Expression of the Viral Genome in Host Cells (How do viruses express their genomes?)

Expression of the Viral Expression of the Viral Genome in Host CellsGenome in Host Cells(How do viruses express their (How do viruses express their

genomes?)genomes?)

Review of Replication, Review of Replication, Transcription, and Transcription, and TranslationTranslation These three processes are separated in These three processes are separated in

time and space in eukaryotic hosts.time and space in eukaryotic hosts. In prokaryotic hosts all three of these In prokaryotic hosts all three of these

processes can occur simultaneously.processes can occur simultaneously. ReplicationReplication

The The DNA polymerasesDNA polymerases that elongate the chain that elongate the chain have proofreading or editing functions. have proofreading or editing functions. ProofreadingProofreading removes inappropriate nucleotide removes inappropriate nucleotide

monophosphates at the 3’ end.monophosphates at the 3’ end.

ReplicationReplication In double stranded linear DNA, 1 end of In double stranded linear DNA, 1 end of

each strand has a free 5’ carbon and 1 each strand has a free 5’ carbon and 1 end has a free 3’OH group. end has a free 3’OH group. The two strands are in the opposite orientation The two strands are in the opposite orientation

with respect to each other (with respect to each other (antiparallelantiparallel).). Adenines always basepair with thymines Adenines always basepair with thymines

(2 hydrogen bonds) and guanines always (2 hydrogen bonds) and guanines always basepair with cytosines (3 hydrogen basepair with cytosines (3 hydrogen bonds)bonds)

The Structure of DNAThe Structure of DNA

Polymerase template and Polymerase template and primer requirementsprimer requirements

DNA polymerase cannot initiate DNA polymerase cannot initiate synthesis on its own. It needs a synthesis on its own. It needs a primerprimer to prime or start the reaction.to prime or start the reaction.

DNA synthesisDNA synthesis Synthesis can occur only in the Synthesis can occur only in the 5’ to 3’5’ to 3’

direction.direction.

DNA synthesisDNA synthesis Remember that DNA replication is Remember that DNA replication is

semiconservative:semiconservative:

Semiconservative DNA Semiconservative DNA replicationreplication

ReplicationReplication Replication of the bacterial chromosomeReplication of the bacterial chromosome

Begins at the Begins at the origin of replicationorigin of replication and and proceeds in both directions. Synthesis proceeds in both directions. Synthesis continues until the whole chromosome is continues until the whole chromosome is copied.copied.

Bacterial chromosome Bacterial chromosome replicationreplication


The rate of replication is controlled at the level of The rate of replication is controlled at the level of initiationinitiation. The polymerase doesn’t go faster to . The polymerase doesn’t go faster to increase the replication rate.increase the replication rate.

Remember that DNA polymerase requires a primer to Remember that DNA polymerase requires a primer to initiate synthesis and that it can only synthesize in the initiate synthesis and that it can only synthesize in the 5’ to 3’ direction. 5’ to 3’ direction.

Remember also that the two strands are antiparallel; Remember also that the two strands are antiparallel; one is in the 5’ to 3’ direction, while its one is in the 5’ to 3’ direction, while its complementary strand is in the 3’ to 5’ direction. complementary strand is in the 3’ to 5’ direction.

This results in one strand (using the 3’ to 5’ template) This results in one strand (using the 3’ to 5’ template) being synthesized continuously (leading strand) and the being synthesized continuously (leading strand) and the other strand (using the 5’ to 3’ template) being other strand (using the 5’ to 3’ template) being synthesized discontinuously (lagging strand).synthesized discontinuously (lagging strand).


Leading strand synthesisLeading strand synthesis – – an RNA polymerase an RNA polymerase called a primasecalled a primase synthesizes a small piece of synthesizes a small piece of complementary RNA 1-5 nucleotides long to complementary RNA 1-5 nucleotides long to prime the continuous 5’ to 3’ synthesis, by prime the continuous 5’ to 3’ synthesis, by DNA DNA polymerase IIIpolymerase III, of the leading strand. Note that , of the leading strand. Note that the template is being exposed in the 3’ to 5’ the template is being exposed in the 3’ to 5’ direction as the replication fork opens up.direction as the replication fork opens up.

Leading strand synthesisLeading strand synthesis


Lagging strand synthesisLagging strand synthesis – As the replication fork opens up, – As the replication fork opens up, the template for the lagging strand is being exposed in the 5’ the template for the lagging strand is being exposed in the 5’ to 3’ direction. to 3’ direction.

But synthesis cannot occur in the 3’ to 5’ direction. But synthesis cannot occur in the 3’ to 5’ direction. Therefore, the lagging strand is made discontinuously in Therefore, the lagging strand is made discontinuously in

fragments of approximately 1000 nucleotides. fragments of approximately 1000 nucleotides. Each fragment is started by a Each fragment is started by a primase primase which synthesizes which synthesizes

a stretch of 1-5 nucleotides of RNA. a stretch of 1-5 nucleotides of RNA. This serves as a primer for This serves as a primer for DNA polymerase IIIDNA polymerase III to extend. to extend. The fragments that are made are called The fragments that are made are called Okazaki Okazaki

fragments.fragments. In both the leading and the lagging strands, the RNA made In both the leading and the lagging strands, the RNA made

by the primase is eventually digested away by the 5’ to 3’ by the primase is eventually digested away by the 5’ to 3’ exonuclease function of exonuclease function of DNA polymerase IDNA polymerase I. .

DNA polymerase I then fills in, with DNA, the gap made by DNA polymerase I then fills in, with DNA, the gap made by the RNA digestion and the fragments are joined by DNA the RNA digestion and the fragments are joined by DNA ligase.ligase.

Lagging strand synthesisLagging strand synthesis

Replication forkReplication fork

ReplicationReplication Replication of eukaryotic DNAReplication of eukaryotic DNA

Eukaryotic DNA is linear and much longerEukaryotic DNA is linear and much longer In order to replicate rapidly, it must have In order to replicate rapidly, it must have

many simultaneous replication forks. many simultaneous replication forks. Therefore there is an origin of replication Therefore there is an origin of replication every 10-1000 um along the DNA.every 10-1000 um along the DNA.

DNA ReplicationDNA Replication The requirement for a primer for DNA synthesis creates a The requirement for a primer for DNA synthesis creates a

problem for achieving complete replication of linear problem for achieving complete replication of linear genomes genomes

There is no mechanism to fill in the gap left when the RNA There is no mechanism to fill in the gap left when the RNA primer is removed at the 5’ end of the leading strandprimer is removed at the 5’ end of the leading strand

When the replication fork reaches the other end, a similar When the replication fork reaches the other end, a similar problem arises with the lagging strand.problem arises with the lagging strand.

Further replication would result in smaller and smaller Further replication would result in smaller and smaller strands of DNAstrands of DNA

Eukaryotic cells solve this problem through the use of Eukaryotic cells solve this problem through the use of telomeres at the ends of their linear chromosomes. They telomeres at the ends of their linear chromosomes. They replicate outside the normal replication process, thus replicate outside the normal replication process, thus preserving the normal chromosome length.preserving the normal chromosome length.

How do viruses solve this problem?How do viruses solve this problem?

End Replication ProblemEnd Replication Problem

Review of Replication, Review of Replication, Transcription, and Transcription, and TranslationTranslation TranscriptionTranscription – In transcription, a strand of – In transcription, a strand of

RNA is synthesized from a portion of the RNA is synthesized from a portion of the cell’s DNA which has opened up via the cell’s DNA which has opened up via the activity of an activity of an endonucleaseendonuclease.. Transcription requires Transcription requires RNA polymeraseRNA polymerase and a and a

supply of RNA nucleotides.supply of RNA nucleotides. RNA polymerase can synthesize RNA only in the RNA polymerase can synthesize RNA only in the 5’ to 5’ to

3’3’ direction. direction. RNA polymerase does not have proof-reading function RNA polymerase does not have proof-reading function RNA polymerase does not require a primer to initiate RNA polymerase does not require a primer to initiate

synthesissynthesis

TranscriptionTranscription Only one strand of the DNA serves as a template Only one strand of the DNA serves as a template

for the RNA synthesis. This is called the for the RNA synthesis. This is called the sense sense strandstrand and the other strand is called the and the other strand is called the anti-anti-sense strand.sense strand.

The region on the DNA where the RNA polymerase The region on the DNA where the RNA polymerase binds to begin transcription is called the binds to begin transcription is called the promoterpromoter..

The region on the DNA where transcription ends is The region on the DNA where transcription ends is called the called the terminatorterminator and at this point RNA and at this point RNA polymerase and the newly formed RNA fall off.polymerase and the newly formed RNA fall off.

The DNA double helix then reforms.The DNA double helix then reforms.

TranscriptionTranscription

Prokaryotic TranscriptionProkaryotic Transcription Prokaryotic transcription – one RNA Prokaryotic transcription – one RNA

polymerase makes all three types of RNA polymerase makes all three types of RNA (mRNA, tRNA, and rRNA)(mRNA, tRNA, and rRNA) Structural genes for metabolic pathways are Structural genes for metabolic pathways are

physically contiguous and under the control of a physically contiguous and under the control of a single set of controlling elements (an single set of controlling elements (an operonoperon).).

The transcription product is polycistronic mRNA The transcription product is polycistronic mRNA with no 5’ cap or 3’ poly A tail. with no 5’ cap or 3’ poly A tail.

Transcription and translation are tightly coupled.Transcription and translation are tightly coupled.

Simultaneous Transcription and Simultaneous Transcription and TranslationTranslation

Eukaryotic transcriptionEukaryotic transcription Eukaryotic transcription – each type of RNA is Eukaryotic transcription – each type of RNA is

made using a different RNA polymerasemade using a different RNA polymerase No operons; each gene is regulated by its own No operons; each gene is regulated by its own

controlling elementscontrolling elements The transcription product is a monocistronic mRNA that The transcription product is a monocistronic mRNA that

has a 5’ 7-methylguanosine cap and a 3’ poly A tail, and has a 5’ 7-methylguanosine cap and a 3’ poly A tail, and that it is usually a spliced product of the initial RNA that it is usually a spliced product of the initial RNA transcript.transcript.

Transcription and translation are separated in both time Transcription and translation are separated in both time and space.and space.

Translation requires identification of the Translation requires identification of the initiation initiation codoncodon, AUG. A ribosome binding site must also , AUG. A ribosome binding site must also be present. Termination of translation occurs at a be present. Termination of translation occurs at a nonsense or stop codonnonsense or stop codon, UAG, UAA, or UGA., UAG, UAA, or UGA.

Amplification steps in Viral Amplification steps in Viral ReplicationReplication

DNA viruses – can either bring in their own DNA DNA viruses – can either bring in their own DNA polymerase or they can use the host cell DNA polymerase or they can use the host cell DNA polymerase for replication of the viral genome. polymerase for replication of the viral genome.

Similarly, they can bring in their own RNA Similarly, they can bring in their own RNA polymerase for transcription or they can use polymerase for transcription or they can use the RNA polymerase of the host cell.the RNA polymerase of the host cell.

• In order for the virus to use the host cell In order for the virus to use the host cell machinery for replication and transcription, the machinery for replication and transcription, the viral genome must contain the appropriate viral genome must contain the appropriate controllingcontrolling sequences. sequences.

Amplification steps in Viral Amplification steps in Viral ReplicationReplication RNA viruses – Neither prokaryotic nor RNA viruses – Neither prokaryotic nor

eukaryotic cells contain eukaryotic cells contain RNA dependent RNA RNA dependent RNA polymerasespolymerases, therefore, all RNA viruses must , therefore, all RNA viruses must bring in their own enzymes for replication of bring in their own enzymes for replication of their genomic RNA and for transcription.their genomic RNA and for transcription. Positive strand RNA viruses only need to bring in Positive strand RNA viruses only need to bring in

the gene that encodes the the gene that encodes the RNA dependent RNA RNA dependent RNA polymerase (replicase)polymerase (replicase) enzyme. enzyme.

Negative strand RNA viruses must bring in both Negative strand RNA viruses must bring in both the gene that encodes the replicase enzyme and the gene that encodes the replicase enzyme and a copy of the enzyme (i.e. the protein) itself. a copy of the enzyme (i.e. the protein) itself. WHY?WHY?

Expression of the viral Expression of the viral genomegenome For both DNA and RNA virusesFor both DNA and RNA viruses

Genes expressed before genomic replication are Genes expressed before genomic replication are the the early genes.early genes.

Genes expressed after genomic replication are the Genes expressed after genomic replication are the late genes.late genes.

The early gene products are usually involved in The early gene products are usually involved in helping the virus to establish control of the host helping the virus to establish control of the host cell and in getting the viral genome ready for cell and in getting the viral genome ready for replication.replication.

The late gene products are usually structural The late gene products are usually structural proteins or proteins involved in maturation and proteins or proteins involved in maturation and release of the virus.release of the virus.

Expression of the Viral Genome in Host Cells (How do viruses express their genomes?)

Documents

Transcript of Expression of the Viral Genome in Host Cells (How do viruses express their genomes?)