CSB351Y1 Lecture 12: Lecture 12
15 Feb 2012
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LECTURE 12
THURSDAY OCTOBER
VIRUS USING REVER TRANSCRIPTASES
Page 87. We were studying viruses which use reverse transcriptase (still in the plant
system). In the notes it says that plant reteroviruses are not classified anymore as
reteroviruses, but a bit later you will understand what the difference is. True reteroviruses
now the genome has to be RNA (like HIV) and they have to integrate into the
chromosome to be classified as a true reteroviruse. So cauliflour mosaic virus, is a DNA
virus (not RNA) so does not integrate into the chromosome but it uses reverse
transcriptase (like HIV) and has its own reverse transcriptase. The same is with Hepatitis
B is a DNA virus, uses reverse transcriptase but does not integrate with the DNA. So the
old classification (was used in the title) because some of these notes are older than us!.
Page 89, top diagram. So cauliflower mosaic viruses is a double stranded circular DNA
genome. They have certain number of genes (he went through them last lecture) (the
capsule protein, the reverse transcriptase which takes a chunk of the genome and the
arrow indicates the orientation of transcription and the major point of this virus is that is
has the 35S promoter. This is the strongest promoter ever to be isolated in plants so its
highly used). So the virus has a capsule protein, reverse transcriptase (which is
responsible for replication of the virus and if you understand this example, you will
understand how HIV replicates). So inclusion body protein is not essential (so you can
remove it during recombination). As any plant virus (no esxception) all plant viruses have
to have a transport of cell to cell movement protein (and this protein is responsible of
allowing the DNA and protein of the viruses to go from cell to cell and remember in the
plant cell they have cell wall & plasmotismata for communication between cells). Also,
this has a small protein responsible for aphid transmission (aphid is a small insect which
helps it get transported). They have a DNA binding protein which is likely involved in
replication (binds to DNA and probably unwinds the DNA).
Supl. To page 89, top figure. Similar to other diagram but we will focus on this.
Remember he told us the genome is nicked (double stranded circular DNA but it has two
nicks, the diagram shows 3 but only should be 2, one has one nick on it means that the
DNA is broken in one spot and the other strand has two nicks on it which means the
DNA is not covalently closed circular DNA; it has 1 nick on one strand and 2 nicks on
the other strand). Question to ask, he said there should be 2 nicks and that the diagram is
wrong, but in the end he said there should be 1 nick on one strand and 2 nicks on the
other, contradiction.
Supl. To page 89, bottom figure The virus just to see how it works, the virus first enters
into the cell (attaches to them) and ends up in the nucleus of the cell. In the nucleus of the
cell, it does not integrate into the chromosome (that’s why we call it episomal, which
means it is in the nucleus but not integrated as part of the chromosome). In the nucleus
whats going to happen is that the 2 nicks are fixed to give you a perfect double stranded
circular DNA. Then using the DNA dependent RNA polymerase of the cell (which
normally transcribes the genes of the chromosomal DNA) but since this one has a very
sexy sequence on the DNA (which is the 35S promoter), it attracts the polymerase more
than anything else. The polymerase opens and unwinds that region of DNA and bind to
that promoter and start transcription from that region to produce a full size RNA (going
all around the circle) which will give you a 35S RNA (mean going around the entire 8000
nucleotide which is a full size mRNA). That mRNA gets out of the nucleus to go into the
cytoplasm to start getting translated. ‘this diagram is confusing because you see the
ribosome are translating the RNA and according to this diagram, the reverse transcription
is occurring in the cytoplasm, but normally this happens inside the nucleus’. You have a
mRNA comes out and produces all of the proteins (capsule, reverse transcriptase…) and
then they start doing their functions. You have a mRNA covering the whole genome the
reverse transcriptase enzyme is going to take the mRNA and convert it into 1 cDNA copy
and make double stranded DNA out of it. This is how the virus replicates. Double
stranded DNA transcribed into mRNA reverse transcribe into single stranded DNA
then double stranded DNA and you have the genome DNA back again.
Page 89, bottom figure and in addition I believe he drew a figure on the overhead which
he referred to, can also refer to supl. To page 89 figure. Now the details of it, how the
reverse transcriptase really works. If you understand this, you understand the replication
of HIV and Hepatitis B. This is going to show you strictly the reverse transcription.
Double stranded DNA fixed inside the nucleus is then transcribed into 35 SRNA. First
reverse transcriptase is an enzyme that reads RNA but it synthesizes complementary
DNA. (remember the DNA dependant RNA polymerase leaves a DNA which is
transcription hence calling it reverse transcription from RNA DNA). To do this
reverse transcriptase you require a primer (a primer is something where you get the
double stranded RNA to sit on it) so reverse transcription in vivo use a transfer tRNA for
the cell (HIV will use LYS tRNA and this guy will you MET tRNA, other use different)
(tRNA is 80-100 nucleotide long, 1 for each amino acid) (this made association with
MET tRNA during evolution). Look, not far from the 5’ end of that 35 SRNA (few
hundred nucleotide down) the RNA of the virus (coming form the DNA) has that
sequence, which is 16-17 nucleotide long which is exactly complementary to the MET
tRNA of the cell (matches perfectly which means that is how the MET tRNA binds to
that because it has a matching, and this is part of the genetic makeup of the virus and is
cellular). The reverse transcriptase which is coming from the translation of the RNA
(remember the RNA is translated and the gene for it is on the RNA so the viral reverse
transcriptase is made) is going to bind to the tRNA. From that point and up it starts
copying the RNA into cDNA (complementary DNA which means cg, at). Then it
say “yes I copied this into DNA but arrives at the end which is a 5’ end of the RNA”.
That’s the first problem (first stop so how would you copy the rest of the RNA?) (drew
something on the board). Reverse transcriptase the enzyme has 3 functions. 1) reverse
transcription (reads out RNA and gives cDNA. 2)it has RNase H activity (RNase H is a
ribonuclease which means it will chew the RNA only when it is Hybridized, so it will eat
the RNA not the DNA so you will degrade the RNA). What’s going to happen when you
remove the RNA, you will have the first jump means (remember) this is a R sequence
and you have it down there as well. You made cDNA, RNA is gone. This other end will
hybridize with the DNA (that’s why they keep repeats on both sides) and now you have
the first big jump and the reverse transcriptase continues the synthesis, remove the RNA
Document Summary
We were studying viruses which use reverse transcriptase (still in the plant system). In the notes it says that plant reteroviruses are not classified anymore as reteroviruses, but a bit later you will understand what the difference is. True reteroviruses now the genome has to be rna (like hiv) and they have to integrate into the chromosome to be classified as a true reteroviruse. So cauliflour mosaic virus, is a dna virus (not rna) so does not integrate into the chromosome but it uses reverse transcriptase (like hiv) and has its own reverse transcriptase. B is a dna virus, uses reverse transcriptase but does not integrate with the dna. So the old classification (was used in the title) because some of these notes are older than us!. So cauliflower mosaic viruses is a double stranded circular dna genome. This is the strongest promoter ever to be isolated in plants so its highly used).
