several thousand
unannotated transcripts in the genomes of
model organisms
BIOLOGICA
L
I M P O R TA N
CE
of RT
A. In Antiviral Tdrugs
h e N e w
 As HIV uses C oreverse
r o n a v i
transcriptase to rcopy
u s its
genetic material and generate
new viruses (part of a
retrovirus proliferation circle),
specific drugs have been
designed to disrupt the
process and thereby suppress
Collectively,
its growth. these drugs are known as RT
inhibitors and include the nucleoside and
nucleotide analogues zidovudine (trade name
Retrovir), lamivudine (Epivir) and tenofovir
(Viread) , as well as non-nucleoside inhibitors,
B. In Molecular biology and
T h e N e w
C o r o n a v i
recombinant DNA technology
r u s

1. cDNA formation
RT is used to obtain
DNA copies of RNA
molecules. i.e., cDNAs
 2. Reverse Trancription
-Polymerase Reaction
T h e N e w
C o r o n a v i
(RT-
PCR)
 RT is commonlyr uused
s in research to
the polymerase chain reaction technique
apply

to RNA in a technique called reverse

transcription polymerase chain reaction
(RT-PCR).
 The classical PCR technique can be
applied only to DNA strands, but, with the
help of RT, RNA can be transcribed into
DNA, thus making PCR analysis of RNA
 Reverse transcriptase
T h e N e w
is used also to
create cDNA libraries
C o r o n a vfrom
i mRNA.
r u s
 Reverse transcriptase has also been
employed in insulin production.
 The commercial availability of reverse
transcriptase greatly improved
knowledge in the area of molecular
biology, as, along with other enzymes,
it allowed scientists to cloning,
sequencing, and characterise RNA.
Thank
Yo u ! !