Transcript Expression of the Viral Genome in Prokaryotic Hosts

Expression of the Viral
Genome in Host Cells
(How do viruses express their
genomes?)
Review of Replication,
Transcription, and Translation
 These three processes are separated in time and
space in eukaryotic hosts.
 In prokaryotic hosts all three of these processes
can occur simultaneously.
 Replication
 The DNA polymerases that elongate the chain have
proofreading or editing functions.
 Proofreading removes inappropriate nucleotide
monophosphates at the 3’ end.
Replication
 In double stranded linear DNA, 1 end of each
strand has a free 5’ carbon and 1 end has a
free 3’OH group.
 The two strands are in the opposite orientation
with respect to each other (antiparallel).
 Adenines always basepair with thymines (2
hydrogen bonds) and guanines always
basepair with cytosines (3 hydrogen bonds)
The Structure of DNA
The Structure of DNA
Polymerase template and primer
requirements
 DNA polymerase cannot initiate synthesis on
its own. It needs a primer to prime or start
the reaction.
DNA synthesis
 Synthesis can occur only in the 5’ to 3’
direction.
DNA synthesis
 Remember that DNA replication is
semiconservative:
Semiconservative DNA
replication
Replication
 Replication of the bacterial chromosome
 Begins at the origin of replication and proceeds in
both directions. Synthesis continues until the
whole chromosome is copied.
Bacterial chromosome
replication
Bacterial chromosome
replication
 The rate of replication is controlled at the level of initiation.
The polymerase doesn’t go faster to increase the replication
rate.
 Remember that DNA polymerase requires a primer to initiate
synthesis and that it can only synthesize in the 5’ to 3’
direction.
 Remember also that the two strands are antiparallel; one is
in the 5’ to 3’ direction, while its complementary strand is in
the 3’ to 5’ direction.
 This results in one strand (using the 3’ to 5’ template) being
synthesized continuously (leading strand) and the other strand
(using the 5’ to 3’ template) being synthesized discontinuously
(lagging strand).
Bacterial chromosome
replication
 Leading strand synthesis – an RNA polymerase called a
primase synthesizes a small piece of complementary RNA
1-5 nucleotides long to prime the continuous 5’ to 3’
synthesis, by DNA polymerase III, of the leading strand.
Note that the template is being exposed in the 3’ to 5’
direction as the replication fork opens up.
Leading strand synthesis
Bacterial chromosome
replication
 Lagging strand synthesis – As the replication fork opens up, the
template for the lagging strand is being exposed in the 5’ to 3’
direction.
 But synthesis cannot occur in the 3’ to 5’ direction.
 Therefore, the lagging strand is made discontinuously in
fragments of approximately 1000 nucleotides.
 Each fragment is started by a primase which synthesizes a
stretch of 1-5 nucleotides of RNA.
 This serves as a primer for DNA polymerase III to extend.
 The fragments that are made are called Okazaki
fragments.
 In both the leading and the lagging strands, the RNA made by
the primase is eventually digested away by the 5’ to 3’
exonuclease function of DNA polymerase I.
 DNA polymerase I then fills in, with DNA, the gap made by the
RNA digestion and the fragments are joined by DNA ligase.
Lagging strand synthesis
Lagging strand synthesis
Replication fork
Replication fork
Replication
 Replication of eukaryotic DNA
 Eukaryotic DNA is linear and much longer
 In order to replicate rapidly, it must have many
simultaneous replication forks. Therefore there is
an origin of replication every 10-1000 um along
the DNA.
DNA Replication
 The requirement for a primer for DNA synthesis creates a
problem for achieving complete replication of linear genomes
 There is no mechanism to fill in the gap left when the RNA
primer is removed at the 5’ end of the leading strand
 When the replication fork reaches the other end, a similar
problem arises with the lagging strand.
 Further replication would result in smaller and smaller strands
of DNA
 Eukaryotic cells solve this problem through the use of
telomeres at the ends of their linear chromosomes. They
replicate outside the normal replication process, thus
preserving the normal chromosome length.
 How do viruses solve this problem?
End Replication Problem
Review of Replication,
Transcription, and Translation
 Transcription – In transcription, a strand of
RNA is synthesized from a portion of the
cell’s DNA which has opened up via the
activity of an endonuclease.
 Transcription requires RNA polymerase and a
supply of RNA nucleotides.
 RNA polymerase can synthesize RNA only in the 5’
to 3’ direction.
 RNA polymerase does not have proof-reading
function
 RNA polymerase does not require a primer to
initiate synthesis
Transcription
 Only one strand of the DNA serves as a template for
the RNA synthesis. This is called the sense strand
and the other strand is called the anti-sense strand.
 The region on the DNA where the RNA polymerase
binds to begin transcription is called the promoter.
 The region on the DNA where transcription ends is
called the terminator and at this point RNA
polymerase and the newly formed RNA fall off.
 The DNA double helix then reforms.
Transcription
Transcription
Prokaryotic Transcription
 Prokaryotic transcription – one RNA
polymerase makes all three types of RNA
(mRNA, tRNA, and rRNA)
 Structural genes for metabolic pathways are
physically contiguous and under the control of a
single set of controlling elements (an operon).
 The transcription product is polycistronic mRNA
with no 5’ cap or 3’ poly A tail.
 Transcription and translation are tightly coupled.
Simultaneous Transcription and
Translation
Eukaryotic transcription
 Eukaryotic transcription – each type of RNA is made
using a different RNA polymerase
 No operons; each gene is regulated by its own controlling
elements
 The transcription product is a monocistronic mRNA that has a 5’
7-methylguanosine cap and a 3’ poly A tail, and that it is usually
a spliced product of the initial RNA transcript.
 Transcription and translation are separated in both time and
space.
 Translation requires identification of the initiation codon,
AUG. A ribosome binding site must also be present.
Termination of translation occurs at a nonsense or stop
codon, UAG, UAA, or UGA.
Amplification steps in Viral
Replication
 DNA viruses – can either bring in their own DNA

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polymerase or they can use the host cell DNA
polymerase for replication of the viral genome.
Similarly, they can bring in their own RNA
polymerase for transcription or they can use the
RNA polymerase of the host cell.
In order for the virus to use the host cell
machinery for replication and transcription, the
viral genome must contain the appropriate
controlling sequences.
Amplification steps in Viral Replication
 RNA viruses – Neither prokaryotic nor eukaryotic
cells contain RNA dependent RNA polymerases,
therefore, all RNA viruses must bring in their
own enzymes for replication of their genomic
RNA and for transcription.
 Positive strand RNA viruses only need to bring in the
gene that encodes the RNA dependent RNA
polymerase (replicase) enzyme.
 Negative strand RNA viruses must bring in both the
gene that encodes the replicase enzyme and a copy
of the enzyme (i.e. the protein) itself. WHY?
Expression of the viral genome
 For both DNA and RNA viruses
 Genes expressed before genomic replication are the
early genes.
 Genes expressed after genomic replication are the
late genes.
 The early gene products are usually involved in
helping the virus to establish control of the host cell
and in getting the viral genome ready for replication.
 The late gene products are usually structural proteins
or proteins involved in maturation and release of the
virus.